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Carcinogenesis |
Carcinogenesis Laboratory, Departments of Microbiology and Biochemistry, and the Cancer Center, Michigan State University, East Lansing, Michigan 48824-1302
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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radiation
(6)
that were found to form tumors in athymic mice had
lost p53 transactivating ability. What is more, these cell
strains showed changes in karyotype (4
, 5)
. The product of the p53 gene is a potent transcription factor involved in the transcriptional control of genes that play critical roles in cell cycle control, DNA repair, and apoptosis (see Refs 7 , 8 ) and is inactivated in >50% of all human tumors (9 , 10) . In addition, inactivation of p53 has resulted in the progression of mammalian fibroblasts in culture to a transformed phenotype (11 , 12) .
Alkylating agents covalently attach alkyl groups to cellular macromolecules. Reaction of a methylating agent with DNA results in the formation of up to 13 different adducts (13) , with methylation of the O6 position of guanine being the most potentially mutagenic lesion (14, 15, 16) , inducing primarily G:C to A:T transitions (16, 17, 18) . Repair of O6-MeG in human cells is accomplished primarily through the action of AGT, which transfers the methyl group from O6-MeG in DNA to an interior cysteine residue of AGT (19, 20, 21) and inactivates the AGT protein. O6-BzG can serve as a substrate analogue for AGT (22) , resulting in the transfer of the benzyl group from O6-BzG to the same cysteine of AGT involved in removal of the methyl group from O6-MeG. Therefore, cells in culture can be depleted of AGT by addition of O6-BzG to the medium, allowing one to manipulate the capacity of the cells to repair O6-MeG and therefore to determine the cellular effects of this particular adduct.
The present study asked four questions: (a) whether the simple methylating agent MNU can transform MSU-1.1 cells to focus formation; (b) whether O6-MeG plays a causal role in this transformation; (c) whether MNU treatment results in the malignant transformation of MSU-1.1 cells; and (d) whether mutations in the p53 gene play a role in focus formation and/or transformation. For this purpose, two populations of MSU-1.1 cells were prepared, one group that was depleted of AGT activity by pretreatment with O6-BzG and another not depleted, exposed to MNU, and assayed for focus formation. Representative focus-derived cell strains were assayed for p53 transactivating ability and for tumorigenicity. A single, brief exposure of the cells to MNU induced a dose-dependent increase in focus formation, and pretreatment with O6-BzG significantly decreased the dose of MNU needed to induce such foci, indicating that O6-MeG is the lesion principally responsible for this transformation. A substantial fraction of the focus-derived cell strains formed tumors in athymic mice, and all but one of these strains lacked transactivating ability. The latter was heterozygous for p53 transactivation ability, but the cells from the tumors produced by that strain contained two mutant p53 alleles. Evidence suggests that the MSU-1.1 population used for these studies contained a very low frequency of cells that were heterozygous for a specific p53 mutation, and that MNU-induced homologous mitotic recombination generated cells with that mutation in both alleles. The data also suggest that loss of p53 transactivating ability allows these cells to form foci. The results indicate that loss of p53 transactivating ability is not sufficient to cause malignant transformation of MSU-1.1 cells, but they strongly suggest that it greatly facilitates such transformation.
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MATERIALS AND METHODS |
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Carcinogen Treatment.
Cells to be treated were plated in complete medium at
104
cells/cm2. After 
16
h, the medium was removed, and the cells were rinsed twice with PBS and
covered with Eagle’s MEM lacking serum and buffered with 20
mM HEPES (pH 7.25; treatment medium). MNU (Sigma, St.
Louis, MO) was dissolved in anhydrous DMSO immediately before use. The
appropriate amount of the MNU-DMSO solution was added to each dish to
give the designated concentrations. The total concentration of DMSO in
the medium was <0.5%. Control populations were treated the same way
but received DMSO only. The cells were incubated for 30 min at 37°C
in a humidified incubator with 5% CO2, after
which the treatment medium was removed, the cells were rinsed twice
with PBS, and complete medium was added.
Elimination of AGT Activity Using O6-BzG.
O6-BzG was dissolved in DMSO at a
concentration of 25 mM and stored at -20°C
under nitrogen gas. For the population of cells to be depleted of AGT
by O6-BzG, the compound was added at a
final concentration of 25 µM 2 h before
treatment with MNU. For this population,
O6-BzG (25 µM)
was also added to the treatment medium, and at the end of the MNU
treatment, these cells were refed with complete medium containing
O6-BzG. After 48 h, the medium
for these cells and also for the other population was changed to
complete medium lacking O6-BzG.
