Affiliations of authors: W.-D. Chen, M. R. Aminoshariae, Department of Medicine, Case Western Reserve University and University Hospitals of Cleveland, OH; J. R. Eshleman, Department of Pathology, The Johns Hopkins University, School of Medicine, Baltimore, MD; A.-H. Ma, N. Veloso, Environmental Health Sciences Department, Case Western Reserve University; S. D. Markowitz, Department of Medicine and Ireland Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, and Howard Hughes Medical Institute, Cleveland; W. D. Sedwick, Department of Medicine and Ireland Cancer Center, Case Western Reserve University and University Hospitals of Cleveland; M. L. Veigl, Ireland Cancer Center, Case Western Reserve University and University Hospitals of Cleveland.
Correspondence to: Sanford D. Markowitz, M.D., Ph.D., Howard Hughes Medical Institute, U.C.R.C. #2, Suite 200, 11001 Cedar Rd., Cleveland, OH 44106 (e-mail: sxm10{at}po.cwru.edu).
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ABSTRACT |
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INTRODUCTION |
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Mammalian cells defective in a DNA repair mechanism are usually more vulnerable to killing by cytotoxic agents (16-18). In contrast, mismatch repair-defective cells often are resistant to killing by cytotoxic agents. Such cells are dramatically refractory to killing by DNA-methylating agents (19-23). In addition, colon cancers with microsatellite instability demonstrate high levels of resistance to 5-fluorouracil (24) and lower degrees of resistance to cisplatin and 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]-pyridine (PhIP) (25-27). Likewise, cell lines derived from mismatch repair-deficient knockout mice are more resistant to the cytotoxic effects of prolonged low levels or acute high levels of ionizing radiation (28,29). To meet an obvious need for agents that are more toxic for cancers with microsatellite instability, we hypothesized that the vulnerability of mismatch repair-defective cells to frameshift mutations might translate into an enhanced susceptibility to the cytotoxicity of potent frameshift-inducing agents. Therefore, we have examined survival following treatment with ICR191 of the repair-deficient human colon carcinoma cell HCT116 versus its repair-reconstituted derivative HCT116+C3.
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MATERIALS AND METHODS |
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Preparation of ICR191. The acridine mutagen ICR191 (Sigma Chemical Co., St. Louis, MO) was dissolved at 4 mg/mL in 0.01 M HCl, adjusted to 400 µg/mL in MEM without serum, filtered under sterile conditions, and diluted to the appropriate concentration in MEM with additives for the experiment. Fresh drug was prepared immediately before each experiment.
Evaluation of the cytotoxicity of ICR191. ICR191-induced cytotoxicity was evaluated by a clonogenic assay, which assesses cell killing. In this experiment, cells were seeded at 500 cells per well of a six-well dish. Then 12-16 hours later, the medium was removed and replaced with serum-free medium containing ICR191 and incubated for 2 hours at 37 °C. After the 2-hour treatment with ICR191, the drug-containing medium was removed and the cultures were returned to MEM with serum, incubated at 37 °C for 7-10 days, and then evaluated for colony number after visualization with methylene blue and automated counting on an Alpha Innotech Imaging System (Alpha Innotech Corp., San Leandro, CA).
Vector preparation. Three vectors were prepared in our laboratory for this study.
The second-generation pCAR vector, pCMV-CAR, a gift from Dr. Bert Vogelstein (The Johns
Hopkins University, Baltimore, MD), was modified to create these vectors. This
second-generation 14.6-kilobase vector, described by Parsons et al. (30),
contains the complete coding region of ß-galactosidase and the eukaryotic promoter derived
from cytomegalovirus, both of which allow direct visualization of mammalian cells expressing
this gene product. The vector also contains an ampicillin-resistance gene and a leu2 promoter
that permit selection of the vector and production of ß-galactosidase in bacteria. In the three
vectors constructed for this study, the CA repeat sequence in the pCAR vector has been replaced
with a 10-base homopolymeric run of G residues 45 bp downstream from the ATG start site for
the ß-galactosidase gene. This region of these three vectors is shown in Fig. 1. One of these vectors is designed to place the ß-galactosidase messenger RNA
template strand 1 bp out-of-frame in the [-1] direction and requires a
[+1] frameshift for expression of ß-galactosidase [designated the
out-of-frame (+1) vector]. A second vector is designed to place the start site codon
[+1] bp out-of-frame and requires a [-1] frameshift for
expression of ß-galactosidase [designated the out-of-frame (-1) vector].
