DOE
EPA
Department
of
Energy
Lawrence Livermore Laboratory
University of California
Livermore CA 94550
UCID-18599
United States
Environmental Protection
Agency
Office of Energy, Minerals, and
Industry
Washington DC 20460
FPA 600 7-79-173
August 1979
Research and Development
Mutagenicity
Testing in
Mammalian Cells
Multiple Drug-
Resistance Markers
Interagency
Energy/Environment
R&D Program
Report
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/7-79-173
August 1979
MUTAGENICITY TESTING IN MAMMALIAN CELLS:
Multiple Drug-Resistance Markers
June H. Carver, Gerald M. Adair, and Daniel L. Wandres
Biomedical Sciences Division
Lawrence Livermore Laboratory
University of California
Livermore, California 94550
Department of Energy Contract No. W-7405-ENG-48
EPA-IAG-D5-E681-AN and AO
U.S. D.O.E. PROJECT DIRECTOR U.S. E.P.A. PROJECT OFFICER
G. Stapleton G. Rausa
Office of Health Effects Research Office of Energy, Minerals and
Asst. Sec. for Environment Industry
U.S. Dept. of Energy U.S. Env. Prot. Agency
Washington, D.C. 20545 Washington, D.C. 20460
-------
FOREWORD
Short-term toxicologic testing, particularly methods for detecting
genetically active substances, has become a keystone of the Environmental
Protection Agency's efforts to detect and evaluate environmental hazards.
Among the most serious hazards to man's future are agents that damage the
genome, or the information bank of cells and organisms, by mechanisms that
can increase the future population load of inherited structural defects and
genetic diseases or that can increase the incidence of cancer in the
current generation. Tests for environmental mutagens play a dual role by
virtue of their ability to detect mutagens and their presumptive ability to
detect carcinogens, owing to abundant laboratory evidence that most known
carcinogens respond positively to tests for mutagens.
The Biomedical Sciences Division of the Lawrence Livermore Laboratory
has for the past five years been heavily engaged in the development and
application of short-term tests for genetic toxicology. Specific examples
are as follows:
• basic studies of classes of mutations in mammalian cells in
culture and methods for selective recovery of mutant cells;
• development of suitable strains of cells for detection of forward
mutations at multiple loci, and of detailed experimental protocols
for mutagen detection;
• development and application of multiple modes of testing in vitro
and in vivo for chromosomal damage and misrepair in the form of
sister chromatid exchange, together with simultaneous correlation
with mutation induction;
• development and application of highly sensitive tests for injury
to sperm cells in the male testis and to oocytes in the juvenile
female ovary;
• development of automated cytochemical methods for detection of
rare events of mutation and malignant transformation of cells in
living animals, including man;
• application of cytogenetic and automated cytopathologic methods to
workforce populations engaged with energy technology, and
-------
• in collaboration with our Environmental Sciences Division,
application of a battery of the foregoing short-term tests to
complex effluents from coal gasification and oil shale retorting
technologies.
The following report describes the development and partial validation
of one of the major tests in our genetic toxicology test battery. A strain
of cultured Chinese hamster ovary (CHO) cells has been selected that is
capable of recognizing mutations in four independent genes. All parameters
that must be controlled for reproducible quantitative assays have been
validated with a set of direct-acting mutagens. Work now in progress will
extend the assay to chemicals requiring metabolic activation and to
partially fractionated complex effluents. This assay is a leading
contender among reliable mammalian cell mutagenesis tests that should be
applied when appropriate to confirm the bacterial screening tests, and to
verify that the genetic damage suggested by a screening test also occurs in
the cells of higher organisms.
Frederick T. Hatch, M.D., Ph.D.
Section Leader for Cell Biology and
Mutagenesis
Lawrence Livermore Laboratory
111
-------
DISCLAIMER
This report has been reviewed by the Lawrence Livermore Laboratory,
University of California, and the U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
IV
-------
PREFACE
Short-term mutagenesis assays are expected to play a major role in
identifying potentially toxic or mutagenic agents in the environment. A
tier concept has evolved that relates the extent and rigor of the screening
protocols to the expected level or extent of human exposure. While the
first tier uses lower organisms for rapid, cost-effective prescreening,
mammalian cells are desirable at the second level of testing because
microbial and mammalian cells differ in their uptake and metabolic
modification of many mutagens, and there are marked differences in the
structure, organization and repair of procaryotic and eucaryotic genomes.
Moreover, mammalian assay systems evaluate different portions of the genome
and assay different types of genetic damage. Assays for forward mutation
at several loci might be expected to respond to most types of genetic
damage, minimizing false positive and negative tests. The latter point is
of major importance; while many pure compounds have been tested, the
frequency of false positives and negatives is virtually unknown for complex
organic mixtures or crude effluent samples.
As an rn vitro test system, Chinese hamster ovary (CHO) cells combine
the favorable growth characteristics, e.g., high plating efficiency and
rapid growth in either monolayer or suspension culture, necessary for
large-scale cost-effective mutagen screening, with a near-diploid genome
that facilitates genetic analysis. Direct drug-resistance selection
procedures, such as those for mutants with alterations in genes coding for
various salvage enzymes in purine or pyrimidine metabolism, allow
quantitative mutagenicity testing at specific well-defined genetic loci.
We have developed a CHO cell line that can be monitored for forward
mutations simultaneously at four gene loci: the autosomal recessive aprt
(adenine phosphoribosyltransferase) and _tk (thymidine kinase) genes, the
widely used X-linked hgprt (hypoxanthine-guanine phosphoribosyltransferase)
gene, and the co-dominant gene for Na-K-ATPase. This multiple-marker assay
can now be used to assess two important areas: (1) determination of the
quantitative dose-response relationships for toxicity and mutagenicity
observed with compounds requiring metabolic activation and known to be
present in environmental pollutant mixtures, and (2) development of methods
to allow activation and assay of crude mixtures and effluents after only
limited fractionation or purification.
-------
ABSTRACT
As a first step in the development of a multiple-marker, mammalian-cell
mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO)
cell line that is heterozygous for both the adenine
phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci.
Presumptive aprt +'~ heterozygotes with intermediate levels of APRT
activity were selected from unmutagenized CUO cell populations on the basis
of resistance to low concentrations of the adenine analog, 8-azadenine. A
functional aprt +'~ heterozygote with ~50% wild-type APRT activity was
subsequently used to derive sublines that were also heterozygous for the tk
locus. Biochemical and genetic characterization of one such sub line,
CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and
^k loci. Chinese hamster ovary (CHD) cell lines heterozygous at both the
adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci
were used for single-step selection of spontaneous and induced mutants
resistant to 8-azaadenine (AAr), 6-thioguanine (TGr), ouabain (OUAR),
or 5-fluorodeoxyuridine (FUdRr). Mutation data are reported for direct
mutagens (EMS, ethyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-
nitrosoguanidine; NQO, 4-nitroquinoline-l-oxide) and promutagens (DMN, di-
methylnitrosamine; BP, benzo(a)pyrene) activated by rat liver homogenates.
Significant dose-dependent increases in mutation frequency were observed
for all four genetic markers after treatment of CHO-AT3-2 cells with ethyl
methanesulfonate. Critical plating densities were established for AAr,
TGr, and FUdRr; no density dependence was observed for OUAR.
Expression of mutant phentotypes after mutation induction with EMS, DMN, or
BP was optimal after 2 to 4 d for AAr, 6 to 8 d for TGr, 3 d for
OUAR, and 1 to 3 d for FUdRr. The induced mutant frequencies as a
function of relative cell survival after treatment with EMS, DMN, or BP
showed locus-specific differences in sensitivity. Of 59 clonal isolates
resistant to AA and assayed for APRT activity, 87% had < 5% wild type
activity; of 30 TGr clones assayed, 83% had < 5% wild type HGPRT activity.
Of 42 FUdRr clones assayed, 98% had < 1% wild type TK activity. Fifty additional
clones selected in medium containing FUdR displayed cross resistance to
5-bromodeoxyuridine (BUdR) and trifluorothymidine (TFT) and all were sen-
sitive to HAT (hypoxanthine-amethopterin-thymidine) medium. The jjc locus
showed the largest mutational response as a function of cell survival after
mutagen treatment. The rapid expression kinetics for FUdRr and the
possibility that the locus detects a broader spectrum of genetic lesions
than the other drug-resistance markers are discussed in terms of a
sensitive screening assay for detecting potential mutagens.
VI
-------
This report was submitted by the Lawrence Livermore Laboratory (Dept.
of Energy, Contract No. W-7405-ENG-48) in partial fulfillment of
Interagency Agreements IAG-D5-E681-AN and AO under the sponsorship of the
U.S. Environmental Protection Agency. This report covers a period from
January, 1976 to January, 1980; work is still ongoing.
vii
-------
CONTENTS
PAGE
Foreword ii
Disclaimer iv
Preface v
Abstract vi
Contents viii
Lis t of Figures ix
List of Tables xi
List of Abbreviations xii
Acknowledgment xiii
1. Introduction 1
2. Conclusions and Recommendations 3
3. Materials and Methods 5
4. Experimental Procedures 9
5. Results 12
6. Discussion 44
References 52
Glossary 61
Vlll
-------
List of Figures
Number Page
1 Derivation of aprt*' heterozygous cell lines .................. 12
2 Derivation of tk+/~ heterozygous cell lines .................... 16
3 Increase in induced frequency of FUdRr, TGr, AAr, and
OUAR mutants with increasing concentration (mM) of EMS ...... ... 22
4 Reconstruction experiments using two aprt*' , hgprt"
lines with increasing numbers of IB-2 cells ................... 24
5 The observed frequency of spontaneous mutants selected in
AG, AA, TG, QUA, FUdR, TFT, or FUdR with increasing density
of IB-2 cells ................................................. 25
6 Decreasing relative survival (S) of CHO-IB-2 or CHO-AT3-2
cells with exposure dose (M) of direct mutagens MNNG, 4-NQO,
and EMS [[[ 28
7 Decreasing relative survival (S) of CHO-AT3-2 cells with
increasing S-9 or microsome concentrations
(mg/ml total protein) ........................................ 30
8 Induced AAr mutant frequency as a function of expression
time before selection with AA ................................ 32
9 Induced TGr mutant frequency as a function of expression
time before selection with TG ................................ 34
10 Induced FUdRr or OUAR mutant frequency as a function of
expression time before selection with FUdR or QUA ............ 36
lla Increase in induced frequency of AAr mutants with decreasing
relative survival after treatment with DMN, BP, or EMS ....... 38
lib Increase in induced frequency of TGr mutants with decreasing
-------
lie Increase in induced frequency of OUAR mutants with decreasing
relative survival after treatment with DMN, BP, or EMS 40
lid Increase in induced frequency of FUdRr mutants after optimal
expression time with decreasing relative survival after
treatment with DMN, BP, or EMS 41
12 Activities of HGPRT, APRT, or TK (relative to wild type) for
spontaneous and induced mutants 43
-------
List of Tables
Number Page
1 Forward mutation frequencies of parental and heterozygous
cell lines 14
2 Luria-Delbruck fluctuation analysis of azaadenine-resistance
in IB-2 cells 15
3 Luria-Delbruck fluctuation analysis of thymidine analogue
resistance in AT3-2 cells 18
4 Complementation analysis of thymidine analog-resistant strains.. 19
5 Segregation analysis of heterozygosity in CHO-AT3-2 20
6 Induced mutant frequency of CHO-IB-2 cells treated with
direct mutagens 29
XI
-------
List of Abbreviations
ct-MEM alpha-minimum essential eagle medium
AA 8-azaadenine
AG 8-azaguanine
AMP adenosine monophosphate
APRT adenine phosphoribosyltransferase, EC 2.4.2.7
ATP adenosine triphosphate
BP benzo(a)pyrene
BUdR 5-bromodeoxyuridine
CdR deoxycytidine
CHO Chinese hamster ovary
DEAE diethylamino ethyl cellulose
DFCS dialyzed fetal calf serum
DMN dimethylnitrosamine
EDTA ethylenediamine tetraacetic acid
EMS ethyl methanesulfonate
PCS fetal calf serum
FUdR 5-fluorodeoxyuridine
HAT hypoxanthine-amethopterin-thymidine
HGPRT hypoxanthine guanine phosphoribosyltransferase, EC 2.4.2.8
IMP inosine monophosphate
MNNG N-methyl-N'nitro-N-nitrosoguanidine
4-NQO 4-nitroquinoline-l-oxide
Na+-
K+ATPase Na+- and Reactivated, Mg++-dependent ATPase, EC 3.6.1.3
QUA ouabain octahydrate
PBS phosphate-buffered saline
PE mean plating efficiency
PEG polyethylene glycol
PRPP 5-phosphoribosyl-l-pyrophosphate
S mean relative survival; surviving cell fraction
S.D. standard deviation
SEM standard error of the mean
TG 6-thioguanine
TFT trifluorothymidine
TK thymidine kinase, EC 2.7.1.21
WT wild type cells
Xll
-------
ACKNOWLEDGMENTS:
The authors thank Drs. W.C. Dewey, T.B. Shows, and R.G. Fenwick for
providing several cell lines used in this study, and Drs. E.W. Branscomb,
L.H. Thompson, M.M. Moore-Brown, and D. Clive for helpful discussions
and/or the provision of unpublished manuscripts relevant to this work. We
are particularly grateful to Dr. F.T. Hatch for invaluable discussion and
guidance during the course of this study. We also thank E.P. Salazar and
M.G. Knize for technical assistance.
Kill
-------
SECTION 1
INTRODUCTION
In vitro mutagenesis assays are expected to play a major role in
identifying carcinogenic or mutagenic agents in the environment (8,
18,65). Because mechanisms for the generation and repair of
mutagen-induced damage may vary considerably in procaryotic and eucaryotic
systems, mammalian mutagenesis assays are needed to confirm and extend the
microbial data. The use of multiple genetic markers may increase the
reliability of such mutagenesis assays for those compounds having diverse
types of interaction with genetic material.
The most thoroughly investigated mutant phenotypes of mammalian cells
are drug-resistant mutants with presumptive alterations in genes coding for
salvage enzymes in the purine and pyrimidine metabolic pathways (1,5,
10,13,14,17,22,24,25,26,35,38,45,44,49,52,58,69,70,74,75,78,80,86,92,96)
Operationally, the definition of a drug resistant phenotype is a mutant's
ability to survive in the presence of cytotoxic purine or pyrimidine
analogs. Analogs frequently used include 6-thioguanine (TG) or
8-azaguanine (AG), which are metabolized by the salvage enzyme,
hypoxanthine guanine phosphoribosyltransferase (HGPRT), or 8-azaadenine
(AA) or 2,6-diaminopurine (DAP), which are metabolized by adenine
phosphoribosyltransferase (APRT). Thymidine analogs such as
5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), or
trifluorothymidine (TFT) selectively kill cells with thymidine kinase (TK)
activity. Ouabain (OUA), which specifically inhibits active transport by
Na+-K+ ATPase, is also used to select drug-resistant mutants (6,19,27).
The presence of more than one functional allele for autosomal loci in
mammalian cells makes direct, single-step selection of recessive,
•drug-resistance phenotypes impractical as a means of assaying induced
mutations. Mammalian cell mutagenesis studies have, inmost cases, used
resistance to either 6-thioguanine (TG) or 8-azaguanine (AG) as a
convenient, well-characterized, and selectable genetic marker to measure
forward mutation at the X-linked hypoxanthine-guanine phosphoribosyl
transferase (hgpjrt) locus (1,15,16,23,30,37,38,44-46,50,58,72-76,83,92,96).
Systematic determination of optimal conditions for the phenotypic
expression and selection of TGr mutants in Chinese hamster cells
(15,16,37,48,72,73,74,75,83,92,96) as well as the development and
application of a practical, in vitro CHO/HGPRT mutagenesis assay system by
Hsie and co-workers (29,30,44-47,72-76), has facilitated quantitative
analysis of mutation induction by a wide range of physical and chemical
-------
agents. Comparable selection of APRT- or TK-deficient mutants requires the
prior derivation of cells heterozygous at the autosomal recessive aprt and
tk loci (17,25,26,52,80,86). Ouabain (QUA) resistance is a genetic marker
that is codominantly expressed and can be used to measure mutation
induction inmost manualian cells (5,6,19,27,90). Since the types of
mutational damage, as well as the mechanisms by which they are produced,
may vary considerably for different mutagens, specific genetic loci may
differ markedly in their mutability by a particular agent. Mutagenesis
assay systems employing multiple genetic markers may be more effective in
detecting a broader spectrum of mutagenic and potentially carcinogenic
agents. Extensive validation and application of the L5178Y TK+/~ mouse
lymphoma mutagenesis assay developed by Clive and co-workers (21-23,25,
26,67), which permits quantitation of forward mutation at the autosomal
thymidine kinase (tk) locus, has revealed apparent differences in the
mutagenic potential of certain agents at the hgprt and _tk_ loci. Several
chemicals that are only weakly or non-mutagenic at the hgprt locus appear
to be strongly mutagenic at the ^k locus (26). Operationally, the
relatively short time required for phenotypic expression of induced
mutations at the tk locus (10,26,67,85) facilitates its use in mutagenesis
assays. Since technical disadvantages limit the utility of the TGr
marker in the L5178Y TK+/~ mouse lymphoma system (22,23,26,58,67), we
were encouraged to develop a CHO multiple-marker mutagenesis assay system
that could use both the hgprt and _tk loci and would combine and expand the
attributes of the two major ^ri vitro mammalian-cell mutagenesis assay
systems currently being used.