Cytotoxicity Assay.
Immediately after treatment, cells were rinsed with PBS, dislodged with
trypsin, suspended in complete medium with or without
O6-BzG, and plated at densities
designed to yield 40 clones per 100-mm-diameter dish. At a minimum,
three dishes were used per group. All cells were refed with complete
medium at 48 h after treatment and after 1 week. After 2 weeks,
the cells were stained with crystal violet. The clones were counted,
and the surviving fraction was calculated using the cloning efficiency
of the treated cells and expressed as a fraction of the cloning
efficiency of untreated cells.
Focus Assay.
The focus assay was performed essentially as described
(6)
. Briefly, cells to be assayed for the ability to form
foci were kept in exponential growth after MNU treatment, subculturing
as needed. After 8 days, the cells were dislodged with trypsin, pooled,
and suspended in complete medium supplemented with 20 mM
HEPES (pH 7.5) and with the serum level reduced to 0.5% supplemented
calf serum. For each dose, 106 cells were assayed
at 5 x 104
cells per
100-mm-diameter dish. Cells were refed weekly with this medium. After
4–6 weeks, the dishes were scanned for foci, i.e., densely
piled, clonal proliferations of cells exhibiting an altered morphology
on a confluent monolayer, visible when the dishes were illuminated from
beneath with a focused beam of light. Representative foci were isolated
using trypsin and subcloned twice to eliminate background,
non-focus-forming cells. The remaining cells in the dishes used to
assay for focus formation were fixed with methanol and stained with
methylene blue, and the foci were counted to determine the frequency.
p53 Transactivation Ability Assay.
A transgenic yeast assay designed by Schärer and Iggo
(23)
was used essentially as described (24)
with the exceptions noted. Briefly, total RNA was isolated from the
cell strain to be assayed using the RNAzol B method (TEL-TEST,
Friendswood, TX) according to the manufacturers’ instructions. RT of
mRNA was performed using 2.5 µg of total RNA denatured at 65°C for
15 min in a 20-µl volume containing 50 mM Tris-HCl (pH
8.3), 75 mM KCl, 3 mM
MgCl2, 10 mM DTT, a 500
nM concentration of each dNTP, 400 ng of
oligo(dT)12–18, and 40 units of murine leukemia
virus reverse transcriptase (Life Technologies) and incubated at
37°C for 90 min. The p53-specific primers used for PCR
were described previously (23)
. The RT reaction product
was diluted 1:10 in water, and 5 µl were used as a template in a
50-µl reaction containing 10 mM KCl, 6
mM
(NH4)2SO4,
20 mM Tris-HCl (pH 8.2), 2
mM MgCl2, 0.1% Triton
X-100, 10 µg/ml BSA, a 400 nM concentration of
each dNTP, a 400 nM concentration of each primer,
and 2.5 units of Pfu polymerase (Stratagene, La Jolla, CA).
The PCR reaction was carried out in a HyBaid (Woodbridge, NJ)
thermocycler for 40 cycles (94°C for 30 s, 55°C for 30 s,
and 72°C for 90 s), and the amplified p53 cDNA
product was purified using a silica gel column (Qiagen, Valencia, CA).
A gapped plasmid carrying a selectable marker (leucine autotrophy) and
the purified PCR product were electroporated into yeast using the
Cell-Porator system (Life Technologies) set at 400 V, 10 µF, and low
resistance. Yeast that incorporated a p53 cDNA into the
gapped plasmid by homologous recombination were selected. These yeast
cells also contained a resident plasmid carrying a
p53-responsive reporter gene. Yeast cells that incorporate
p53 cDNA from a wild-type allele of the human cells can be
identified by the expression of this reporter gene, which requires
wild-type p53 for transcriptional activation. Because of its
design, the assay can only detect inactivating mutations located within
exons 4–10, but it offers the advantage of assaying each allele
separately and, therefore, can determine that a cell is heterozygous
for p53. However, the assay cannot reveal that a cell is
hemizygous for p53.
Nucleotide Sequencing of the p53 Gene.