A control vector was also constructed, in which the run of G residues is in-frame, allowing
constitutive synthesis of ß-galactosidase following transfection into human colon cell lines
or Escherichia coli (designated the in-frame vector).
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Transfection of ex vivo treated vector DNA into bacteria. A total of 0.1
µg of purified vector DNA (treated or untreated) was transfected into Subcloning Efficiency
DH5competent E. coli cells (Life Technologies, Inc. [GIBCO BRL],
Gaithersburg, MD) to determine the mutational effect of ICR191 on the out-of-frame vectors. We
determined the number of successful transformants by using the in-frame vector and monitoring
colony growth on Luria Broth-ampicillin plates containing X-gal (i.e.,
5-bromo-4-chloro-3-indolyl ß-D-galacto-pyranoside). Colonies with frameshifts
in the out-of-frame vector targets were visualized as blue colonies on these X-gal-containing
plates.
Transfection of vectors into colon cancer cell lines and visualization of ß-galactosidase production. Both the in-frame and out-of-frame vectors were transfected into colon cancer cells (seeded at 1.2 x 105 cells per well of a six-well dish) with the use of a Fugene procedure. FugeneTM 6 Transfection Reagent (Boehringer Mannheim Corp., Indianapolis, IN) was employed to facilitate transient transfection of the vectors. Fugene (4 µL) was added directly to 100 µL of Opti-MEM (Life Technologies, Inc.) without antibiotics and incubated at room temperature for 5 minutes. Vector DNA (1.5 µg) was then added to the mixture, followed by a 15-minute incubation at room temperature. The transfection mix was subsequently added to a well of a six-well dish, which contained a subconfluent culture of human colon cancer cells, and was incubated at 37 °C in a 5% CO2 atmosphere overnight. The following day, the transfection mix was removed, each well was washed with MEM, and fresh MEM with additives was added to the wells. Two or three days later, the medium was removed, the wells were washed with PBS, and the plates were incubated at 4 °C for 5 minutes in fixative solution (2% formaldehyde-0.2% glutaraldehyde in PBS). For detection of ß-galactosidase-dependent staining, the wells were washed three times with PBS, and the staining solution (0.5 mg/mL X-gal in PBS containing 20 mM potassium ferricyanide, 20 mM potassium ferrocyanide, and 2 mM MgCl2) was added. Cells were incubated at 37 °C for 2-3 hours in this staining solution, followed by an overnight incubation at 4 °C, which allowed visualization of cells producing ß-galactosidase. The ß-galactosidase-producing blue cells were then quantified microscopically.
In vivo treatment of cells with ICR191 to evaluate inducible mutation frequency. Human colon cancer cells were seeded at 1-1.5 x 105 cells per well in six-well dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ) and incubated at 37 °C for 1 day. The cells were then either transfected with the appropriate vector or, in the case of the controls, subjected to a mock transfection. One day after transfection, the transfection mix was removed and the medium was replaced with serum-free medium containing ICR191. After 2 hours at 37 °C, the drug was removed and the cultures were returned to MEM with serum, incubated at 37 °C for 2 days, and then analyzed for mutation as described below.