In this study, we describe the derivation of a CHO cell line that is
heterozygous for both the adenine phosphoribosyltransferase (aprt) and
thymidine kinase (tk) loci. This subline allows single-step selection of
autosomal recessive AAr or FUdRr mutant phenotypes as well as the more
commonly used OUAR or TGr genetic markers. Biochemical and genetic
characterization of the heterozygous cell line, and dose response data for
mutation induction at these four genetic loci by the direct-acting mutagen,
ethyl methanesulfonate (EMS), are presented. We also establish optimal
conditions for the phenotypic expression and selection of AAr and FUdRr
mutants of CHO cells and present biochemical validation of the mutant
phenotypes. Optimal drug concentrations, cell plating densities, and
expression time requirements are determined for all four drug-resistance
markers. Mutation data are reported for direct mutagens (EMS, MNNG, NQO)
and promutagens requiring metabolic activation (DMN, BP). Finally, we
discuss the role and expected use of the multiple-marker mutagensis assay
to yield increased sensitivity and reliability in detecting genetic damage
induced by complex pollutant mixtures of environmental concern.
-------
SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The primary objective of this program has been to develop, validate,
and apply multiple-marker, in vitro assays using mammalian cells to
quantify the mutagenic effcts of pollutants arising from energy extraction,
conversion, and use. The multiple-marker system, which was initially
validated with data from standardized dose-response protocols, is now being
applied to analyzing complex organic mixtures from crude effluents.
Because most of the suspected mutagens associated with energy by-products
and effluents require metabolic activation, activation techniques that can
be coupled with the in vitro mammalian assay are being optimized.
Experiments are being designed to compare the relative efficacy of organ
homogenates (S9 or microsomes) versus cell-mediated activation and also to
test whether our system will yield data at multiple gene loci which are
proportional to the relative potency of compounds in other in vitro and in
vivo systems. To further validate the system, paired compounds
(carcinogenic and noncarcinogenic) have been tested. In this study
(MRC/ICI/NIEHS, Mut. Res. 54: 203, 1978), results from the multiple marker
assay are being compared with results from a wide assortment of test
systems, including human cells. The simultaneous use of multiple markers
is expected to test differences in sensitivity or mechanism of damage at
the genetic loci involved, and such a system may increase the generality
and reliability of mutagenesis assays for compounds having diverse
interaction with mammalian genetic material.
Since the mutagenesis assay at mutiple gene loci may be a sensitive
detector of genetic damage induced by complex pollutants of environmental
concern, it should be applied to evaluating potential health effects
associated with effluents from a wide variety of sources. To determine the
versatility and general applicability of the multiple marker approach to
studies of fractions from crude effluents, chemicals representative of many
different classes should be tested. Such compounds, e.g., aromatic amines,
alkyl halides, polycyclic aromatic hydrocarbons, nitrogen-containing
heterocyclics, aliphatics, and aromatics, are frequently found in pollutant
samples and have a broad spectrum of potentially hazardous interactions
with mammalian DNA. Many problems are expected in exposing crude effluents
to mammalian cells with an ir» vitro activation system (general toxicity,
inhibitory or synergistic effects among mutagens, chemical transformation);
in vitro mammalian bioassays must be adapted to accept crude mixtures and
fractionated samples. These assays for forward mutation at several gene
loci are expected to show a response to a broad variety of genetic damage,
-------
minimizing false positive and false negative tests. The latter point is
important because while many pure compounds have been tested, the frequency
of false positive and negative results is not presently known for complex
organic mixtures or effluent samples.
-------
SECTION 3
MATERIALS AND METHODS
Cells and culture conditions. The parental cell line for these studies
was a Chinese hamster ovary (CHO) cell line designated CHO-CIOA, obtained
from Dr. W.C. Dewey. One of the aprt *' heterozygotes we derived from
this line, CHO-IB-2, was subsequently used to derive sublines that were
also heterozygous for the ^k locus. One such subline, designated
CHO-ATS-2, has been further characterized and used in a series of
mutagenesis experiments described in this and succeeding papers (3,16;
Adair & Carver, manuscript in preparation). Other Chinese hamster cell
lines used in this study were the thymidine kinase-deficient strains DON-a3
(obtained from Dr. T.B. Shows), and RJK-92, originally designated 462-10
(obtained from Dr. R.G. Fenwick).
Cells were routinely maintained at 37°C in a-MEM (K.C. Biological,
Inc.) supplemented with antibiotics (penicillin/streptomycin) and 10% fetal
calf serum (K.C. Biological, Inc.). Cell numbers were determined with a
Coulter electronic particle counter. Cultures were periodically tested and
confirmed to be free of mycoplasma. Under standard growth conditions, the
doubling times (T£) of our stock CHO-IB-2 and CHO-AT3-2 cell lines ranged
from 14 to 16 h in monolayer culture and 14 to 18 h in suspension culture.
The doubling time of CHO-IB-2 in suspension culture containing dialyzed
fetal calf serum (DECS) was often 20-22 h with substantial lag time after
dilution; CHO-AT3-2 displayed a consistent doubling time under all culture
conditions, with no significant delays after dilution.
Selection of presumptive heterozygotes for the aprt locus. Presumptive
aprt *l~ heterozygotes with intermediate levels of APRT activity were
selected on the basis of their resistance to low concentrations of AA.
Unmutagenized CHO-CIOA cells and cells from six clonal sublines of CHO-CIOA
were plated at 10^-10^/100 mm dish, in a series of selection media
containing from 3.0 to 5.0 yg/ml AA. The selection media were changed
after one week. Background growth of cells was excessive at the lower AA
concentrations, but discrete colonies were obtained in most of the cultures
at 4.5 to 5.0 vig/ml AA. Considerable clonal variation in the frequency of
these low-level AAr colonies was observed. A large number of colonies
were isolated, plated into fresh selection medium containing 5 ug/ml AA to
test whether they were stably resistant, and then secured in liquid
nitrogen.
-------
Isolation of HATr revertants of TK-deficient mutants. TK-deficient
mutants of CHO-IB-2 were selected in 150 ug/ml BUdR. Cells from eight
spontaneous BUdRr strains with barely-detectable levels of TK activity
( <1% that of wild-type) were subsequently plated into HAT medium (a-MEM +
FCS plus 100 yM hypoxanthine, 4pM amethopterin, 50 uM thymidine), to
select for revertants which had regained partial TK activity. Cells that
lack TK activity are unable to utilize exogenous thymidine and are killed
in the presence of amethopterin. Seven of the eight ^k ~'~ strains
yielded no spontaneous revertants in initial experiments (revertant
frequencies < 3 x 10~7). To obtain HATr revertants, monolayer cultures
of each of the eight ^k "' strains were treated with EMS (150 pg/ml, 16
hours), subcultured at 2-day intervals for 8 days to allow fixation and
phenotypic expression of induced mutations, and then plated into HAT
selection medium (5x10^ cells/100-mm dish, 12 replicate dishes/culture).
Five of the eight lines yielded HATr colonies, at frequencies ranging
from 3 x 10~7 to 6 x 10~5.
Preparation of liver homogenate (S-9 and microsomes). Eight week old
outbred male rats (Simonsen albino, Sprague-Dawley derived) were injected
i.p. with Aroclor 1254 (200 mg/ml in corn oil) at 500 nig/kg body weight.
Four to five d later, they were sacrificed by immobilization in C02
atmosphere followed by arterial bleeding. Livers from > 6 rats were
dissected aseptically, washed with cold, sterile PBS, placed in PBS
(3 mis PBS/ gm wet liver weight), and homogenized with a Polytron
homogenizer (Brinkmann Instruments) at setting 4 for 1 m. The homogenate
was centrifuged at 9000 x g for 15 m at 4°C and the supernatant (S-9) was
quickly frozen and maintained at -80°C for < 6 m. For the preparation of
microsomes (u), the S-9 supernatant was immediately centrifuged at
100,000 x g for 1 h. The pellet was gently homogenized, resuspended in
sterile PBS + 10% glycerol, and maintained at -80°C. Protein
concentration was determined by the method of Lowry (63).
Preparation of cell extracts. Aliquots of 2xl07 cells were taken
from suspension cultures of mutant or wild-type cells in exponential growth
phase. Cells were collected by low-speed centrifugation, washed by
resuspension in cold (0-4°C) phosphate-buffered saline (PBS), and
recentrifuged. Each cell pellet was resuspended in 1.0 ml of cold
extraction buffer (50 mM KH2P04, 10 mM 2-mercaptoethanol, 1.0% Triton
X-100, adjusted to pH 7), and the cell suspensions were sonicated on ice
using a Branson model S125 ultrasonic cell disruptor with a microprobe
tip. Cells were lysed with four 5-second bursts (power setting 4), with
10-second cooling intervals. Resulting cell lysates were cleared of debris
by centrifugation for 30 minutes at 12,000 x g (0-4°C), and the
supernatants were immediately frozen and stored at -80°C. Protein
concentrations were determined by the method of Lowry £t al. (63) using
crystallized bovine serum albumin as a standard.
Adenine phosphoribosyltransferase assays. Adenine
phosphoribosyltransferase (APRT) activities were assayed using the
radioisotopic/DEAE filter disc method of Fenwick and Caskey (37), which
measures the rate of conversion of ^C-adenine to ^C-AMP by APRT.
Each 60 wl reaction mixture contained 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2,
-------
2 mM 5-phosphorylribose-l-pyrophosphate (dimagnesium salt), 0.2 mM
8~ C-adenine (60 Ci/mol), and 0-48 pg extract protein. All assays were
run in duplicate. Background determinations were obtained by substituting
extract buffer for cell extract. Reactions were initiated by adding cell
extract to prewarmed reaction mixtures. Assay mixtures were incubated at
37°C, and 20 P 1 samples were taken after 15 and 30 minutes. Reactions
were terminated by spotting the samples directly onto 2.4 cm DE-81 DEAE
filter discs (Whatman) that had been pre-treated with 50 U1 of 100 mM EDTA,
pH 7.0. After the samples had dried, the discs were washed four times wth
20 ml volumes of 1:1 methanol-I^O, once with H20, and once with 95%
ethanol, to remove unreacted l^C-adenine. The amount of ^C-AMP bound
to the discs was subsequently counted by liquid scintillation. Reaction
rates were constant with time for at least 30 minutes and proportional to
the amount of extract protein in the reaction mixture over the range
0-48 pg protein. APRT activities of aprt +/~ heterozygotes and AAr
mutants have been normalized to the mean specific activity of wild-type
CHQ-CIOA cells, 4.3 nmol/min/mg protein.
Thymidine kinase assays. Thymidine kinase (TK) activities were assayed
using the DEAE-disc method described by Roufa et^ aK (80), which measures
the rate of phosphorylation of ^C-thymidine by TK. Each 50 p 1 reaction
mixture contained 200 PM Tris-HCl, pH 8.0, 2 mM MgCl2, 5 mM ATP, 40 PM
2-l^C-thymidine (58 Ci/mol), and 20-80 pg extract protein. All assays
were run in duplicate. Background determinations were obtained by
substituting extract buffer for cell extract. Reactions were initiated by
adding cell extract to prewarmed reaction mixtures. Assay mixtures were
incubated at 37°C for 10-20 minutes, and reactions were terminated by
dilution with 3 ml of ice-cold 1 M sodium formate, pH 6.6. Diluted
reaction mixtures were immediately filtered through 2.4 cm DE-81 DEAE
filter discs (Whatman), which were rinsed three times with cold 1 mM sodium
formate and allowed to dry before counting by liquid scintillation.
Background counts represented less than 0.1% of the input radioactivity.
TK activities of thymidine analog-resistant mutants and ^k +/-
heterozygotes have been normalized to the mean specific activity for
CHO-IB-2 cells, 171± 15 pmol/min/mg protein.
Chemicals and radioisotopes. All chemicals were obtained from Sigma or
Calbiochem except DMN (Aldrich), 4-NQO (K & K Laboratories), BP (Eastman),
5-phosphorylribose-l-pyrophosphate (P.L. Biochemicals), and polyethylene
glycol 1000 or PEG (J.T. Baker Chemicals Co.). All were stored at < 4°C.
The mutagens EMS and DMN (aqueous), MNNG (in ethanol), or NQO (in DMSO,
final [DMSO] <0.015%) were prepared and diluted with growth medium
immediately before use. The BP stock (in DMSO) was stored < 6 months at
room temperature; the stock was not filter-sterilized. Purine analogues
(AA, AG, TG) were dissolved in NaOH and adjusted to pH 7.4 with HC1;
ouabain was dissolved in 100°C 1^0 and immediately diluted with growth
medium. The BUdR, FUdR, and TFT stock solutions (aqueous) were stored
frozen at -20°C and protected from light during all procedures. All
solutions (except BP) were sterilized by filtration through a 0.22pm pore
filter (Millipore Corp.) before use. Radioisotopes (8-14C-adenine and
2-14C-Thymidine) were obtained from Amersham.
-------
Statistics. Where shown, error bars represent one standard error of
the mean (SEM). To determine the extent of variance from the mean, both
the standard deviation of the data points (experimental error) and the
counting error (sampling or Poisson error) were calculated; in all cases,
the larger of these values was used to determine the standard error (7).
Straight line fits were made by standard linear regression analysis. The
t test to obtain P values used Scheffe's special d-solution to the
Behrens-Fisher problem (81); for degrees of freedom, a conservative and
minimal value (n-2) was used, with n equal to the number of data points in
the data set with the smallest number of observations.
-------
SECTION 4
EXPERIMENTAL PROCEDURES
Mutagenesis. Approximately 18-24 h before treatment with mutagens,
cells from a recently-thawed culture were plated at 1 to 2 x 10° per T25
flask or 3 to 4 x 10° per T75 flask (Corning). Treatment with direct
mutagens was carried out in medium supplemented with PCS (a-MEM PCS) by
exposing monolayer cultures to fresh medium or fresh medium containing the
mutagen ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), or 4-nitroquinolinel-oxide (NQO). After 16-18 h the
mutagen-containing medium was removed, the cells were rinsed and dispersed
with trypsin, and 3 to 5 x 10" cells were placed into suspension culture
for expression of phenotypes. Treatment with promutagens requiring
metabolic activation involved a 2 h exposure in a-MEM (serum-free)
containing S-9 or microsomes plus the following co-factors: NADPH, 0.37
mM; NADH, 0.93 mM; NADP, 0.87 mM; glucose-6-phosphate, 6.57 mM. Following
exposure, the cells were washed and allowed a 1 h recovery period in a-MEM
PCS. Approximately 1 to 2 x 10" cells were carried forward in monolayer
culture for expression. After three days incubation, 3 to 5 x 10" cells
were place into suspension culture for continued phenotypic expression (up
to 10 d) and subsequent replating for mutant selection.
After mutagen treatment, aliquots of cells were plated at 200 to 3000
cells/60-mm dish (six replicates per plating) to determine the surviving
cell fraction (S). Following 7 d of incubation, surviving colonies were
fixed with methanol, stained with 0.5% crystal violet, and those colonies
containing 100 or more cells were counted. The plating efficiencies of
untreated controls in a-MEM PCS ranged from 0.65 to 0.90; in medium
supplemented with extensively dialyzed PCS (a-MEM DFCS, K.C. Biological,
Inc), plating efficiencies ranged from 0.55 to 0.75.