The sequence of the p53 RT-PCR product was determined by
automated dye terminator sequencing (Applied Biosystems Division,
Perkin-Elmer, Foster City, CA). The primers, specifically designed to
bind to p53 cDNA and used for sequencing, are shown in Table 1
.
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Restriction Enzyme Analysis.
Analysis of p53 RT-PCR product involved digestion of 1 µg
of purified p53 coding sequence in a 20 µl reaction volume
using NlaIII restriction enzyme (New England Biolabs,
Beverly, MA) following the manufacturers’ recommended procedure. The
entire reaction was resolved on a 3% agarose gel containing 0.5
µg/ml EtBr, and the DNA was visualized using UV light.
Southern Blotting Analysis.
DNA was isolated using the Puregene kit (Gentra Systems, Research
Triangle Park, NC). DNA (15 µg) was digested with the designated
restriction enzyme (Boehringer Mannheim, Indianapolis, IN) (5 U/µg
DNA) following manufacturers’ instructions. The reactions were carried
out at 37°C for 16 h, and the products were purified by
phenol:chloroform extraction. Purified digest (10 µg) was resolved on
a 1% agarose gel by electrophoresis at 35 V (constant) for 16–18 h.
DNA was transferred to a Zeta Probe membrane (Bio-Rad, Hercules, CA),
crosslinked using the UV Stratalinker 2400 (Stratagene, La Jolla, CA),
and analyzed. For RFLP analysis, probe pUC10–41 (American Type Culture
Collection, Rockville, MD), which is homologous to D17S71, and pYNZ22.1
(ATCC, Rockville MD), which is homologous to D17S5, were used. To
determine the number of p53 alleles, p53 DNA
(RT-PCR product) was used as a probe for p53, and an intron
1 DNA sequence of the HPRT gene (26)
was used
as a probe for the HPRT gene, which served as a loading
control. The random primer labeling of probes and hybridization
conditions used were as described (27)
. Blots were exposed
to PhosphoImaging cassettes, and the data were analyzed using Image
Quant 3.1 software (Molecular Dynamics, Sunnyvale, CA).
Microsatellite Analysis.
The sequences for the primer pairs (Whitehead Institute database) used
to amplify the region surrounding the microsatellite repeats are given
in Table 2
. PCR was performed using 250 ng genomic DNA in a 25 µl mixture
containing: 50 mM Tris-Cl (pH 9.0); 20 mM
(NH4)2SO4;
1.5 mM MgCl2; 400 nM of
each dNTP; 150 ng of each primer; and 2.5 U Tfl polymerase
(Epicentre Technologies, Madison, WI). Amplification was performed
using a HyBaid thermocycler (Woodbridge, NJ) and using a PCR profile of
94°C for 3 min, followed by 35 cycles of 94°C for 10 s, X°
for 10 s and 72°C for 15 s. The value "X" depended upon
the specific primer set used. These are listed in Table 2
. The PCR
products were analyzed by 15% nondenaturing PAGE [100 V (constant)
for 16–18 h]. The bands were stained with EtBr and visualized using
UV illumination.
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RESULTS |
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, with both sets of cells, there was a dose-dependent
decrease in survival, but this was significantly greater in the cells
depleted of AGT activity, indicating that
O6-MeG is the principal cytotoxic
adduct formed. The cells exhibited a corresponding dose-dependent
increase in focus induction, and
O6-BzG pretreatment significantly
enhanced the frequency of this induction (Fig. 1B
).
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7 x 107 cells, and injected into mice as described
(106 cells per site). The foci used for these
assays were taken from experiments in which the frequency of foci was
at least four times higher than background; the majority of them were
from experiments yielding frequencies 6–30 times higher than the
background frequencies. A total of 35 independent focus-derived cell
strains were assayed for the ability to form tumors in athymic mice, 28
from MNU-treated populations and 7 from nontreated control populations.
The nontumorigenic MSU-1.1 parental cells were also tested. The data
for MSU-1.1 cells are shown in Table 3
. They included results from a large series of experiments over several
years. As shown in Table 3
, 10 of 35 strains (28.6%) formed tumors
that reached a diameter of 1 cm in a relatively short period (6–16
weeks). One of these strains, MA0-1, was from a focus taken from an
untreated population. An additional cell strain, MA2-1, derived from a
focus from an MNU-treated group, gave rise to tumors after a somewhat
longer period (11–31 weeks). All of the tumors were malignant.