Mutant Selection. To quantitate mutation induction by the replating
method, mutagenized or untreated (control) cell populations were incubated
in monolayer and/or suspension culture to allow expression of mutant
phenotypes. Cell populations treated with direct mutagens were maintained
for 7-10 d in suspension culture; those exposed to promutagens requiring
metabolic activation were maintained for the first three d in monolayer
culture and subsequently transferred to suspension culture for 4-7 d
additional expression time. After expression, the cells were resuspended,
plated, and incubated as follows:
-------
Mutant Concentration of # Cells Plated per # Days Incu-
Phenotype Selecting Drug ()jg/ml) 100-mm petri dish bation at 37°
Mr
AGr
TGr
OUAR
FUdRr
BUDRr
TFTr
80
40
4
2200
2
150
3
(0
(0
(0
(3
(0
(0
(0
.59
.26
mM)
mM)
.024 mM)
.00
mM)
.008 mM)
.49
.01
mM)
mM)
3
3
2
8
3
3
3
X
X
X
X
X
X
X
io5
io5
IO5
IO5
IO4
io4
IO4
12-14
16-18
12-14
10-12
12-14
12-12
12-14
Twelve to twenty-four replicate 100-mm dishes containing 16-20 ml of drug-
selection medium were plated for each selecting drug.
With the CHO-AT3-2 cell line, the colony size in extensively dialyzed
serum was enhanced by the addition of deoxycytidine at 10-20 pg/ml (0.076
mM). Media containing DFCS was used for mutant selection with AA, TG, and
FUdR and included deoxycytidine at 20 ug/ml; FUdR selection also included
uridine at 10 Pg/ml (0.04 mM) to reduce incorporation of FUdR metabolites
into RNA (42,43).
To quantitate mutation induction by the in situ method, mutagenized
cells were plated in 100-mm petri dishes at approximately 1.2 x 10^
cells/dish and incubated until the number of cells had increased to the
equivalent of five to six doublings; at that time, the medium was carefully
removed and fresh medium containing the appropriate selecting drug was
added. The relative increase in the number of cells during the expression
period was monitored by counting (after dispersing with trypsin) replicate
cultures that had been inoculated at the same density as in the mutation
experiment. The increase was also determined by plating 100-mm dishes with
approximately 4 x 10^ cells in 100-mm dishes and counting the number of
cells per colony-forming group at appropriate intervals (13). The maximum
density of cells in the control was about 4 to 8 x 10^ cells per 100-mm
dish (i.e., approximately 0.75 to 1.5 x 10^ cells/cm2 or~10^
colonies). The actual cell density in mutagenized cultures was somewhat
lower and dependent upon the dose of mutagen.
To determine the plating efficiency (PE) at the time of mutant
selection, 200 to 400 cells plated' in 60-mm dishes (six replicates per
condition) were counted. The observed mutant frequency represents the mean
number of mutant colonies per dish divided by the number of viable cells,
i.e., the number of cells plated per dish, corrected for PE. The induced
10
-------
mutant frequency is the difference between the observed mutant frequency of
treated cultures and the observed spontaneous mutant frequency of controls.
Luria-Delbruck fluctuation analysis. The pattern and rate of
appearance of drug-resistant variants in cultures was determined by
fluctuation analysis (11,62,64). Thirty-two independent replicate cultures
were initiated from inocula of 50 or 100 cells. The cultures were grown
for 9-10 days (approximately 14-16 doublings) and plated into medium
containing the appropriate selection drug. The distribution of
drug-resistant colonies was analyzed by the methods of Luria and Delbruck
(64) and Lea and Coulson (62) to obtain estimates of the apparent
spontaneous mutation rates for the aprt and ^k loci in CHO-IB-2 or
CHO-AT3-2 cells.
Cell hybridization. Cell hybrids were obtained by PEG-induced fusion,
using the procedure of Davidson and coworkers (33) as modified by Adair e_t
al. (4). PEG-treated cultures were incubated overnight in a-MEM + PCS,
then trypsinized and replated into HAT medium to select for complementing
cell hybrids. Cells that lack TK activity are unable to utilize exogenous
thymidine and are killed in the presence of amethopterin. Complementation
in cell hybrids between two thymidine analog-resistant mutants would allow
colony formation in HAT selection medium, and would indicate that the two
mutants did not share the same genetic defect. Cell hybrids between
HGPRT-deficient TGr cells and each thymidine analog-resistant strain were
included in complementation analysis experiments to determine whether the
TK-deficient mutant phenotypes were dominant or recessive, and as a
positive control for complementation. Controls for reversion for each
mutant strain were also included, but no revertants were observed in
complementation analysis experiments.
Segregation analysis. Utilizing one of the TK-deficient, BUdRr
strains we had previously isolated from CHO-IB-2, we selected a spontaneous
AAr mutant that lacked both APRT and TK activities. Cells from this
aprt '", tk ~/~ strain were subsequently hybridized by PEG-induced
fusion with a TGr subline of CHO-AT3-2 or TGr subline of wild-type
(tk +/+) CHO, to test whether CHO-AT3-2 cells were indeed functionally
hemizygous at both the aprt and ^k loci. Hybrids were selected and grown
to mass culture in HAT medium. An inoculum of 3x10^ HATr cells of each
hybrid were then grown for approximately ten generations in the absence of
further selection and plated into medium containing either 80 yg/ml AA or
150 Mg/ml BUdR. Spontaneous nondisjunction involving the segregation or
loss of dominant wild-type alleles for the aprt or tk loci from such
hybrids will result in reexpression of recessive AAr or BUdRr
phenotypes. The frequencies of such drug-resistant segregants should be
much higher in hybrids between mutant X heterozygotes than in hybrids
between mutant X wild-type cells; only one wild-type allele would have to
be lost for reexpression of the mutant phenotype in the former case, while
two wild-type alleles would have to be lost in the latter. Thus, by
comparing the drug-resistant segregant frequencies in the above hybrids, we
could determine whether CHO-AT3-2 cells were indeed heterozygous at both
the aprt and tk loci.
11
-------
SECTION 5
RESULTS
Isolation and characterization of aprt+'~ heterozygotes. Presumptive
heterozygotes for the autosomal aprt locus were selected from unmutagenized
populations of CHO-CIOA or clonal sublines, on the basis of resistance to
low levels of AA (Fig. 1). This approach was originally used by Jones and
Sargent (52) to isolate aprt heterozygotes from a different CHO strain, but
a subsequent attempt to isolate heterozygotes using similar approaches was
unsuccessful (34). As reported by Jones and Sargent, resistance to AA is
correlated with a loss of APRT activity and occurs in two discrete steps.
The concentration of AA necessary to reduce CHO-CIOA survival to 10%
(DIQ) was approximately 2.5 iig/ml, compared to 7-11 Pg/ml reported by
Jones and Sargent. Using concentrations of 4-5 pg/ml AA, which reduced
CHO-CIOA survival to approximately 10~^, we were able to obtain
aprt +/~ heterozygotes with intermediate levels of APRT activity (Fig.
1). Eleven out of twelve presumptive heterozygotes, confirmed to be stably
resistant to 5 ug/ml AA and subsequently assayed for APRT activity, had
specific activities ranging from 47% to 78% that of wild-type CHO-CIOA.
Three of these presumptive heterozygotes (designated IIA-1, IB-2, IID-4)
with approximately 50% wild-type APRT activity were subsequently employed
in second step selections for spontaneous and EMS-induced mutants resistant
to higher levels of AA (Fig. 1). Mutants resistant to 20-80 ug/ml AA were
isolated and assayed for APRT activity. Fifty-five of the fifty-nine AAr
colonies assayed (93%) had <12% wild-type APRT activity. No significant
differences in residual APRT activity were observed for mutants selected at
20, 40, or 80 ug/ml AA. On the basis of its favorable growth and plating
chacteristics, one aprt +'~ strain, CHO-IB-2, was chosen for further
genetic analysis and use in mutagenesis experiments.
Forward mutation at the aprt locus. Spontaneous and EMS-induced
forward mutation frequencies for the aprt locus for wild-type CHO-CIOA-19
and the aprt +'~ heterozygote, CHO-IB-2, are shown in Table 1. Observed
frequencies of mutants resistant to 80 yg/ml AA were at least several
orders of magnitude higher for the aprt +'~ heterozygote than for
wild-type, aprt +'+ cells. High frequencies of AAr mutants were
induced by treating CHO-IB-2 cells with known direct-acting mutagens such
as EMS (Table 1), indicating that this subline and the AAr marker would
be practical for mutagenesis assays. Most of the AAr mutants assayed had
< 5% wild-type APRT activity. The spontaneous forward mutation rate at the
aprt locus in CHO-IB-2 cells was determined by Luria-Delbruck fluctuation
analysis to be approximately 3x10"? per cell per generation (Table 2).
This value agrees closely with the rate reported by Jones and Sargent (52).
12
-------
Wild-type
aprt+/+
CHO-CIOA (parental line)
APRT activity: 1.00
CIOA-19 (clone of CHO-CIOA)
APRT activity: 1.00
(relative to CHO-CIOA: 1.05)
Presumptive
aprt+/-
Heterozygotes
Presumptive
aprt~/-
Homozygotes
IIA-1
0.51 ± 0.04
Spontaneous
< 0.05(4)
Induced
IB-2
0.52 ± 0.02
Spontaneous
< 0.05(10)
0.49(1)
Induced
IID-4
0.54 ± 0.02
Spontaneous
< 0.05(5)
Induced
< 0.05(6) < 0.05(22) < 0.05(4)
0.10(1) ~ 0.09(1) ~ 0.45(1)
0.42(1) 0.12(2)
0.59(1)
Figure 1. Derivation of aprt*' heterozygous cell lines, with
subsequent single-step selection of spontaneous and
EMS-induced mutants resistant to AA (aprt~'~). Relative
APRT activities are shown, with the number of clones tested
in parentheses.
Derivation of heterozygotes for the tk locus. Although aprt +/~
heterozygotes had been readily selected on the basis of their resistance to
intermediate levels of AA, a similar approach for isolating tk_ +'~
heterozygotes was unsuccessful. Direct, single-step selection of tk +'~
heterozygotes in intermediate concentrations of BUdR was impractical due to
excessive background growth of wild-type cells. Attempts to isolate
tk +'~ heterozygotes using another thymidine analog, TFT, yielded
unstable drug-resistant variants at high spontaneous frequencies (2).
Thus, we were forced to employ an indirect approach similar to that of
Clive £t £1. (22), first isolating rare J* ~'~ mutants of CHO-IB-2 on the
basis of their resistance to high levels of BUdR, and subsequently
selecting HATr revertants of those cells (Fig. 2). We recovered BUdRr
colonies from unmutagenized CHO-IB-2 cell populations in three of five
selection experiments, at an overall frequency of 2 x 10~' (Table 1).
All eight spontaneous BUdRr strains assayed, as well as 32/32
mutagen-induced BUdRr or FUdRr isolates, had barely detectable levels
of TK activity ( <1% that of wild-type). All of these ^k ~/~ mutants
were stable, HAT-sensitive, and cross-resistant to BUdR, FUdR, and TFT.
When 8 Jtk ~/~ mutants were subjected to EMS mutagenesis and plated into
HAT medium, five of the eight strains yielded HATr colonies, at
frequencies ranging from 3 x 10~7 to 6 x 10~5. To determine whether
13
-------
Table 1
Forward mutation frequencies of parental and heterozygous cell lines
Cell line
Selecting Drug Spontaneous
EMS-induced3
CHO-CIOA-19
parental line
+/+ +/+
(aprt > tk )
AA
BUdR
FUdR
_7
3.0 x 10
_7
9.0 + 6.4 x 10 '
-7
5.0 x 10
9.6 +
1.4 +
3.7 +
b
5.2 x 10~6
__b
0.3 x 10~
,b
1.3 x 10~°
IB-2 AA
aprt heterozygote BUdR
(aprt + , tk +^+) FUdR
3.7 + 0.4 x 10
-5
2.4 + 0.8 x 10
-7
7.0 + 7.0 x 10
-8
6.6 + 0.5 x 10
2.3 + 1.0 x 10
5.0 + 5.0 x 10
-4
AT3-2 AA
aprt, tk heterozygote BUdR
(aprt +/~, tk +/~) FUdR
2.7 + 0.2 x 10
-5
2.8 + 0.2 x 10
-3
1.6 + 0.2 x 10
-3
1.5 + 0.1 x 10
-3
6.1 + 0.3 x 10
-3
Expression time 7-8d.
b
100-125 pg/ml, 16-18 hr exposure, S'
C160 ug/ml, 16 hr exposure, S ~0.35.
420 ug/ml, 16 hr exposure, S ~0.30.
0.65 - 0.70.
14
-------
Table 2
Luria-Delbruck fluctuation analysis of
azaadenine-resistance in IB-2 cells
*J
Replicate cultures
Initial number of cells per replicate
Final number of cells per replicate
32
100
1.75 x 107
1
100
1.75 x
107
Cells tested per sampleb; (# samples) 3.6 x 106 (1) 3 x 105 (48)
Number of mutants per sample:
Range
Median
Mean
Variance
Ratio: Variance/Mean
Null fraction
Mutation rate calculated by:
d
Mean method
g
Median method
p
Null fraction method
Q
Maximum likelihood method
0-89
1
3.7
244
65.5
0.47
_7
3.6 x 10 '
_7
3.2 x 10
_7
2.7 + 0.6 x 10
-7
2.4 _+ 0.5 x 10
0-2
0
0.31C
0.38C
1.23°
0.85
Replicate cultures initiated by innoculating Corning T-25 flasks with 100
cells were subsequently transfered to T-75 flasks.
Cells were plated at 3 x 10 /100-mm dish in medium containing 80 pg/ml
AA and incubated for 12 d to allow colony formation.
CThese numbers may be underestimated due to limitations of sample size
which resulted in many plates with no mutant colonies.
^ef. 11.
eRef. 62.
15
-------
these colonies actually represented ^k +/~ revertants, a total of 33
HATr colonies were isolated, grown to mass culture, and assayed for TK
activity. TK specific activities ranged from 7-184 pmol/min/mg protein
(compared to 171 +_ 15 pmol/min/mg protein for wild-type CHO-IB-2), with
most of the revertants having 10-30% wild-type TK activity. Further
biochemical characterization of several of these presumptive tk +'~
heterozygotes (Adair, manuscript in preparation) revealed differences in rn
vitro thermolability and substrate concentration optima for TK, suggesting
that they indeed represent structural gene mutations. One of these
tk +/~ heterozygotes, CHO-AT3-2, was chosen for further genetic analysis
and use in mutagenesis assays.
Wild-type
tk+/+
CHO-IB-2
TK activity: 1.00 ±0.09
Presumptive
tk-'-
Homozygotes
Presumptive
tk+/-
Heterozygotes
Presumptive
tk-/~
Homozygotes
SBUdR'-2
<0.01
I
AT2-4
0.49 ± 0.06
I
Spontaneous
< 0.01 (18)
SBUdRr-3
<0.01
AT3-2
0.36 ± 0.04
I
Spontaneous
< 0.01 (23)
0.15(1)
Figure 2. Derivation of tk+'~ heterozygous cell lines by isolating
tk~/~ mutants of CHO-IB-2, with subsequent EMS mutagenesis
and HAT selection of tk+'~ revertants. Spontaneous mutants
resistant to FUdR were recovered in a single step; relative
TK activities are shown, with the number of clones tested in
parentheses.
16
-------
Forward mutation at the tk locus. Although BUdRr or FUdRr, ^
mutants were rarely recovered from uninutagenized CHO-IB-2 populations
(Table 1), such variants were readily obtained from CHO-AT3-2 at very high
spontaneous frequencies (2-3 x 10"-*). To determine whether expression of
^k ~/~, thymidine analog-resistant phenotypes in CHO-AT3-2 indeed
represented spontaneous, randomly occurring "mutational" events, and
whether the high frequencies of these variants in unmutagenized populations
reflected a high spontaneous rate of mutation at the tjk locus, we performed
a Luria-Delbruck fluctuation analysis experiment. Cells from each
replicate culture were plated into media containing BUdR, FUdR or TFT,
allowing us to determine the spontaneous rate and pattern of occurrence of
drug-resistance for each selection agent. In each case, as shown by data
in Table 3, the variance for replicate, independent cultures was
significantly greater than that for multiple platings of a single culture.
These results indicate that Jtk ~/~, thymidine analog-resistant variants
arose spontaneously and randomly in CHO-AT3-2 populations, suggesting that
acquisition of the thymidine analog-resistant phenotype represented a
"mutational" event occurring prior to drug selection and was not merely an
adaptive response to the selecting drug. Calculation of apparent forward
mutation rates by several methods (Table 3) indicated that ^k ~/~,
thymidine analog-resistant variants arise at a rate of 2-3 x 10~^ per
cell per generation. These estimated mutation rates are approximately
three orders of magnitude higher than those for single-step, forward
mutation at the hgprt or aprt loci in the same cell line (Table 2 and
unpublished results), and are simlar for BUdR, FUdR, and TFT selections.
Complementation analysis of thymidine analog-resistant strains. The
thymidine analog-resistant strains we have isolated characteristically lack
appreciable TK activity and thus are killed in HAT selection medium. To
determine whether the HAT-sensitive phenotypes of these mutants were
dominant or recessive, and to determine whether all shared the same genetic
deficiency, a series of mutants were subjected to complementation analysis
by fusion in pairwise crosses. Complementation analysis of BUdRr,
FUdRr, and TFTr derivatives of CHO-AT3-2 with other TK-deficient mutant
strains (Table 4), indicated that all of these mutants behave recessively
and fail to complement each other or other tk ~'~ Chinese hamster cell
lines such as DON-a3 or RJK-92 (V-79 462-10). Thus, all of these mutants
belong to a single complementation class and presumably reflect mutational
lesions at the same genetic locus. The DON-a3 cell line has been
previously used to map this presumptive, structural gene locus for TK in
both the mouse (60) and human (36) karyotypes.
Segregation analysis of heterozygosity in CHO-AT3-2. Further evidence
that CHO-AT3-2 cells were indeed heterozygous at both the aprt and Jrk loci
was obtained by comparing the frequencies of AAr or BUdRr segregants
from mutant X CHO-AT3-2 and mutant X wild-type cell hybrids. For both
loci, the frequencies of drug-resistant segregants from mutant X CHO-AT3-2
hybrids were at least 2-3 orders of magnitude greater than those from
mutant X wild-type cells (Table 5), as would be expected in the case of a
double heterozygote with a single wild-type allele at both the aprt and ^k
loci.
17
-------
Table 3
Luria-Delbruck fluctuation analysis of tbymidine
analogue resistance in AT3-2 cells
oo
Replicate cultures
Initial cell number per replicate
Final cell number per replicate
Cells tested per sample (# samples)
Number of mutants per sample:
Range
Median
Mean
Variance
Ratio: Variance/Mean
Mutation rate calculated by:
Mean method3
Median method
c
Accumulation with time , exp A
exp B
31 1
50 50
2.7 x 107 2.7 x 107
9.0 x 104(1) 9.0 x 104(12)
138-293
180
188.6
1597
8.47
2.1 +
3.5 +.
2.7 x
1.7 x
BUdRr
144-210
174
173.1
254
1.47
0.5 x 10-4/cell/gen
0.4 x 10~4/cell/gen
10~4/cell/gen
—L
10 /eel I/ gen
83-225
124
134.6
1266
9.41
1.6 1
2.6 +
1.8 x
1.2 x
FUdRr
111-150
120
40.9
321
2.61
0.4 x 10~4/cell/gen
0.3 x 10~4/cell/gen
10~4/cell/gen
— L
10 Vcell/gen
TFTr
77-205
127
122.7
852
6.53
1.5 +
2.6 +
1.8 x
1.2 x
99-141
117
120.0
206
1.71
0.3 x 10~4/cell/gen
0.3 x 10~4/cell/gen
10~4/cell/gen
—L
10 /cell/gen
64.
bRef. 62.
°Ref. 82. Experiment A and B separated by three additional days of expression time.
-------
DON-a3
RJK-92
SBUdRr-l
SBUdRr-2
SBUdRr-3
ETFTr-2
BUdRr AT3-2
FUdRr AT3-2
TFTr AT3-2
STGr-l
Table 4
Complementation analysis of thymidine analog-resistant strains8
BUdSr FUdRr TFl'r
DON-a3b RJK-92C SBUdRr-l SBUdRr-2 SBUdRr-3 ETFTr-2 AT3-2 AT3-2 AT3-2 STGr-ld
Complementation (+) or noncomplementation (-) based upon growth of hybrids in HAT selection medium.
TK-deficient strain of DON cells, isolated by Westerveld et^ al_. (98).
CTK-deficient strain of V-79 cells (originally designated 462-10), characterized by Roufa £t al_. (80).
HGPRT-deficient strain of CHO isolated on the basis of its resistance to 4pg/ml TG.
-------
Table 5
Segregation analysis of heterozygosity in CHO-AT3-2
Frequency of drug-resistant
o
segregant colonies
Hybrid
AA1
BUdRr
AAr, BUdRr Mutant
(aprt'/', tk~'~)
Expt. 1
Expt. 2
X Wild-type CHO
+/+ +/+
(aprt , tk )
4.8 + 1.5 x 10~6 4.3 + 4.3 x 10~7
7.2 + 1.7 x 10~6 13.4 + 7.7 x 10~7
AAr, BUdRr Mutant X CHO-AT3-2
(aprt'7", tk"7") (aprt+/~, t
Expt. 1
Expt. 2
1.4 + 0.1 x 10
0.7 + 0.1 x 10
-3
-3
2.5 + 0.5 x 10
5.0 + 0.7 x 10
-4
-4
Spontaneous frequencies determined after~10 population doublings, by plating
hybrid cells into media containing the appropriate selecting drug. Segregant
frequencies have been corrected for P.E. (0.50-0.65).
20
-------
Assay of specific locus mutation using multiple markers. Using
CHO-AT3-2 cells, it is possible to quantitate mutation induction at the
autosomal aprt and ^k loci, as well as in the genes involved in
QUA-resistance or at the X-linked hgprt locus. We have defined optimal
conditions for the selection of TGr, AAr, OUAR, and BUdRr or
FUdRr mutant phenotypes. Establishment of selection parameters such as
maximal cell plating densities and expression kinetics for each drug
selection marker are described (16). Maximal TGr mutant frequencies are
attained by 6-8 days of expression and plateau at that level. Full
expression of AAr or OUAR mutants requires only 2-4 days, with no
apparent loss of mutant cells by longer expression times. Expression
kinetics for resistance to FUdR are more complex with mutagen-specific
differences in optimal expression time (16; Adair & Carver, manuscript in
preparation). Dose response data for mutation induction after 8 d
expression by the direct-acting mutagen, EMS, in the genes for APRT, TK,
HGPRT, and Na+/K+ ATPase (OUAR) in CHO-AT3-2 cells are presented in
Fig. 3. Significant dose-dependent increases in mutation frequency were
observed for all four drug-resistance markers; in each case there is a
linear dose-response relationship between mutation frequency and EMS
concentration. The slopes for mutation induction as a function of applied
molar dose were: FUdRr, 1.31 + 0.04 x 10~3/mM; AAr, 4.14 + 0.05 x
10~4/mM; TGr, 5.21 +0.14 x 10~4/mM; OUAR, 1.27 + 0.09 x 10-4/mM.
These data indicate that the gene for TK may be much more mutable than the
genes for APRT, HGPRT, or Na+/K+ ATPase (OUAR).
21
-------
600
400 —
I
300 —
a>
I
•*
I
200 —
100 —
[EMSl.mM
Figure 3. Increase in induced frequency of FUdRr (-*-), TGr(—* )AAr
(--•—), an(j QUA^ ( v ) mutants with increasing concentration (mM) of EMS.
Different experiments are represented by open and closed symbols. The linear
regression of the straight line fit (Y = bg + b^ X, where X= EMS , mM)
has the following parameters (+ 1 S.D.):
FUdR
TGr:
AA1
QUA
R
0
-0.04 + 0.11 x 10
-2.96 +_ 2.47 x 10
0.02 + 1.37 x 10
bfl = -1.65 + 2.38 x 10
-3
-5
«
i
-5.
i
-5
; b = 1.31 + 0.04 x 10
; b = 5.21 + 0.14 x 10
= 4.14 + 0.05 x 10
, = 1.27 + 0.09 x 10
-3
-4
-4
-4
22
-------
Cell Density Analysis. The number of wild type cells plated affects
the survival of drug-resistant mutant cells in the selecting drug. As
previously reported (13), reconstruction experiments with wild type CHO
cells showed that a critical density for AGr mutant recovery in the
selecting drug could be as low as 3 to 4 x 104 cells/cm2. For CHO-IB-2,
a similar reconstruction experiment (Fig. 4) indicated that the mean
critical density (MCD5Q, or density of cells yielding 50% mutant
survival) for recovery of an aprt ~'~, hgprt" mutant was approximately
1.3 x 104 cells/cm2 in 80 ug/ml AA and 3.0 x 104 cells/cm2 in
40 ug/ml AG. The recovery of two such spontaneous aprt '~, hgprt~
mutants was reduced by 50% at approximately 7.5 x 10-* cells/cm* in
4 vig/ml TG.
Reconstruction experiments such as these measure the response of a
single mutant phenotype, i.e., one prototype mutant clone isolated from a
selection experiment and grown to a population sufficiently large for
testing. However, a number of independent mutational events, and thus a
variety of mutant phenotypes, contribute to the frequency of spontaneous
mutants. In Fig. 5, an experiment designed to test the optimal plating
density for maximum recovery of spontaneous mutants is shown. The observed
spontaneous mutant frequencies of AAr, AGr, TGr, OUAR and
FUdRr/BUdRr/TFTr are compared as a function of the number of
CHD-AT3-2 or CHO-IB-2 cells plated. The cell density ranged from 1 x 105
to 1 x 106 cells per plate (1 x 104 to 1 x 105 cells per plate for
pyrimidine analogs), corresponding to approximately 1.9 x 10-* to 1.9 x
104 cells/cm2 and 1.9 x 102 to 1.9 x 10^ cells/cm2, respectively.
In Fig. 5a, recovery of AAr mutants decreased with increasing cell
density (50% decrease at about 6.5 x 10^ cells/plate) corresponding to
the metabolic cooperation observed in Fig. 4. The experiments described
(Table 6, Figs. 8, lla) used 80 pg/ml AA and 3 x 105 cells/plate, a drug
concentration and cell density that allows maximal recovery of spontaneous
mutants. As shown in Fig. 5a, selection with either 30 or 40 gg/ml AG was
not affected by cell concentrations up to 1 x 10° cells/plate
(1.85 x 104 cells/cm2). The dose response data described in Table 6
were obtained with 40 pg/ml AG and 3 x 105 CHO-IB-2 cells/plate.
For both CHO-AT3-2 and CHO-IB-2, the optimal cell density for recovery
of TGr mutants (Fig. 5b) is approximately 1 to 2 x 10^ cells/plate;
mutant recovery was reduced by 50% at 8 to 9 x 10-* cells/cm2. No
significant increase in recovery was seen at either 5 x 104 or 7.5 x 104
cells/plate (data not shown). The data in Table 6 and Figs. 9 and lib
correspond to selection with 4 yg/ml TG and 2 x 10^ cells/plate. For
OUAR mutants, no density effect was observed up to 8 x 105 CHO-IB-2
cells/plate (Fig. 5b). This was tested not only with new spontaneous
mutants but also in a reconstruction experiment with 200 OUAR
cells/plate; the CHO-IB-2 cell density ranged from 1 x 105 to 4 x 106
cells per plate. The resulting plating efficiency (data not shown) showed
no significant change as a function of the number of CHO-IB-2 cells,
although smaller colonies were observed when 2 and 4 x 106 cells were
plated. The frequencies of OUAR mutants shown in Table 6, Figs. lOc and
lie correspond to selection with 3mM OUA and 8 x 105 cells/plate.
23
-------
106
Wild type cells/plate
10'
Fig. 4 Reconstruction experiments using two aprt~/~, hgprt" sublines
derived from CHO-IB-2 cells (one line selected first in AA and then in TG,
open symbols, and one line selected in AA followed by AG, closed symbols).
The resistant population was plated in 100-mm petri dishes at different
IB-2 cell densities (O= 40 pg/ml AG, D= 80 yg/ml AA, and A= 4 ug/ml TG).
24
-------
10
-4
.
O>
0.
u
0>
10
-5
t
a .
AA80
AA40
£ 10~4
c
CD
.Q
O
10
-5
_:£__?_ 5 AG30
$-!-§--- -5— -5-
AG40
I I I I
1 23456789 10
Wild type cells/plate, X 105
5a
Fig. 5 The observed frequency of spontaneous mutants selected from
increasing density of CHO-IB-2 cells (closed symbols) or CHO-AT3-2 cells
(open symbols). (a) 30 ug/ml AG (—•--) or 40 pg/ml AG (—O—); 40 pg/ml
AA ( -•—); or 80 wg/ml AA (—P— ). (b) 4 wg/ml TG (-A-^ , —A— )
or 3 mM QUA (-»-). (c) 150 wg/ml BUdR (-o—); 3 y g/ml TFT (—O—);
2 ug/ml FUdR (—A—). Error bars are 1 SEM.
25
-------
8
_£
.O
u
Q>
3 ««-5
ff
(D
*J
i
"S
.O
O
10-
10
-6
I I I I
123456789
Wild type calls/plate, X 105
10
5b
8
8
.O
O
•s -_ •»
d mutant frequency per vk
* —
3 C
J.
I I I I I I
" Q 5 Q - - Pi
y :: Q y Q
- S ^ T 9 ? 0
-1- T A A X .1
- £ * ~ 4
1 1 1 1 1 1
1 1 1
BUdR 150
TFT 3
FUdR2
I ! I
I-
c .
4
i
234567
Wild type cells/plate, X 104
10
5c
26
-------
The high spontaneous frequency of tk'~ mutants allows platings below
the range where metabolic cooperation is thought to occur. In Fig. 5c the
frequency was constant over the range of 1 x 10^ to 1 x 10^ cells/plate
(2 x 102 to 2 x 103 cells/cm2); data in Figs. lOa, lOb, and lid are
the result of 3 x 104 cells/plate in 2 ug/ml FUdR.
Multiple Marker Dose Response for Direct Mutagens. The relative
survival (S), or the fraction of surviving CHO-IB-2 and CHO-AT3-2 cells as
a function of applied mutagen dose of MNNG, NQO, or EMS, is given in Fig. 6.
Table 6 shows the induction of mutants at three genetic markers (AAr,
AGr/TGr, and OUAR). Detailed dose response data on EMS and MNNG were
previously reported for CHO cells (29,30,45); our emphasis was primarily to
establish empirically how data for these widely-used, direct-acting mutagens
varied as a function of cell line and different culture conditions during
expression. Results in Table 6 are pooled from experiments carried out
over 1 y using a single lot of FCS; data for experiments using a different
lot of serum were consistently elevated and are not shown.
When shown as a function of relative cell survival for all three
markers, the response in Table 6 was not always linear at lower cell
survival (<0.50). The nonlinearity might reflect unequal growth rates of
mutant and wild type cells after higher mutagen doses, resulting in an
apparent decline in mutant frequencies. Obtaining mutation data by the
replating protocol assumes that both mutant and wild type cells grow at
approximately the same rate during expression in suspension culture. We
tested this assumpution by performing parallel experiments employing either
(a) expression of cells in suspension and replating, or (b) in situ
expression and selection of cells plated at a low cell density (to avoid
metabolic cooperation). Results from the in situ experiment are also shown
in Table 6. Differences in the AAr mutant frequencies in the replating
and iji situ protocols after 5 to 6 doublings (in situ) or 5 to 7 doublings
(replating) were significant only for mutants induced by EMS. However,
both the TGr and AGr mutant frequencies for in situ selection were
significantly higher for both EMS- and MNNG-induced mutants.
Colony-splitting occurring at the time drug medium was added is a
possibility, but the correlation of MNNG- and 4-NQO-induced AAr mutants
selected either rn situ or replated indicates that the magnitude of
colony-splitting was variable and small in magnitude, if it occurred at
all. Therefore, at least for CHO-IB-2 mutants induced by EMS and MNNG, a
subpopulation of HGPRT" mutants may be at a growth disadvantage in
suspension. The growth disadvantage could account for the nonlinearity
observed at low survival, where extended growth delays resulting from
toxicity obscure the fate of the surviving mutant subpopulation. The
delays may negate the assumption that mutant and wild type cells have
similar growth properties over the expression period.
27
-------
1.00p
10
Exposure dose, M
Fig. 6 Decreasing relative survival (S) of CHO-IB-2 (•,*,•) or CHO-AT3-2
(T, *) cells with exposure dose (M) of direct mutagens MNNG, 4-NQO, and
EMS. Solid and open symbols represent separate experiments. CHO-IB-2
cells were treated for 18 h, CHO-AT3-2 cells for 16 h.
Expression may also be carried out in monolayer culture; with expres-
sion in monolayer culture, linearity of the dose response has been re-
reported under conditions of low survival for some mutanges (29,30) but not
others (61,62). The multiple marker mutagenesis assay reported here
requires a large number of cells per dose point, e.g. about 5 x 10? for
spontaneous and low dose determinations, if optimal statistical param-
eters are desired. The need to maintain cell density below a critical
level in monolayer culture during a prolonged expression period (1,13)
makes monolayer expression impractical for these large-scale experiments.
Transfer of the cells to suspension culture for two or more doublings
before drug selection appears to be sufficient to minimize cell-to-cell
interaction before selection (Carver, unpublished data).