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for
p53 transactivating ability. Parental MSU-1.1 cells were
also tested. The p53 status results are included in Table 3
.
Of the 28 strains derived from MNU-induced foci, 14 (50%) had lost all
p53 transactivating ability. Ten of these 14 were strains
that had proven able to produce tumors rapidly. Because the other four
did not form tumors, loss of p53 activity is not sufficient
to convert the cells into tumor-forming cells. One MNU-induced
focus-derived strain, i.e., MA2-1, the one that produced
tumors only after a relatively long latency, was heterozygous for
p53 activity, but analysis of cells derived from a tumor
formed by strain MA2-1 revealed that the tumor-derived cells no longer
contained a wild-type p53 allele. Of the seven foci-derived
strains from nontreated populations, only the one that formed tumors
(MA0-1) had lost p53 transactivating ability (Table 3)
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Nature of the Mutation in the p53 Gene.
Nucleotide sequencing of the coding region of the p53 gene
from several representative cell strains that expressed only mutant
p53 showed that each contained an A:T to G:C transition at
the first position of codon 215, which changes the amino acid from
serine to glycine. Finding a common mutation was highly unexpected,
because each strain had been derived from a focus that developed in an
independent population of cells treated with MNU, and the frequency of
such foci was significantly higher than background. Furthermore, the
coding region of p53 has hundreds of sites where a mutation
can eliminate its transactivating function (7
, 10)
.
In addition, an A:T to G:C transition is not commonly induced by
methylating agents (15
, 17)
. This strongly suggested that
the focus-derived strains that exhibited complete loss of
p53 transactivating ability arose as the result of an effect
of MNU on a subpopulation of cells in the target population that
already contained the A:T to G:C transition at codon 215 in
p53.
The A:T to G:C base change at codon 215 creates a recognition site for
the restriction enzyme NlaIII. Therefore, we used
restriction enzyme digestion and gel electrophoresis to rapidly screen
for the presence of this specific base substitution in additional
focus-derived cell strains that the yeast assay indicated were devoid
of p53 transactivating ability. NlaIII
restriction of wild-type p53 gives three fragments of
lengths 477, 414, and 173 bp and five fragments <30 bp. The A:T to G:C
transition at codon 215 results in the 173-bp fragment being further
digested to yield fragments of 107 and 66 bp (see Fig. 2
). Analysis using NlaIII digestion confirmed that all
focus-derived cell strains that exhibited loss of wild-type
p53, as determined by the yeast assay, including the
heterogygous strain, MA2-1, contained the identical codon 215 mutation.
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Southern blotting analysis of EcoRI-digested DNA was carried
out, using an RT-PCR product from p53 mRNA as the probe for
the p53 gene. The HPRT gene, used as a loading
control for the DNA blots, was probed with an intron 1 segment of that
gene (26)
. The blot was initially probed for
p53 and then stripped and reprobed for HPRT.
MSU-1.1 cells are derived from a male donor and contain a single X
chromosome (1)
. Because HPRT is located on the
X chromosome, it represents a single-copy gene for these male cells.
The intensities of the resulting bands from the different probes were
normalized using DNA from parental MSU-1.1 cells as the standard. The
results from the comparative Southern analysis showed that all 15
focus-derived cell strains that expressed only mutant p53
contained two copies of p53. These data are included where
appropriate in the characterization of the strains in Table 3
.
Investigation of parental MSU-1.1 cells revealed two informative RFLP
markers on the p arm of chromosome 17. One locus, located telomeric to
p53 and detected by the pYNZ22.1 probe, produces multiple
bands in the range of 0.5–1.3 kb in MspI-digested DNA. The
other locus, centromeric to p53 and detected by the pUC10-41
probe, yields bands of 2.4 and 1.9 kb in MspI-digested DNA.
Using these markers, we examined each of the focus-derived cell strains
that had lost p53 transactivating function. Three patterns
of LOH were found: loss of the telomeric marker, loss of both markers,
and no LOH (data not shown). A representative RFLP Southern blot is
shown in Fig. 3
.