Metabolic Activation. At a dose of 9 mM DMN, maximal induction of
mutation for all four markers was obtained with concentrations < 1.6 mg/ml
S-9 protein; a similar increase in DMN-induced mutants as a function of
cell survival was observed when microsomal activation (<0.75 mg/ml) was
used. When S-9 protein concentrations were > 2.0 mg/ml S-9 or > 1.0 mg/ml
microsomes, toxicity was not increased (Fig. 7) and the induced mutant
frequency (data not shown) remained level or decreased.
28
-------
TABLE 6 Induced mutant frequency of CHO-IB-2 cells
treated with direct mutagens
pg/ml
EMSb
(In Situ)
N00b
(In Situ)
(In Situ)
MNNGb
(In Situ)
100
125
175
225
275
125
0.30
0.40
0.50
0.55
0.65
0.70
0.65
0.70
0.07
0.08
0.09
0.11
0.14
0.11
S
0.74
0.30
0.17
0.04
0.02
0.30
0.96
0.83
0.77
0.66
0.49
0.15
0.49
0.15
0.75
0.44
0.26
0.12
0.05
0.12
AAr x 10
2.4
7.3
9.7
7.4
11.3
20.0
0.1
0.6
2.2
3.5
5.4
6.2
3.0
5.6
1.1
2.2
2.2
2.7
2.8
5.9
1 °
± °
± °
± °
± °
+ 3
± °
± °
± °
± °
± °
± °
+ 1
+ 2
1 °
± °
± °
± °
± °
+ 2
-4
.2
.3
.5
.4
.6
.8
.01
.04
.2
.4
.4
.4
.2
.2
.1
.1
.2
.2
.2
.4
AGr x
4.7
14.1
20.5
108.8
0.2
1.5
2.6
10.1
16.8
19.0
18.8
35.8
0.8
6.7
7.1
7.1
7.7
49.5
-4 r -4
10 TG x 10
+ 0.2
+ 0.8
± 1-2
-
-
+ 16.3
+ 0.0
+ 0.1
+ 0.2
+ 0.8
± 1-1
+ 2.0
+ 5.6
+ 10.7
+ 0.1
+ 0.4
+ 0.5
+ 0.5
+ 0.7
+ 14.9
1.1
5.0
7.0
7.7
8.4
34.8
0.4
0.7
1.7
3.1
4.9
6.3
9.3
10.4
0.6
0.8
1.0
1.1
6.8
+ 0.1
± 2-5
+ 0.4
+ 0.3
+ 0.4
+ 2.6
+ 0.31
+ 0.5
+ 0.2
+ 0.3
+ 0.5
+ 0.4
+ 1.2
+ 2.1
+ 0.1
+ 0.1
+ 0.1
i o.i
•f 2.6
OUAR
2.5
10.0
9.6
8.9
13.1
0.5
1.5
3.0
3.9
5.4
0.3
1.6
2.1
2.4
x 10~5
+ 0.2
+ 0.5
+ 0.5
+ 0.5
+ 0.6
-
+ 0.04
+ 0.1
-
+ 0.3
+ 0.4
+ 0.5
_
_
+ 0.05
-
+ 0.1
+ 0.2
+ 0.2
"•
aSpontaneous frequencies were as follows: M 1.2 x 10 ; AG 1.0 x 10 ;
TGr 4.6 x 10~5j OUAR 5.1 x 10~6.
^Treatment time was 18 h.
29
-------
1.00
5
E
3
(£
0.10
2.00 [S-9]
1.00 l/i]
Fig. 7 Decreasing relative survival (S) of CHO-AT3-2 cells with increasing
S-9 (open symbols) or microsome (y, solid symbols) concentrations (.mg/ml
total protein). The cells were treated for 2 h with the promutagens DMN at
9 mM (o,«) and BP at 0.04 mM (A,A).
When activation of BP by S-9 at concentrations of 0.50 to 2.0 mg/ml was
compared to activation by microsomee at 0.25 to 1.0 mg/ml, the data
indicated that BP was most effectively metabolized to mutagenic products by
microsomes at < 0.50 mg/ml; microsome concentrations> 0.50 mg/ml
consistently lowered the optimal yield of BP-induced mutants. Although we
observed only minimal induced mutation from BP activated by S-9 at
> 0.5 mg/ml, recent experiments have indicated that BP is efficiently
converted to mutagenic metabolites by S-9 at < 0.1 mg/ml, followed by a
decrease at higher concentrations of S-9 protein.
Determination of Expression Time. Following induction of (genotypic)
mutation, a period, i.e., expression time, is required to allow maximal
phenotypic expression before drug selection. In these experiments, the
mutations were expressed during cell growth in suspension culture to
minimize cell-to-cell contact and to provide uniform growth conditions.
The CHD-AT3-2 subline had a doubling time of 14 h in suspension culture,
and the spontaneous frequency of all four drug-resistant loci remained
reasonably constant (S.D. of 1 60%) over a 10 d period. The relative
survival for CHO-AT3-2 exposed to EMS is shown in Fig. 6. Fig. 7 shows the
relative survival (S) of CHQ-AT3-2 cells as a function of increasing
30
-------
S-9 or microsome concentration; promutagens coupled with metabolic activion
were DMN (9 mM) and BP (0.04 mM).
In the multiple-marker system, expression of AAr mutants induced by
EMS was complete by 2 d (Fig. 8a). The BP-induced AAr mutant frequency
(Fig. 8b) appeared to be maximal by 4 d, declining slightly but not signi-
ficantly over the next 6 d. The DMN-induced AAr mutant phenotypes (Fig.
8c) were maximally expressed by 3 d, and a plateau in induced mutant
frequency was maintained through 10 d. The longer expression time needed
for complete expression of TG resistance (compared to AG resistance) has
been documented previously (28). Mutants induced by EMS showed full
TG-resistance by 6 d (Fig. 9a). The frequency of TGr mutants in response
to BP was also maximal by 6 d, with no increase observed during the
following 4 d (Fig. 9b). The DMN-induced mutant frequency for TGr shown
in Fig. 9c increased up to 8 d and had plateaued by 10 d.
In Fig. lOa, OUA^ mutants were fully expressed by 3 d and remained
reasonably constant over the next 7 d. With CHO-IB-2 cells, we observed a
pronounced decline in the number of MNNG-induced OUA^ mutants after 10-12
d (data not shown). The expression of FUdRr mutants is shown in Figs.
lOb and lOc. Mutants induced by EMS were maximal at 2-3 d, then declined.
However, the response to BP declined after 1 d. The frequency of mutants
induced by DMN was maximal at 3 d, declining significantly at 4 d and then
remaining constant over the 10 d period. Response at the ^k locus following
exposure to several mutagens has shown maximal expression at 1-4 d (Adair
and Carver, in preparation).
Multiple Marker Dose Response for Optimal Expression. In Figs, lla-lld,
the induced mutant frequency per viable cell is fitted as a linear function
of the response to mutagen dose in units of the logarithym of decreasing
cell survival. The slope of the linear function can be used to compare the
mutagenic potency at equivalent cell survival. If the AAr response (Fig.
lla) after BP treatment is compared to that for DMN, i.e. a ratio of the
slopes, then DMN and BP are comparable mutagens at the aprt locus
(P >0.80). In terms of equal cell survival, EMS is about seven times more
potent than DMN or BP. In Fig. lib, the response of TGr mutants induced
by BP is shown. The response is slightly less than that for DMN (P < 0.06
that the mutagens are comparable in potency). Similar to AAr, the TGr
response for EMS is about six fold greater than that for DMN or BP. In
Fig. lie, BP and DMN are similar in potency (P~0.30) for inducing OUAR
mutants. The response for EMS at this locus is about seven times greater
than that of DMN and BP.
The above data have compared the mutant frequency observed at optimal
(7-8 d) expression. For the tk locus, the data are compared in Fig. lid
for optimal but varying expression, i.e. 1 d for BP, 3d for DMN and EMS.
At early expression, DMN and BP are comparable mutagens; however, EMS is
slightly less effective than DMN and BP at the tk locus.
31
-------
r
f
t
EMS
1
456
Expression, days
8
10
8a
Fig. 8 Induced AAr mutant frequency per viable cell (CHO-AT3-2) as a
function of expression time before selection with 80 ug/ml AA. Error
bars are 1 SEM. The AA spontaneous frequency (+_ 1 S.D.) was
1.35 +_ 0.85 x 10 . The mutagen concentrations and corresponding
fraction of surviving cells at time zero were as follows:
(a) EMS: 2.4 mM, 0.53 {-£-); 2.9 mM, 0.40 ( A )•
3.2 mM, 0.33 ( --A-- ); 3.4 mM 0.24 (—«—).
(b) BP: 0.04 mM, 0.2 mg/ml S-9, 0.90 (—O—);
0.04 mM, 0.4 mg/ml S-9, 0.76 ( « );
0.04 mM, 0.6 mg/ml S-9, 0.69 (--•").
(c) DMN: 9 mM, 0.5 mg/ml S-9, 0.94 (—o—);
9 mM, 1.0 mg/ml S-9, 0.80 ( O );
9 mM, 1.5 mg/ml S-9, 0.55 (—•--).
32
-------
in
o
f
S
3
u
TJ
BP
I I
1 I
I I
I I
456
Expression, days
9 10
8b
9
in
X
c
Q>
§• 6
1
i
fe
•o
0
C
1 1 1 1 1 1 1 1 1
DMN j
T, ,- 1
* f *
/
/ IT
- ' j n
;/,r-r
//
''J\ \ S ? ^
)123456789
Expression, days
-
__c
n
r
K
8c
33
-------
4 5
Expression, days
8
10
Fig. 9 Induced TGr mutant frequency per viable cell (CHO-AT3-2) as a
function of expression time before selection with 4 pg/ml AA. All symbols
as in Fig. 8- Spontaneous TGr frequency was 2.52 _+ 1.46 x 10~5.
34
-------
in
I,
21
18
15
9
•o
I I I I
I I
BP
4567
Expression, days
21
18
X
fr 15
g
a
•a
I I I
DMN
Q FJ
123456
Expression, days
9b
I I I I I
8 9 10
9c
35
-------
3U
40
in
o
x 30
o
c
0)
3
Sf 20
«^
4->
«2 J
3
E
1 1 l l i
H- T !
1 =
I \
i J
E(V
—
••
rf
III!
i \
k 1
> -3
IS
—
•>
i
O
o
•a
3 -
2 -
1 -
DMN, BP
l
456
Expression, days
8
lOa
Fig. 10 Induced FUdRr or OUAR mutant frequency per viable cell
(CHO-AT3-2) as a function of expression time before selection with 2 jj g/ml
FUdR or 3 mM QUA. All symbols as in Fig. 8. Spontaneous frequency was
2.44 + 0.81 x 10~6 for OUAR and 2.39 + 0.86 x 10"3 for FUdRr.
36
-------
234567
Expression, days
XTT UNll\
r------
1 * _i_
* $
456
Expression, days
8
10
lOc
37
-------
0.5
0.10
Relative Survival
lla
Fig. lla Increase in induced frequency of AAr mutants (7-8 d
expression) with decreasing relative survival or fraction of surviving
CHO-AT3-2 cells (S) after treatment with DMN (—-A-.-), BP (-—•—), or
EMS (—•—). Open and closed symbols represent different experiments.
The linear regression of the straight line fit (y = DQ + b^ X, where
X = log S ) has the following parameters: (_+ 1 S.D.):
DMN: bQ = -0.27 + 0.86 x 10~5,
BP: bQ = -1.75 + 1.57 x 10~5,
EMS: bQ = 1.72 + 1.62 x 10~4,
= 4.83
0.28 x 10~4;
"4
= 5.04 + 0.92 x 10
= 3.33 + 0.77 x 10~3.
38
-------
Relative Survival
0.10
lib
Fig. lib Increase in induced frequency of TGr mutants (7-8 d expression)
with decreasing relative survival or fraction of surviving CHO-AT3-2 cells
(S) after treatment with DMN, BP, or EMS. All symbols as in Fig. lla.
Regression parameters are:
DMN: bQ = 0.26 +_ 0.92 x 10~5, b
BP:
EMS: b
b0 = -1.69
1.58 x 10
1.55 + 1.95 x 10
~4
= 7.66 + 0.35 x 10'4;
L = 5.45 + 0.85 x 10~4;
= 4.43 + 0.93 x 10"3.
39
-------
1.00
0.50
0.10
Relative survival
lie
Fig. lie Increase in induced frequency of OUA^ mutants (7-8 d
expression) with decreasing relative survival or fraction of surviving
CHD-AT3-2 cells (S) after treatment with DMN, BP, or EMS. All symbols as
in Fig. lla. Regression parameters are:
DMN:
BP:
EMS:
bQ = 0.22 + 2.1 x 10
"6
1.63 + 0.12 x 10
-4.
b0 = 0.12 + 3.22 x 10~6, \>l = 1.42 + 0.17 x 10~4;
x 10"5, bj =
°'25
10
40
-------
900 -
X
fr
600 -
1
cc
3
300
1.00
0.50
0.10
Relative survival
lid
Fig. lid Increase in induced frequency of FUdRr mutants after optimal
expression time with decreasing relative survival or fraction of surviving
CHD-AT3-2 cells (S) after treatment with DMN, BP, or EMS. All symbols as
in Fig. lla. Regression parameters are:
DMN (3 d):
b0 = 0.47 ± 0.34 x
= 1>93 0.26 x 1Q-2.
~3
BP (1 d): bQ = 0.47 + 0.34 x 10,
EMS (3 d): bQ = 0.14 + 0.07 x 10~3,b1
= 1.93
0.26 x 10~2;
1.38 + 0.20 x 10
~2
41
-------
Validation of Mutant Phenotypes. As shown in Fig. 12a, of the 61 AAr
clonal isolates assayed for APRT activity, 96% (22/23) of the spontaneous
mutants and 82% (31/38) of the mutants from mutagenized cultures had < 5%
wild type activity; only 4 AAr clones had APRT activity approaching those
of the heterozygous parental cells. Of the 54 AGr clones assayed for
HGPRT activity, 89% (16/18) of the spontaneous mutants and 86% (31/36) of
the mutants from mutagenized cultures had < 30% wild type activity; 4 AGr
clones had HGPRT activities similar to those of CHO-IB-2 and CHO-AT3-2.
The mean residual HGPRT activity of TGT mutants was much lower than that
of AGr mutants. None of the 30 TGr mutant clones assayed had WT HGPRT
activity, with 94% (17/18) of the spontaneous mutants and 67% (8/12) of the
induced mutants having < 5% WT activity.
In Fig. 12b, the residual TK activity of spontaneous and induced
mutants is shown. Because the spontaneous frequency of FUdRr is high, no
attempt was made to distinguish between spontaneous and induced mutants.
Both classes had virtually no detectable TK activity; a single isolated
mutant appeared to have 15% of WT TK activity, but the line was lost before
the assay could be repeated. All other mutants have had < 1% WT activity.
After treatment with DMN or BP, fifty induced mutant clones resistant to
2 y g/ml FUdR were isolated. The clones were grown 12-16 generations in the
absence of FUdR, then tested for FUdRr, BUdRr, TFTr, and HAT8. All
fifty clones displayed cross-resistance to the three thymidine analogs
(P.E. 0.40-0.70) and showed complete sensitivity to HAT (P.E. < 10"^).
-------
30
3j 20
ID
I
I 10
I • • ' ' I ' • • ' I ' • • ' I
Spontaneous AG' LI
TG' 0
I i ' ' ' I ' ' ' ' I ' ' ' ' I
Induced AG' 0
TG' 0
• i i i n m i i n~
30
S
20
g
o
O 10
Spontaneous AA'
25 50 75 100 0 25 50 75 100
% Wild type activity % Wild type activity
48
0)
(Q
y,
(9
S
§24
ii i i | i i i i | i i i i | i i i
Spontaneous and induced
™
-
£
V
FUdRr D
BUdRr 0
TFTr •
o m
1 1
-
^~
-
-
—
25 50 75
% Wild type activity
100
Fig. 12 Activities of HGPRT, APRT, or TK (relative to wild type) for
spontaneous and induced mutants. Mutant clones were isolated, grown 10-12
generations in the absence of selecting drug, and assayed for in vitro
activity of the appropriate enzyme, (a) Mutants resistant to 40 Mg/ml AG
or 4 ug/ml TG or 80 pg/ml AA; (b) mutants resistant to 2 ug/ml FUdR,
150 yg/ml BUdR, and 3 ug/ml TFT.
43
-------
SECTION 6
DISCUSSION
As an in vitro test system, CHO cells combine a near diploid karyotype
that facilitates genetic and cytogenetic analysis with the favorable growth
characteristics, e.g., high plating efficiency and rapid growth in either
monolayer or suspension culture, necessary for large-scale, cost-effective
mutagen screening. The CHO-AT3-2 subline, which is also heterozygous at
both the aprt and tk loci, was developed to allow single-step selection of
autosomal recessive AAr and FUdRr mutant phenotypes, as well as the
more commonly used X-linked TGr and co-dominant OUAR genetic markers.
Although functionally heterozygous aprf1"' strains of CHO had been
isolated previously (17,52), optimal conditions for the phenotypic
expression and selection of AAr mutants were not established, and the
strains were not extensively used for quantitative mutagenesis assays.
Only limited data have been reported in the literature regarding the
optimizations of selection parameters and the induction of APRT-deficient
mutants by known mutagens (17,34,52,87,92). Presumptive J:k+/~ revertants
of TK-deficient mutants have been isolated in several cell lines
(3,10,22,80,85). Extensive validation and application of the L5178Y
_tk~/~ mouse lymphoma system for mutagenesis assays (20,22,24,25) prompted
our efforts to use the tk locus in a CHO cell mutagenesis assay system.
The CHO-AT3-2 subline facilitates simultaneous, quantitative mutagenicity
testing at four specific gene loci.
Derivation of heterozygous cell lines. Phenotypic expression of
single-step forward mutations at the autosomal aprt and tk loci, and use of
the AAr and FUdRr genetic markers for mutagenesis assay, first required
the derivation of a CHO subline that was heterozygous for these loci.
Using the approach of Jones and Sargent (52), an aprt +/~ heterozygote
with approximately 50% wild-type APRT activity (CHO-IB-2) was selected from
an unmutagenized CHO cell population. The aprt +/~ CHO-IB-2 cells were
subsequently used to derive sublines that were also heterozygous for the tk
locus. Since direct, single-step selection of _tk +'~ heterozygotes
proved to be impractical (2,22,67), such heterozygotes were obtained by
first isolating rare, BUdRr, ttc ~/~ mutants of CHO-IB-2, and
subsequently selecting HATr revertants which had intermediate levels of
TK activity. A similar approach was used by Clive and co-workers (22) to
obtain the tk +/~ heterozygote employed in their L5178Y TK +'~ mouse
lymphoma mutagenesis assay system.
Biochemical and genetic characterization. The CHO-AT3-2 cells are
heterozygous for both the aprt and ^k loci. Consistent with this premise
44
-------
were: (a) the manner in which the CHO-AT3-2 cell line was derived (Fig. 1
& 2); (b) this line's intermediate levels of APRT and TK activity (Fig. 1 &
2); (c) the high frequencies at which APRT- or TK-deficient mutants were
obtained from unmutagenized CHO-AT3-2 cell populations in single-step
selections with AA or FUdR (Table 1, Fig. 3); and (d) forward mutation
rates at these autosomal gene loci that were greater than or similar to
those at the X-linked hgprt locus (Tables 2 & 3). Spontaneous and
EMS-induced frequencies of AAr and BUdRr or FUdRr mutants were at
least 2-3 orders of magnitude higher in CHO-AT3-2 cells than in wild-type,
aprt +/+ and tk +/+ CHO cells (Table 1). Segregation analysis (Table 5)
confirmed that CHO-AT3-2 cells were indeed functionally hemizygous for the
genes for APRT and TK.
Biochemical validation of drug resistant mutant phenotypes revealed
that fifty-one of fifty-nine clones selected on the basis of their
resistance to AA had < 5% of wild-type APRT activity. APRT-deficient
mutants have been selected from several other cells lines (17,33,52,87,-
92,93), and the aprt locus has been mapped in both the mouse (59) and human
(93) karyotypes. Functionally heterozygous, aprt +'~ strains have not
been previously utilized for quantitative mutagenesis assay, and only
limited data has been reported in the literature regarding the induction of
AA-resistance by known mutagens (15,16,52,92). Dose-dependent increases in
the frequency of AAr colonies (~10 fold over background) after treatment
of an aprt +'~ strain of CHO cells with EMS were reported by Jones and
Sargent (52), but expression kinetics for the AAr marker were not
determined. Having established optimal conditions for the phenotypic
expression and selection of AAr mutants in our system (15,16), we can
directly compare induced mutation frequencies at the aprt and hgprt loci,
and assess the relative sensitivity of the genes for APRT and HGPRT to
mutation by various physical and chemical agents. For the direct-acting
mutagen, EMS, induced mutation frequencies for AA-resistance were only
slightly lower than those for TG-resistance (Fig. 3). However, we have
observed mutagen-specific differences in the relative mutability of the
aprt and hgprt loci using other mutagens (15,16; Adair & Carver, manuscript
in preparation).
Although TK-deficient strains have been isolated from a number of
different cell lines (10,22,55-57,80,85,86), many of these variants were
obtained by prolonged, multi-step selection with BUdR, and are of uncertain
genetic origin. The presumptive structural gene locus for TK has been
mapped in both the mouse (60) and human (36,66) karyotypes, using
TK-deficient mutants of mouse and Chinese hamster cells. Derivation of a
^k *' heterozygote of L5178Y mouse lymphoma cells by Clive and
co-workers (22), and extensive validation and application of this system
for autagenesis assay (21-23,25,26,67), has prompted efforts to utilize the
tk locus in other mammalian cell mutagenesis assay systems as well (16,85).
We have isolated TK-deficient mutants of CHO cells using BUdR, FUdR, or
TFT as the selecting agent. All but one of forty-three thymidine
analog-resistant clones isolated from CHO-AT3-2 or CHO-IB-2 had < 1% of
wild-type TK activity. All of the TK-deficient mutants we have tested were
HAT-sensitive and cross-resistant to BUdR, FUdR, and TFT. Further
45
-------
biochemical and genetic characterization of several TK-deficient mutants
and HATr revertants with intermediate levels of TK activity provided
evidence that they probably represent mutations in the structural gene for
TK (Adair, manuscript in preparation). The thymidine analog-resistant,
TK-deficient mutant phenotypes were recessive, and complementation analysis
(Table 4) indicated that all of the BUdRr, FUdRr, or TFTr clones
tested represented the same complementation group, reflecting mutation at
the same genetic locus. Furthermore, non-complementation of the thymidine
analog-resistant phenotype in hybrids with TK-deficient DON-a3 cells
identified this genetic locus as the presumptive structural gene locus for
TK, previously mapped in both the mouse and human genomes using the DON-a3
strain (36,60).
Most previous studies have utilized BUdR as the selecting agent for
TK-deficient mutants (10,21-23,25,26,40,55-57,80). The killing of
TK-positive wild-type cells by this drug is rather slow, and several cell
divisions can occur before cell death (2,42,67,94). FUdR rapidly inhibits
cell division (42,94), effectively eliminating background growth of
wild-type cells; unlike BUdR, it is apparently not mutagenic in mammalian
cells (50). Both analogs are readily phosphorylated by TK, but FUdR is not
further metabolized to the di- or tri-phosphates, or incorporated into DNA
(42,43). FUdMP is a potent, irreversible inhibitor of thymidylate
synthetase (43,79). The observed cross-resistance of TK-deficient mutants
to BUdR, FUdR, and TFT, along with the non-complementation of TK-deficient
mutants regardless of which drug was used for their selection (Table 4) and
the similar rates of spontaneous mutation for resistance to each drug in
CHO-AT3-2 (Table 3), together suggest that BUdR and FUdR select for the
same class of TK-deficient mutant phenotypes. Therefore, we have used FUdR
as the selecting agent for such mutants in these CHO-AT3-2 multiple-marker
mutagenesis experiments.
The ^k locus appears to be considerably more susceptible to spontaneous
mutation than the genes for APRT, HGPRT, or Na+/K+ ATPase (OUAR) in
Chinese hamster cells. Background frequencies of BUdRr or FUdRr
colonies for unmutagenized CHO-AT3-2 cell populations were consistently
1.5 - 3.0 x 10~-\ Similar spontaneous frequencies have been reported for
BUdRr variants in several presumptive ^k +'~ heterozygotes of another
Chinese hamster cell line (80). Luria-Delbruck fluctuation analysis
(Table 3) indicated that the high frequencies of thymidine analog-resistant
variants in CHO-AT3-2 cell populations reflect a very high spontaneous rate
of mutation at the j:k locus. Stable, thymidine analog-resistant variants
arise spontaneously at a rate of 2-3 x 10~^ per cell per generation; this
rate is approximately three orders of magnitude greater than those for
spontaneous mutation at either the aprt or hgprt loci in the same cell line
(Table 2). Despite the high background frequencies of FUdRr colonies,
significant dose-dependent increases in mutation frequency were observed
with this genetic marker after treatment with known mutagens (Fig. 3).
Furthermore, for most of the mutagens we have tested in the CHO-AT3-2
system, more mutations appear to be induced at the £k locus as a function
of mutagen dose than at either the aprt or hgprt loci, or in the genes
involved in ouabain resistance (16; Adair & Carver, manuscript in
preparation). Clive e_t £l_. (26) reported that the ^k locus was, on the
46
-------
average, nearly 10 times more mutable than was the hgprt locus, for 13
agents tested in the L5178Y TK +/~ mouse lymphoma system. Several
carcinogens that were only weakly or non-mutagenic at the hgprt locus were
found to be strongly mutagenic at the ^k locus. Clive (26) has suggested
that the L5178Y system, utilizing the tk locus, might be allowing the
detection of TK-deficient mutants with gross chromosomal lesions as well as
those generated by base-pair substitutions or small deletions. The
extraordinarily high rate of spontaneous mutation at the ^k locus in
CHO-AT3-2 cells may reflect a position effect or "hot spot" for mutations
or chromosomal events such as mitotic recombination, segregation, or
non-disjunction. Further investigation of this system may provide insight
into the involvement of extra-mutational events in the expression of
genetic damage in somatic cells.
Cell density parameters. A major technical consideration in mutagen
testing assays is the dependence of mutant selection upon wild type cell
density, i.e., fewer mutants can survive in high density cultures because
of metabolic cooperation (contact feeding). Cell density effects have been
previously quantitated for several drug resistance markers in other cell
lines (32,34,52,67,71,78,80,88,92). However, to establish optimal cell
plating density conditions for the selection of AAr, AGr, TGr,
OUA^, and FUdRr mutants in our cell line, the mean critical density
MCD50 (34), of CHO-IB-2 and CHO-AT3-2 cells for each drug resistant
phenotype was determined.
Reconstruction experiments (Fig. 4), and determinations of spontaneous
mutant frequencies as a function of cell plating density (Fig. 5),
indicated that critical cell densities for TG, AG, and AA were 7.5 x 103
cells per cm2, 3.0 x 10^ cells per cm2, and 1.3 x 10^ cells per
cm2, respectively. Our cell density data for AA resistance are very
similar to those reported in a detailed study of CHO-K1 by Dickerman and
Tischfield (34) and do not show the density independence of AAr
previously reported in another CHO cell line (52). Critical densities
ranging from 4 x 102 to 4 x 10^ cells per cm2 for AG-resistance
(32,41,51,71,78, 97), and from 6 x 103 to 1.4 x 106 cells per cm2 has
been reported for TG- resistance (27,31, Corsaro and Migeon as calculated
in 34,58,71). The selection of ^k~'~ phenotypes was not dependent upon
cell density over the range of 2 to 20 x 103 cells per cm2. In
general, cell lines plated in semi-solid agar substrates, e.g. L5178Y, S49,
are not thought to show cell-to-cell cooperation in mutant selections
(27,41). However, a recent report (67), indicated that BUdR (but not TFT)
selection of L5178Y mutant cells in agar showed decreased recovery at 4 x
10^ cells per cm2.
Concentration of Selecting Drug. Cumulative data suggest that AG must
be used at concentrations > 30 ug/ml to select reproducibly bona fide
hgprt" mutants (12,39,41,53,78). Since selection of TGr mutants is
affected by metabolic cooperation at densities approximately 4-5 fold lower
than AGr (Figs. 4, 5a), we initially attempted to optimize the AGr
selection and obtain quantitative, reproducible data. Although mutant
selection can be improved by using 30 to 40 ug/ml AG, selection of hgprt"
mutants with the CHO-IB-2 cell line is more definitive and quantitatively
47
-------
reproducible when TG (2-4 ug/ml) is used as the selecting agent, an
observation consistent with results reported in other cell lines
(47,58,88,95). Furthermore, as shown in Fig. 12, the spectrum of residual
HGPRT activity is much broader for mutants selected by resistance to AG;
the resulting heterogeneity in colony size makes quantitation more
difficult and requires extended incubation of mutant colonies (up to 18 d).
Therefore, we prefer TG for selecting HGPRT-def icient mutants in our
system. Azaadenine at 40 to 80 ug/ml effectively selects APRT-deficient
mutants. While BUdR used at 150 ug/ml avoids most of the background haze
associated with abortive phenotypes, the quantitation of TK-def icient
mutants is more satisfactory with FUdR at 1 to 3 ug/ml; very definitive
data are also obtained with TFT at 1 to 3
Expression Time. Optimization of expression time is a major factor in
the reproducible selection of drug-resistant mutants (48,84,89). Although
expression time is generally expressed either as days or population
doublings (or cell generations), both chronological time and cell division
seem to contribute to expression (32,48,54). The HGPRT activity in a new
presumptive mutant (and consequently its potential sensitivity to cytotoxic
selecting agents) decreases as a function of both degradation of preexisting
enzyme and dilution by cell division (48). Althogh HGPRT is apearently
degraded even in contact-inhibited, nondividing cell populations (48),
reports also indicate that maximal mutation frequencies are not achieved
when cells are maintained in a nondividing state (54). Cox and Masson (32)
presented evidence suggesting that cell doubline parameters may be more
important than chronological time, at least in the expression of
radiation-induced mutation at the hgprt locus. With direct, in situ
expression of mutagenized cell populations, prolonged expression times are
not practical because of cell-to-cell density effects and metabolic
cooperation. With the replating method, the optimal expression time
generally extends over at least several generations (1,13,32,48,69,74,96).
In CHO-AT3-2 cells, the optimal expression time depends on both the
drug-resistant marker and the mutagen used (Figs. 8,9,10). Mutagen-
specific variation in expression time has also been reported for diploid
human lymphoblasts (91), in which MNNG-induced TG resistance was expressed
by 6 generations but cells treated with 1CR-191 required 12 to 16
generations before full expression had occurred. Shapiro and co-workers
(82) reported maximal expression of TGr mutants after 2 to 6 generations,
but expression times from 2 to 20 d have been reported by other
investigators (1,27,32,48,58,74,82,89,91,96). In CHO-AT3-2, 6 to 8 d (8 to
10 generations) were required for maximal expression of TGr mutants
(Fig. 9).
Optimal expression times for AG-resistance ranging from 3 to 7 d have
been reported (32,39,51,69). Khalizev, e± a±. (54) reported that 2 to 3
doublings were sufficient for maximal expression of the AGr mutants
phenotypes in Chinese hamster cells, but our data for CHO-IB-2 indicated
that 5 to 7 doublings were required for optimal expression of AGr mutants
induced by EMS or MNNG (data not shown).
48
-------
In CHO-AT3-2 cells, 2 to 4 d are needed for full expression of AAr
phenotypes (Fig. 8). For QUA resistance, maximal expression reportedly
occurred in 1 to 4 d (27), while in our system 3 d (3 to 4 doublings) were
sufficient for expression of most OUAR mutants (Fig. lOa). The mutant
frequency was constant over the 10 d, whereas Buchwald (9) observed a
decrease in the frequency of OUAR mutants after 2 generations of
expression. For the ^k locus, the optimal conditions for mutant expression
are complex and. mutagen-specific. For the mutagens studied, the maximal
observed mutant frequency varies from 1 to 3 d (Figs. lOb, lOc). Previous
reports indicated 2 to 3 d for mouse lymphoma (26), 3 d for V79 cells (80),
and a constant 2 doublings (generations) for BHK cells treated with EMS
(10).
Optimal expression time clearly varies for different mutagens, and the
time for maximal expression observed for a few standard mutagens like EMS
may be of limited value in predicting the optimal expression time for other
classes of mutagens or for complex mixtures. If quantitative comparisons
are to be made between mutagens, optimal expression time may need to be
established for each mutagen, e.g., BP vs. DMN at the tk locus.
Dose Response Relationships at Different Genetic Markers. When the
observed frequency of AAr, AGr, TGr, and OUAK mutants per viab1e
cell is compared for CHO-IB-2 as a function of molar dose (Table 6), both
NQO and MNNG are more effective than EMS (approximately 200 to 350 fold and
100 to 500 fold, respectively). However, when the induced frequency of
mutants per viable cell is compared for the various markers as a function
of log cell survival, NQO and EMS are 2 to 7 and 3 to 9 fold more mutagenic
than MNNG (Table 6). Thus, as previously suggested (29,77), when analyzed
on the basis of cell survival, MNNG is not a particularly effective mutagen
in mammalian cells.