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). Three are located between p53 and the telomeric RFLP
marker; the other three are located between p53 and the
centromeric RFLP marker. A representative microsatellite analysis gel
is shown in Fig. 5
. Analysis of the 15 focused-derived cell strains using these
informative microsatellite markers revealed seven distinct patterns of
LOH (Fig. 6
). To determine whether the LOH seen in these strains for 17p resulted
from some kind of generalized genomic instability caused by the loss of
p53 function, we examined the status of informative
microsatellite markers on chromosomes 11 and 18 in these focus-derived
cell strains. The data revealed no LOH of these markers for any cell
strain tested (data not shown). The results from the RFLP analysis,
taken together with the comparative Southern blotting data indicating
that focus-derived cell strains that expressed only mutant
p53 contain two copies of p53, support the
hypothesis that homologous recombination was involved in the generation
of the of MNU-induced, focus-derived cell strains that express two
mutant p53 alleles.
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Attempt to Isolate the Preexisting Precursor Containing the
Mutation at Codon 215.
The role that loss of p53 function plays in causing or
allowing cells to form foci is not readily apparent. Because the
majority, 54% (19 of 35), of the focus-derived cell strains assayed
retained wild-type p53, clearly such loss is not required
for focus formation. The fact that 15 of the 35 strains assayed (43%)
lost p53 function raises the question of whether loss of
p53 activity can directly cause MSU-1.1 cells to form foci.
Our data show that assaying MSU-1.1 cells for focus formation provides
an efficient method for identifying cells that lack functional
p53, but that changes other than loss of p53
function may also be involved. However, if loss of p53
transactivating ability did not cause the MSU-1.1 cells to form foci,
and the foci resulted from an additional, as yet unidentified, genetic
change induced by MNU, it is difficult to understand why such a high
proportion of the MNU-induced, focus-derived cell strains were found to
lack wild-type p53. If loss of p53 were not
somehow being "selected for," the frequency of cells containing the
codon 215 mutation that preexisted in the population would have to be
very high indeed, i.e., 43%. We plated parental MSU-1.1
cells at cloning densities and used RT-PCR and NlaIII
digestion to analyze 80 independent clones for their p53
status. None of the 80 exhibited a transition at codon 215. Therefore,
the frequency of cells in that population that contain that codon 215
mutation has to be <1 in 80. We estimate that the frequency is not
lower than 1 in 10,000 cells, because near the beginning of our studies
on the frequency of MNU-induced focus formation, we carried out a
limiting dilution of our MSU-1.1 target cell population to reduce the
background frequency of foci. We plated 10 dishes at 5,000 cells per
dish, expanded these subpopulations, and stored them for future use.
Progeny of five of these limiting dilution sets of cells were assayed
for the frequency of foci, using 3 x 105
from cells from each population. No foci were
found, indicating a background of <1 x 10-6 cells. One of these limiting dilution sets
was then expanded and used as the target population for the MNU
experiments reported here, as well as for the studies with cobalt-60
irradiation referred to above. Each carcinogen treatment yielded cells
able to form foci, and some of these contained the codon 215 mutation.
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DISCUSSION |
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The data in Fig. 1
indicate that MNU plays a role in converting a
substantial number of target MSU-1.1 cells into focus-forming cells.
However, the role of MNU in converting focus-forming cells into
malignant cells appears to be indirect. As shown in Table 3
, the
ability to form foci is not sufficient for tumorigenicity. Neither is
mere loss of p53 transactivating ability, because only 10 of
15 strains that lacked functional p53 were able to form
tumors. Nevertheless, of 35 focus-derived cell strains tested, only
those that had lost this p53 function were able to form
tumors. (In the one case in which the function of only one
p53 allele was lost, the cells derived from the tumors
formed by the latter strain, MA2-1T, no longer contained the wild-type
p53 allele.) These data indicate that although loss of
p53 function is not sufficient for transformation of MSU-1.1
cells into malignant cells, it greatly increases the chance that a cell
will acquire the additional changes required for such transformation.
The fact that tumorigenic cell strains were isolated from foci induced by a much lower dose of MNU in cells from which AGT had been eliminated by pretreatment with O6-BzG than was required for populations not depleted of AGT strongly suggests that O6-MeG adducts played an indirect causal role in the malignant transformation. Unpublished data from this laboratory5 show that the latency period is a function of the number of malignant cells injected into athymic mice. The fact that finding that the tumors formed by cell strain MA2-1, which is heterozygous for p53, exhibited a longer latency period than those from strains totally devoid of p53 transactivating ability, whereas the cells derived from those tumors (MA2-1T) were devoid of wild-type p53 activity, supports the hypothesis that loss of p53 enables cells to acquire the additional changes needed to become malignant. We hypothesize that during the propagation of the heterozygous MA2-1 cells to obtain sufficient cells for injection, a fraction of the population lost the wild-type p53 allele, and their progeny cells gave rise to the tumors that arose after a relatively long latency.