As a function of applied molar dose, the mutational response at the
hgprt locus in CHO-AT3-2 cells had a slope of 5.2 x 10"^ mutants per
viable cell per mM EMS, compared to a slope of 4.3 x 10~^ previously
reported for this locus by Hsie and co-workers (45) at similar 16 h
treatment times. Slopes for AAr, OUAR, and FUdRr mutants after 8 d
expression were 4.1 x 10~^, 1.3 x 10"^, and 1.3 x 10"-*, respectively
(3).
When the mutational response for EMS is plotted as a function of cell
survival, the observed frequency of AAr, TGr, and OUAR, mutants by
EMS decreases at survivals 0.50 (Fig. 11), as was the case in previous
experiments using CHO-IB-2 cells (Table 6). However, induction of FUdRr
mutants by EMS increased linearly as a function of the logarithym of
decreasing survival, as did induction of AAr or TGr mutants by DMN.
Specificity and sensitivity of the multiple markers. Significant
increases in mutation frequency were observed for all four drug resistance
markers following treatment of CHO-AT3-2 cells with EMS, DMN, or BP (Fig.
11), but locus-specific differences in sensitivity to these mutagens were
noted. Under optimal expression conditions, the tk locus consistently
showed the greatest mutational response as a function of cell survival with
49
-------
induction by DMN = BP > EMS. The jtk locus was much more sensitive than the
other loci to mutation induction by DMN and BP, but only slightly more
sensitive to mutation induction by EMS. With DMN and BP, the slopes for
mutation induction for FUdRr (Fig. 11) were about 25 to 40 fold greater
than those for AAr or TGr, and over 100 fold greater than those for
OUAR. With EMS, mutation induction slopes for FUdRr were still about 3
to 4 fold higher than those for AAr or TGr, and about 12 fold higher
than those for OUAR. EMS, however, was much (7 to 9 fold) more effective
in inducing AAr, TGr, and OUAR mutants than either DMN or BP.
Of the four genetic markers employed in CHO-AT3-2 mutagenesis assay
system, the tk locus has consistently shown the greatest mutational
response to positive mutagens (Ref. 3, Fig. 11). The magnitude of induced
mutation at this locus suggest that FUdR selection in the CHO-AT3-2 system
may detect a broader range of genetic lesions than the other drug-resistance
markers. In the L5178Y TK"4"' mouse lymphoma system, the ^k locus appears
to be approximately ten times more mutable than the hgprt locus (26), and
Clive and co-workers have postulated that their system may be detecting
TK-deficient variants arising from gross chromosomal lesions as well as
those generated by base-pair substitution, frame shift, or small deletions.
In our CHO-AT3-2 cells, as in the L5178Y TK+/~ system, phenotypic
expression of mutants resistant to thymidine analogs was rapid, with
maximal frequencies generally attained within 1 to 3 d after mutagen
treatment (Fig. 10). Mutant frequencies typically decline with longer
expresstion times. In both systems, cell isolates resistant to thymidine
analogs have little or no detectable TK activity (22,25) as would be
expected in the case of deletions or gross chromosomal lesions.
The rapid expression kinetics for induced FUdRr mutants may
facilitate the detection of mutants with gross chromosomal anomalies or
large scale DNA damage affecting cell growth rate. Such gross genetic
lesions might not be detected using either the OUAR marker (a functional
Na+-K+-ATPase is required for cell survival) or the TGr marker
(expression of TGr phenotypes requires long expression times). Moreover,
the gene for _tk may be unusually susceptible to mutation. The high
frequencies of FUdRr observed in unmutagenized CHO-AT3-2 cell populations
are the result of an exceptionally high, apparent rate of spontaneous
mutation at the ^k locus (3). The apparent susceptibility to mutation of
the £k locus in CHO-AT3-2 cells may reflect chromosomal events such as
mitotic recombination, segregation, or nondisjunction. Position effects in
lower eucaryotes and "hot spots" in microbial systems are not uncommon.
Since the FUdRr marker may allow detection of a broader range of genetic
lesions than the other drug resistance markers, use of the tk locus in the
CHD-AT3-2 mutagenesis assay enhances the potential sensitivity of the
system.
Validation of mutant phenotypes. As shown in Fig. 12, the spectrum of
residual enzyme activities of AGr mutants is more heterogeneous than that
for TGr mutants. Although many of the mutants selected with AG display
stable characteristics, the observed heterogeneity probably contributes to
technical problems with the selection of AGr phenotypes. There was no
50
-------
statistical difference in the residual enzyme activities in EMS-, MNNG-,
and NQO-induced mutants (data not shown). Mutants selected with FUdR,
BUdR, and TFT appear to have identical enzymatic properties, displaying
< 1% WT TK activity, sensitivity to HAT, and cross resistance to all three
selecting drugs. The results in Fig. 12 indicate that a very high
percentage of the spontaneous and induced drug-resistant clones scored as
mutants have substantially diminished enzyme activity at the selected locus.
The Multiple Marker Mutagenesis Assay System. Systematic determination
of optimal conditions for the phenotypic expression and selection of AAr,
FUdRr, OUAR and TGr mutants (15,16; Adair & Carver, manuscript in
preparation), and the development and application of a multiple-marker
mutagenesis assay system employing CHO-AT3-2 will facilitate quantitation
and direct comparison of the extent of mutational damage at these four
defined genetic loci. The CHO-AT3-2 cells combine favorable growth
characteristics (e.g., short doubling time, excellent cloning efficiency,
and the ability to grow in either monolayer or suspension culture) with a
near diploid genome that facilitates genetic and cytogenetic analysis. As
an in vitro mutagenesis assay syste, the CHO-AT3-2 cell line offers the
unique capability of measuring mutation induction at the autosomal aprt and
tk loci as well as in the genes involved in ouabain resistance or at the
X-linked hgprt locus. The assay thus combines the attributes of the two
major in vitro mammalian-cell mutagenesis systems currently being used
Furthermore, it offers the only CHO assay presently using the J:k locus and
facilitates comparison with results from the L5178Y mouse lymphoma system
(20,22,24,25,26). Such comparisons will be invaluable in determining
species variation among short-term, in vitro mutagenesis assays. To
develop this system and confirm its potential, we have: (a) selected CHO
stock lines heterozygous at the aprt and the ^k loci (necessary for
single-step selection of autosomal recessive mutations), (b) tested stock
cell lines for optimal plating density, expression time, and concentration
of selecting drugs, (c) validated the assay with data for induced mutant
frequencies after treatment with the direct mutagens EMS, MNNG, and NQO and
promutagens DMN and BP, (d) quantified and defined the dose response, and
(e) confirmed the mutational events by assays for enzyme activities in
mutant clones isolated from the above experiments.
Because the mechanisms by which mutational damage is incurred may vary
considerably for different agents, and genetic loci may differ in their
sensitivity to those agents, systems employing multiple markers may be more
effective in detecting potentially mutagenic agents. The operating
parameters for our multiple marker mutagenesis assay are now well-defined
and the system can be applied to testing other known mutagen classes as
well as to screening samples containing mixtures of potentially mutagenic
agents. Coupled with the metabolic activation methods, the system can be
used to screen crude mixtures and effluents of environmental concern.
51
-------
References
1. Abbondandolo, A., Stefania Bonatti, C. Colella, G. Corti, F.
Matteucci, A. Mazzaccaro, and G. Rainaldi, A comparative study of
different experimental protocols for mutagenesis assays with the
8-azaguanine resistance system in cultured Chinese hamster cells,
Mutation Res., 37 (1976) 293-306.
2. Adair, G.M. and J.H. Carver, Unstable, non-mutational expression of
resistance to the thymidine analogue, trifluorothymidine in CHO
cells, Mutation Res., 60 U979) 207-213.
3. Adair, G.M., J.H. Carver and D.L. Wandres, Mutagenicity testing in
mammalian cells. I. Derivation of a Chinese hamster ovary cell
line heterozygous for the adenine phosphoribosyltransferase and
thymidine kinase loci, submitted to Mutation Res.
4. Adair, G.M., L.H. Thompson, and S. Fong, -^H Amino acid selection
of aminoacyl-tRNA synthetase mutants of CHO cells: Evidence of
homo- vs. hemizygosity at specific loci, Somat. Cell Genet. 5 (1979)
329-344.
5. Arlett, C.F., D. Turnbull, S.A. Harcourt, A.R. Lehmann, and C.M.
Colella, A comparison of the 8-azaguanine and ouabain-resistance
systems for the selection of induced mutant Chinese hamster cells,
Mutation Res., 33 (1975) 261-278.
6. Baker, R.M., D.M. Brunette, R. Mankovitz, L.H. Thompson, G.F.
Whitmore, L. Simonvitch, and J.E. Till, Ouabain-resistant mutants of
mouse and hamster cells in culture, Cell, 1 (1974) 9-21.
7. Boag, J.W., The statistical treatment of cell survival data, in: T.
Alper (Ed.), Cell Survival after Low Doses of Radiation: Theoretical
and Clinical Implications, The Instutute of Physics and John Wiley
and Sons, Bristol, 1975, pp. 40-53.
8. Bridges, B.A., The three-tier approach to mutagenicity screening and
the concept of radiation-equivalent dose, Mutation Res., 26 U974)
335-340.
9. Buchwald, M., Mutagenesis at the ouabain-resistance locus in human
diploid fibroblasts, Mutat. Res., 44 (1977) 401-412.
52
-------
10. Caboche, M., Comparison of the frequencies of spontaneous and
chemically induced 5-bromodeoxyuridine-resistance mutations in
wild-type and revertant BHK-21/13 cells, Genetics, 77 U974) 309-322.
11. Capizzi, R.L. and J.W. Jameson, A table for the estimation of the
spontaneous mutation rate of cells in culture, Mutation Res., 17
(1973) 147-148.
12. Carson, M.P., D. Vernick and J. Morrow, Clones of Chinese hamster
cells cultivated in vitro not permanently resistant to azaguanine,
Mutation Res., 24 (1974) 47-54.
13. Carver, J.H., W.C. Dewey and L.E. Hopwood, X-ray-induced mutants
resistant to 8-azaguanine. I. Effects of cell density and
expression time, Mutation Res., 34 (1976) 447-464.
14. Carver, J.H., W.C. Dewey and L.E. Hopwood, X-ray-induced mutants
resistant to 9-azaguanine. II. Cell cycle dose response, Mutation
15. Carver, J.H., D.L. Wandres, G.M. Adair, and E.W. Branscomb,
Mutagenicity testing in mammalian cells: The development of
multiple drug-resistance markers having practical application for
screening potential mutagens, Mutation Res., 53 (1978) 96-97.
16. Carver, J.H., G.M. Adair, and D.L. Wandres, Mutagenicity testing in
mammalian cells. II. Validation of multiple drug-resistance markers
having practical application for screening potential mutagens,
submitted to Mutation Res.
17. Chasin, L.A, Mutations affecting adenine phosphoribosyl transferase
activity in Chinese hamster cells, Cell, 2 (1974) 37-41. Res., 34
(1976) 465-480.
18. Claxton, L.D. and P.Z. Barry, Chemical mutagenesis: An emerging
issue for public health, Amer. J. of Public Health, 67 (.1977)
1037-1042.
19. Cleaver, J.E., Induction of thioguanine- and ouabain-resistant
mutants and single-strand breaks in the DNA of Chinese hamster ovary
cells by 3H-thymidine, Genetics, 87 (1977) 129-138.
20. Clive, D., A linear relationship between tumorigenic potency in vivo
and mutagenic potency at the heterozygous thymidine kinase (.TIC*')
locus of L5178Y mouse lymphoma cells coupled with mammalian
metabolism, 1977 Elsvier/North-Holland Biomedical Press, Progress in
Genetic Toxicology, 1977, pp. 241-247.
21. Clive, D, Recent developments with the L5178Y TK heterozygote
mutagen assay system, Environ. Health Perspect., 6 (1973) 119-126.
53
-------
22. Clive, D., Flamm, W.G., Machesko, M.R., and Bernheim, N.J, A
mutational assay system using the thymidine kinase locus in mouse
lymphoma cells, Mutation Res., 16 (1972) 77-87.
23. Clive, D., G. Flamm, and J.B. Patterson, Specific-locus mutational
assay systems for mouse lymphoma cells, in: A. Hollaender (Ed.;,
Chemical Mutagens, Principles and Methods for their Detection, Vol.
3, Plenum Press, New York, 1973, pp. 79-103.
24. Clive, D. and J.F.S. Spector, Laboratory procedure for assessing
specific locus mutations at the TK locus in cultured L5178Y mouse
lymphoma cells, Mutation Res., 31 (1975) 17-29.
25. Clive, D. and P. Voytek, Evidence for chemically-induced structural
gene mutations at the thymidine kinase locus in cultured L5178Y
mouse lymphoma cells, Mutation Res., 44 (1977) 269-278.
26. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M.
Brown, Validation and characterization of the L5178Y/TK+/~ mouse
lymphoma mutagen assay system, Mutation Res., 59 (1979) 61-108.
27. Cole, J. and C.F. Arlett, Ethyl methanesulphonate mutagenesis with
L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine
and excess thymidine resistance, Mutation Res., 34 (.1976) 507-526.
28. Cole, J. and C.R. Arlett, Methyl methanesulphonate mutagenesis in
L5178Y mouse lymphoma cells, Mutation Res., 50 U978) 111-120.
29. Couch, D.B. and A.W. Hsie, Mutagenicity and cytotoxicity of
congeners of two classes of nitroso compounds in Chinese hamster
ovary cells, Mutation Res., 57 (1978) 209-216.
30. Couch, D.B., N.L. Forbes and A.W. Hsie, Comparative mutagenicity of
alkylsulfate and alkanesulfonate derivatives in Chinese hamster
ovary cells, Mutation Res., 57 (1978) 217-224.
31. Cox, R. and W.K. Masson, The isolation and preliminary
characterisation of 6-thioguanine-resistant mutants of human diploid
fibroblasts, Mutation Res., 36 (1976) 93-104.
32. Cox, R. and W.K. Masson, X-ray-induced mutation to 6-thioguanine
resistance in cultured human diploid fibroblasts, Mutation Res., 37
(1976) 125-136.
33. Davidson, R.L., K.A. O'Malley, and T.B. Wheeler, Polyethylene
Glycol-induced mammalian cell hybridization: Effect of polyethylene
glycol molecular weight and concentration, Somat. Cell Genet., 2
(1976) 271-280.
54
-------
34. Dickerman, L.H. and J.A. Tischfield, Comparative effects of adenine
analogs upon metabolic cooperation between Chinese hamster cells
with different levels of adenine phosphoribosyltransferase activity,
Mutation Res., 49 (1978) 83-94.
35. Duncan, M.E. and P. Brookes, The induction of azaguanine-resistant
mutants in cultured Chinese hamster cells by reactive derivatives of
carcinogenic hydrocarbons, Mutation Res., 21 (1978) 107-118.
36. Elsevier, S.M., R.S. Kucherlapati, G.A. Nichols, R.P. Creagan, R.G.
Giles, F.H. Ruddle, K. Willecke, and J.K. McDougall, Assignment of
the gene for galactokinase to human chromosome 17 and its regional
localization to band q21-22, Nature, 251 (1974) 633-636.
37. Fenwick, R.G. Jr. and C.T. Caskey, Mutant Chinese hamster cells with
a thermosensitive hypoxanthine-guanine phosphoribosyltransferase,
Cell, 5 (1975) 115-122.
38. Fox, M., Factors affecting the quantitation of dose-response curves
for mutation induction in Vyg Chinese hamster cells after exposure
to chemical and physical mutagens, Mutation Res., 29 (1975) 449-466.
39. Fox, M. and M. Radacic, Adaptational origin of some purine-analogue
resistant phenotypes in cultured mammalian cells, Mutation Res., 49
(1978) 275-296.
40. Frantz, C.N., Bromodeoxyuridine resistance induced in mouse lymphoma
cells by microsomal activation of dimethylnitrosamine, J. Toxicol
Environ. Health 2 (1976) 179-187.
41. Friedrich, U. and P. Coffino, Mutagenesis in S49 mouse lymphoma
cells: Induction of resistance to ouabain, 6-thioguanine, and
dibutyryl cyclic AMP, Proc. Natl. Acad. Sci. (U.S.), 74 (1977)
679-683.
42. Fujiwara, Y., Toshikazu, 0., and C. Heidelberger, Fluorinated
Pyrimidines XXXVII, Effects of 5-Trifluoromethyl-2-deoxyuridine on
the Synthesis of Deoxyribonucleic Acid of Mammalian Cells in
Culture, Mol. Pharmacol., 6 (1970) 273-280.
43. Heidelberger, C., Fluorinated Pyrimidines, Prog. Nucl. Acid Res.
Mol. Biol., 4 (1965) 1-50.
44. Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee, The
dose-response relationship for ethyl methanesulfonate-induced
mutations at the hypoxanthine-guanine phosphoribosyl transferase
locus in Chinese hamster ovary cells, Somat. Cell Genet., 1 (1975;
247-261.
55
-------
45. Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee, The
dose-response relationship for ultraviolet-light-induced mutations
at the hypoxanthine-guanine phosphoribosyltransferase locus in
Chinese hamster ovary cells, Somat. Cell Genet., 1 (1975) 383-389.
46. Hsie, A.W., A.P. Li, and R. Machanoff, A fluence response study of
lethality and mutagenicity of white, black, and blue fluorescent
light, sunlamp, and sunlight irradiation in Chinese hamster ovary
cells, Mutation Res., 45 (1977) 333-342.
47. Hsie, A.W., P.A. Brimer, R. Machanoff and M.H. Hsie, Further
evidence for the genetic origin of mutations in mammalian somatic
cells: The effects of ploidy level and selection stringency on
dose-dependent chemical mutagenesis to purine analogue resistance in
Chinese hamster ovary cells, Mutation Res., 45 (1977) 271-282.
48. Huang, S.L., Dilution of hypoxanthine-guanine phosphoribosyl
transferase and other factors affecting the frequency of
6-thioguanine resistance in Chinese hamster lung cells, Mutation
Res., 44 (1977) 119-128.
49. Huberman, E. and L. Sachs, Mutability of different genetic loci in
mammalian cells by metabolically activated carcinogenic polycyclic
hydrocarbons, Proc. Natl. Acad. Sci. (U.S.;, 73 (1976) 188-192.
50. Huberman, E. and C. Heidelberger, The mutagenicity to mammalian
cells of pyrimidine nucleoside analogs, Mutation Res., 14 (.1972)
130-132.
51. Jacobs, L. and R. DeMars, Quantification of chemical mutagenesis in
diploid human fibroblasts: Induction of azaguanine-resistant mutants
by N-methyl-N1-nitro-N-nitrosoguanidine, Mutation Res., 53 (1978)
29-53.
52. Jones, G.E. and P.A. Sargent, Mutants of cultured Chinese hamster
cells deficient in adenine phosphoribosyl transferase, Cell, 2
(1974) 43-54.
53. Jostes, R.F., L.E. Hopwood, W.C. Dewey and G.R. Blackburn, The
dependence of mutation frequency on 8-azaguanine concentration in
control and irradiated CHO cells, Mutation Res., 50 (1978) 433-440.
54. Khalizev, A.E., A.P. Pyzhov and N.I. Shapiro, Spontaneous
mutagenesis in mammalian somatic cells and DNA replication. II.
Expression of gene mutations in cultured cells, Sov. Genet., 12
(1976) 1345-1352.
55. Kit, S., D.R. Dubbs, L.J. Piekarski, and T.C. Hsu, Deletion of
thymidine kinase activity from L cells resistant to
bromodeoxyuridine, Exp. Cell Res., 31 (1963) 297-312.
56
-------
56. Kit, S., D.R. Dubbs, and P.M. Frearson, HeLa cells resistant to
bromodeoxyuridine and deficient in thymidine kinase activity, Int.
J. Cancer, 1 (1966) 19-30.
57. Kit, S., W.C. Leung, and D. Trkula, Properties of thymidine kinase
enzymes isolated from mitochondrial and cytosol fractions of normal,
bromodeoxy-uridine-resistant, and virus-infected cells, in: M. A.
Mehlman & R. W. Hanson (Eds.) Control Processes in Neoplasia,
Academic Press, New York, 1974, pp. 103-145.
58. Knaap, A.G. and J.W.I.M. Simons, A mutational assay system for
L5178Y mouse lymphoma cells using hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) deficiency as marker the
occurrence of a long expression time for mutations induced by x-rays
and EMS, Mutation Res., 39 (1975) 97-110.
59. Kozak, C., E. Nichols, and F.H. Ruddle, Gene linkage analysis in the
mouse by somatic cell hybridization: Assignment of adenine
phosphoribosyltransferase to chromosome 8 and a-galactosidase to the
X chromosome, Somat. Cell Genet., 1 (1975) 371-382.
60. Kozak, C.D. and F.H. Ruddle, Assignment of the genes for thymidine
kinase and galactokinase to Mus musculus chromosome 11 and the
preferential segregation of this chromosome in Chinese hamster/mouse
somatic cell hybrids, Somat. Cell Genet., 3 (1977) 121-133.
61. Krahn, D.F. and C. Heidelberger, Liver homogenate-mediated
mutagenesis in Chinese hamster V79 cells by polycyclic aromatic
hydrocarbons and aflatoxins, Mutation Res., 46 (.1977) 27-44.
62. Lea, D.E. and C.A. Coulson, The distribution of the numbers of
mutants in bacterial populations, J. Genet., 49 (1948) 264-284.
63. Lowry, O.K., N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein
measurement with the folin phenol reagent, J. Bioi. Chem., 193
(1951) 265-275.
64. Luria, S.E. and M. Delbruck, Mutations of bacteria from virus
sensitivity to virus resistance, Genetics, 28 (1943) 491-511.
65. Mayer, V.W. and W.G. Flamm, Legislative and technical aspects of
mutagenicity testing, Mutation Res., 29 (1975) 205-300.
66. Miller, O.J., P.W. Allderdice, D.A. Miller, W.R. Breg, and B.R.
Migneon, Human thymidine kinase locus: Assignment to chromosome 17
in a hybrid of man and mouse cells, Science, 173 (1971) 244-245.
67. Moore-Brown, M.M., D. Clive, B.E. Howard, A.G. Batson and K.O.
Johnson, The utilization of trifluorothymidine (TFT) to select for
thymidine kinase-deficient (TK"1"/") L5178Y mouse lymphoma cells,
in preparation.
57
-------
68. Morrow J., Gene inactivation as a mechanism for the generation of
variability in somatic cells cultivated in vitro, Mutation Res., 44
(1977) 391-400.
69. Myhr, B.C. and J.A. DiPaolo, Requirement for cell dispersion prior
to selection of induced azaguanine-resistant colonies of Chinese
hamster cells, Genetics, 80 (1975) 157-169.
70. Newbold, R.F., P. Brookes, C.F. Arlett, B.A. Bridges and B. Dean,
The effect of variable serum factors and clonal morphology on the
ability to detect hypoxanthine guanine phosphoribosyl transferase
(HPRT) deficient variants in cultured Chinese hamster cells,
Mutation Res., 30 (1975) 143-148.
71. Nikaido, 0. and M. Fox, The relative effectiveness of 6-thioguanine
and 8-azaguanine in selecting resistant mutants from two V79 Chinese
hamster cells in vitro, Mutation Res., 35 (1976) 279-288.
72. O'Neill, J.P. and A.W. Hsie, Chemical mutagenesis of mammalian cells
can be quantified, Nature 269 (1977) 815-817.
73. O'Neill, J.P. and A.W. Hsie, Phenotypic expression time of
mutagen-induced 6-thioguanine resistance in Chinese hamster ovary
cells (CHO/HGPRT system), Mutation Res., 59 (1979) 109-118.
74. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie,
A quantitative assay of mutation induction at the
hypoxanthine-guanine phosphoribosyl transferase locus in Chinese
hamster ovary cells (CHO/HGPRT system): Development and definition
of the system, Mutation Res., 45 U977) 91-101.
75. O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian, P.A.
Brimer and A.W. Hsie, A quantitative assay of mutation induction at
the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese
hamster ovary cells (CHO/HGPRT system): Utilization with a variety
of mutagenic agents, Mutation Res., 45 (1977) 103-109.
76. O'Neill, J.P., J.C. Fuscoe and A.W. Hsie, Mutagenicity of
hetrocyclic nitrogen mustards (ICR compounds) in cultured mammalian
cells, Cancer Res., 38 (1978) 506-509.
77. Orkin, S.H. and J.W. Littlefield, Nitrosoguanidine mutagenesis in
synchronized hamster cells, Exptl. Cell Res., 66(1971) 69-74.
78. Peterson, A.R., D.F. Krahn, H. Peterson, C. Heidelberger, B.K.
Bhuyan and L.H. Li, The influence of serum components on the growth
and mutation of Chinese hamster cells in medium containing
8-azaguanine, Mutation Res., 36 (1976) 345-356.
58
-------
79. Reyes, P. and C. Heidelberger, Fluorinated pyrimidines, XXVI,
Mammalian thymidylate synthetase: Its mechanism of action and
inhibition by fluorinated nucleotides, Mol. Pharmacol., 1 (1965)
14-30.
80. Roufa, D.J., B.N. Sadow and C.T. Caskey, Derivation of TK- clones
from revertant TK+ mammalian cells, Genetics, 75 (1973) 515-530.
81. Scheffe, H., Practical solutions of the Behrens-Fisher problem, J.
Am. Stat. Assoc., 65 (1970) 1501-1508.
82. Shapiro, N.I., A.E. Khalizev, E.V. Luss, E.S. Manuilova, O..N.
Petrova and N.B. Varshaver, Mutagenesis in cultured mammalian
cells. II. Induction of gene mutations in Chinese hamster cells,
Mutation Res., 16 (1972) 89-101.
83. Shaw, C.I. and A.W. Hsie, Conditions necessary for quantifying ethyl
methanesulfonate-induced mutations to purine-analogue resistance in
Chinese hamster V79 cells, Mutation Res., 51 (1978) 237-254.
84. Simons, J.W.I.M., Dose-response relationships for mutants in
mammalian somatic cells in vitro, Mutation Res., 25 (1974) 219-227.
85. Skopek, T.R., H.L. Leiber, B.W. Penman and W.G. Thilly, Isolation of
a human lymphoblastoid line heterozygous at the thymidine kinase
locus: Possibility for a rapid human cell mutation assay, Biochem.
Biophys. Res. Comm., 84 (1978) 411-416.
86. Slack, C., R.H.M. Morgan, B. Carritt, P.S.G. Goldfarb, and M.L.
Hooper, Isolation and characterisation of Chinese hamster cells
resistant to 5-fluorodeoxyuridine, Exper. Cell Res., 98 (1976) 1-14.
87. Taylor, M.W., J.H. Pipkorn, M.K. Tokito, and R.O. Pozzatti Jr.,
Purine mutants of mammalian cells III. Control of purine
biosynthesis in adenina phosphoribosyl transferase mutants of CHO
cells, Somat. Cell Genet., 3 (1977) 195-206.
88. Thacker, J., M.A. Stephens and A. Stretch, Factors affecting the
efficiency of purine analogues as selective agents for mutants of
mammalian cells induced by ionising radiation, Mutation Res., 35
(1976) 465-478.
89. Thacker, J., A. Stretch and M.A. Stephens, The induction of
thioguanine-resistant mutants of Chinese hamster cells by -rays,
Mutation Res., 42 (1977) 313-326.
90. Thacker, J., M.A. Stephens, and A. Stretch, Mutation to
ouabain-resistance in Chinese hamster cells: Induction by ethyl
methanesulphonate and lack of induction by ionising radiation,
Mutation Res., 51 (1978) 255-270.
59
-------
91. Thilly, W.G., J.G. Deluca, H. Hoppe IV and B.W. Penman, Phenotypic
lag and mutation to 6-thioguanine resistance in diploid human
lymphoblasts, Mutation Res., 50 (1978) 137-144.
92. Thompson, L.H., S. Fong, and K. Brookman, Validation of conditions
for efficient detection of HPRT and APRT mutations in suspension
cultured Chinese hamster ovary cells, Mutation Res., in press.
93. Tischfield, J.A. and F.H. Ruddle, Assignment of the gene tor adenine
phosphoribosyltransferase to human chromosome 16 by mouse-human
somatic cell hybridization, Proc. Natl. Acad. Sci. USA, 71 (1974)
45-49.
94. Umeda, M. and C. Heidelberger, Comparative studies of fluorinated
pyrimidines with various cell lines, Cancer Res., 28 (1978).
95. van Zeeland, A.A. and J.W.I.M. Simons, The effect of calf serum on
the toxicity of 8-azaguanine, Mutation Res., 27 (1975) 135-138.
96. van Zeeland, A.A. and J.W.I.M. Simons, Linear dose-response
relationships after prolonged expression times in V-79 Chinese
hamster cells, Mutation Res., 35 (1976) 129-138.
97. van Zeeland, A.A. and J.W.I.M. Simons, The use of correction factors
in the determination of mutant frequencies in populations of human
diploid skin fibroblasts, Mutation Res. 34 (1976) 149-158.
98. Westerveld, A., R.P. Visser, P. Meeva Khan, and D. Bootsma, Loss of
Human genetic markers in man-Chinese hamster somatic cell hybrids,
Nature New Biol., 234 (1971) 20-24.
60
-------
AAr
AGr
aprt+/+
aprt+/~
aprt~/~
C HO- IB- 2
CHO-AT3-2
hgprt"1"
hgprt"
OUAR
FUdRr
PE
S~, cells
WT
GLOSSARY
cell phenotype, resistance to AA
cell phenotype, resistance to AG
cell genotype, with two genes for functional APRT enzyme
cells with only one gene (heterzygous) tor functional APRT
enzyme
cells with no gene for functional APRT enzyme
cell line heterzygous at the aprt locus
cell line heterzygous at the aprt and ^k loci
cell genotype, with a gene (X-linked) for functional HGPRT
enzyme
cells without a gene for functional HGPRT enzyme
cell phenotype, resistance to QUA
cell phenotype, resistance to FUdR
ratio of viable ells to cells plated; mean plating efficiency
surviving cell fraction; mean cell survival relative to
controls
cell phenotype, resistance to TG
cell genotype, with two genes for functional TK enzyme
cell genotype, with only one gene for functional TK enzyme
cells with no gene for functional TK enzyme
wild type cells (aprt"1"/"1", hgprt"1", tk+/+, ATPase with
normal sensitivity to ouabain)
61
-------
TECHNICAL REPORT DATA
(f lease rf.acl Instructions on t/ic reverse before cutnplctinx}
1. REPORT NO.
2.
4. T.TLE AND SUBTITLE Mutagenicity Testin- in Mammalian
Cells. The development and Validati.o'.i of Multiple
Drug-Resistance Markers Having Practical Application
for Screening Potential Mutagens.
3. RECIPIENT'S ACCESSION NO.
0. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO
June II. Carver, Gerald M. Adair, and Daniel L. Wandres
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Lawrence Livermore Laboratory
Biomedical Sciences Division
University of California
Livermore. CA 94550
10. PROGRAM ELEMENT NO.
1NE 625F
11. CONTRACT/GRANT NO.
EPA-IAG-D5-E681-AN and AO
12. SPONSORING AGENCY NAME AND ADDRESS
U. S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Interim 1/76 thru 1/79
14. SPONSORING AGENCY CODE
EPA/600/17
15. SUPPLEMENTARY NOTES
This project is part of the EPA-planned and coordinated Federal Interagency
Energy/Environment R&D Program.
16. ABSTRACT
Chinese hamster ovary (CHO) cell lines heterozygous at the adenine phospho-
ribosyltransferase (aprt) and thymidine kinase (tk) loci were isolated and used for
single-step selection of spontaneous and induced mutants resistant to 8-azaadenine
(AAr), 6-thioguanine (TGr), 5-fluorodeoxyuridine (FUdRr), or ouabain (OUAR). Mutation
data are reported for direct mutagens ethyl methanesulfonate (EMS), N-methyl-N'-nitro-
N-nitrosoguanidine (MNNG), 4-nitroquinoline-l-oxide (NQO) and for promutagens dimethyl-
nitrosamine (DMN) and benzo(a)pyrene (BaP) activated by rat liver homogenates.
Critical plating densities were established for AAr, TGr, and FUdRr. Optimal ex-
pression of mutant phenotypes after mutation induction with EMS, DMN, or BaP were 2 to
4 d for AAr, 6 to 8 d for TGr, 3 d for OUAR, and 1 to 3 d for FUdRr. The induced
mutant frequencies as a function of relative cell survival after treatment with EMS,
DMN, or BaP showed locus-specific differences in sensitivity. Of 61 clonal isolates
resistant to AA and assayed for APRT activity, 87% had < 5% wild type activity; of 30
TGr clones assayed, 83% had<5% wild type HGPRT activity. Of 42 FUdRr clones assayed,
41 had
------- |