The data suggesting that by causing mitotic recombination between homologous chromatids, MNU was indirectly able to cause cells to become malignant may have clinical significance, because alkylating agents are used in chemotherapy (31 , 32) . Chemotherapy protocols try to maximize the cytotoxic effects of specific agents without increasing the chances that therapy-related new malignancies arise. The results of our study indicate that such agents, e.g., those that act through formation of adducts on the O6 position of guanine, not only cannot induce point mutations but also may produce cells that have been converted from a heterozygous state for a particular mutation into a homozygous state by inducing homologous mitotic recombination. Recent evidence that such recombination occurs in carcinogen-treated mice that carry a mutant p53 transgene (33) supports this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by United States
Department of Health and Human Services Grants CA21253 and CA60907 from
the National Cancer Institute and Training Grant ES07255 from the
National Institute of Environmental Health Sciences. 
2 Present address: Chemical Industry Institute of
Toxicology, Research Triangle Park, NC 27709.
3 To whom requests for reprints should be
addressed, at Carcinogenesis Laboratory, Food Safety and Toxicology
Building, Michigan State University, East Lansing, MI 48824-1302.
Phone: (517) 353-7785; Fax: (517) 353-9004; E-mail: maher{at}msu.edu
4 The abbreviations used are: BPDE,
(±)-7ß,8
-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene;
AGT, O6-alkylguanine-DNA alkyltransferase;
EtBr, ethidium bromide; HPRT, hypoxanthine phosphoribosyltransferase;
LOH, loss of heterozygosity; MNU,
N-methyl-N-nitrosourea; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
O6-BzG,
O6-benzylguanine;
O6-MeG,
O6-methylguanine; RT, reverse
transcription.
5 J. J. McCormick, unpublished data.
Received 3/ 9/00. Accepted 5/30/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.. Proc.Natl.Acad.Sci.USA, 89: 2237-2241, 1992.This article has been cited by other articles:
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  P. Lito, B. D. Mets, S. Kleff, S. O'Reilly, V. M. Maher, and J. J. McCormick Evidence That Sprouty 2 Is Necessary for Sarcoma Formation by H-Ras Oncogene-transformed Human Fibroblasts J. Biol. Chem., January 25, 2008; 283(4): 2002 - 2009. [Abstract] [Full Text] [PDF]
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 Z. Lou, S. O'Reilly, H. Liang, V. M. Maher, S. D. Sleight, and J. J. McCormick Down-Regulation of Overexpressed Sp1 Protein in Human Fibrosarcoma Cell Lines Inhibits Tumor Formation Cancer Res., February 1, 2005; 65(3): 1007 - 1017. [Abstract] [Full Text] [PDF]
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 S. W.P. Wijnhoven, E. Sonneveld, H. J.M. Kool, C. M.M. van Teijlingen, and H. Vrieling Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes Carcinogenesis, January 1, 2003; 24(1): 139 - 144. [Abstract] [Full Text] [PDF]
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 G. P. Margison, M. F. Santibanez Koref, and A. C. Povey Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine Mutagenesis, November 1, 2002; 17(6): 483 - 487. [Abstract] [Full Text] [PDF]
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 A. C. Povey, A. F. Badawi, D. P. Cooper, C. N. Hall, K. L. Harrison, P. E. Jackson, N. P. Lees, P. J. O'Connor, and G. P. Margison DNA Alkylation and Repair in the Large Bowel: Animal and Human Studies J. Nutr., November 1, 2002; 132(11): 3518S - 3521. [Abstract] [Full Text] [PDF]
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, cells were
pretreated with 25 µMO6-BzG for 2 h, had
O6-BzG present during the 30-min MNU
treatment, and were maintained in
O6-BzG for an additional 48 h;
•, cells received MNU treatment only. The background frequencies of
foci have been subtracted.
, retention of
heterozygosity at a marker; •, LOH. The number of independent
focus-derived cell strains exhibiting each pattern is as follows:
patterns 2–4, one strain each; patterns
1 and 6, two strains; pattern
5, three strains; pattern 7, five
strains.