Determination of frameshift mutation frequency. Induced frameshift mutations in the human colon cancer cell lines were assessed with the use of the three vectors described above. Since the out-of-frame vectors allowed synthesis of ß-galactosidase only when the reading frame was restored by a frameshift mutation, the number of ß-galactosidase-producing cells in each well transfected with the out-of-frame vector served as an indication of the number of frameshift mutations. Conversely, the number of ß-galactosidase-producing cells in each well transfected with the in-frame vector allowed successful transfection efficiency to be quantified. Since treatment with ICR191 was toxic to both the cells and the vector, the in-frame-vector controls were included in duplicate for every concentration of ICR191 used in the study. After we determined the total number of blue cells per well with the in-frame and out-of-frame vectors, we calculated the frequency of frameshifts induced by a given concentration of ICR191 by dividing the number of ß-galactosidase-producing cells obtained with the out-of-frame vector by the number of ß-galactosidase-producing cells obtained with the in-frame vector for each dose of ICR191. A mock-transfection control was also included at each ICR191 dose to ensure that neither the ICR191 treatment nor the transfection procedure resulted in production of endogenous ß-galactosidase.
Statistical methods. All drug treatments were done in multiple replicates. Results are shown as mean values calculated for each drug dose, with 95% confidence intervals calculated as 1.96 times the standard error of the means.
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RESULTS |
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The inability of mismatch repair-defective cells to repair lesions
causing frameshifts raises the possibility that agents leading to these
lesions may exert more toxicity for mismatch repair-defective cells
than for mismatch repair-proficient cells. To test this hypothesis, we
used a clonogenic survival assay to compare the toxicity of ICR191 in
the HCT116 human colon cancer cell line, which bears only an inactive
mutant hMLH1 mismatch repair gene, with that in HCT116+C3 cells, in
which mismatch repair has been reconstituted with a wild-type hMLH1
gene transferred on an exogenous chromosome 3 (Fig.
2). The results demonstrate that ICR191 is more
toxic to mismatch repair-deficient cells than to mismatch
repair-proficient cells. The LD50 dose of ICR191 (i.e., the
dose at which clonogenic survival fell by 50%) was increased fivefold
from 1 µg/mL in the sensitive HCT116 cells to 5 µg/mL in the more
resistant HCT116+C3 cells. Similarly, the cytotoxicity of a single
5-µg/mL dose of ICR191 increased from LD50 in the resistant
HCT116+C3 cells to LD98 in the sensitive HCT116 cells. In
contrast, a derivative of HCT116+C3 cells, in which the wild-type hMLH1
gene has been lost [clone M2 (21)], demonstrated restored
sensitivity to killing by ICR191, with an ICR191 LD50 dose
decreased back to a level identical to that of the HCT116 cells. The
fivefold increased sensitivity of HCT116 cells to killing by a
frameshift-inducing agent was greater than the twofold change in
resistance that these cells have demonstrated to killing by the
chemotherapeutic agent cisplatin (25,31). While numerically
modest, such changes in drug sensitivity are within the range of
alterations that can directly affect clinical responsiveness of cancers.
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The vector, out-of-frame (+1), carrying a 10-bp
G : C repeat sequence constituted to
facilitate detection of [+1] frameshifts, was used as a target for
the mutational effects of ICR191. After direct exposure of this vector
to varying concentrations of ICR191 between 1 and 10 µg/mL (2-22
µM), the drug-exposed vector was transfected into HCT116 and
HCT116+C3 human colon cancer cells. Concentrations of ICR191 that cause
no expression of mutations in the out-of-frame (+1) vector when
transfected into repair-proficient HCT116+C3 cells induced substantial
numbers of mutations in the out-of-frame (+1) vector when transfected
into mismatch repair-deficient HCT116 cells (Fig. 3).
Since ICR191 was reacted with vector DNA and not directly with HCT116
cells, the increased mutations demonstrated in HCT116 cells reflect an
intrinsic vulnerability of these cells to ICR191-induced mutations and
not an increase in the level of ICR191 drug uptake or accumulation. At
ICR191 concentrations above those shown in Fig. 3
, recovery of mutants
became less efficient as the drug became toxic to vector viability.
This was evident by the decrease in transfection efficiency of the
ICR191-treated control in-frame vector.
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Susceptibility of HCT116 Cells to ICR191-Induced Mutations In Vivo
To confirm that repair-deficient living cells were directly
susceptible to a frameshift mutagen, HCT116 and HCT116+C3 cells were
transiently transfected with the out-of-frame (+1) vector harboring the
[+1] frameshift reporter; 24 hours later, the transfected cells were
treated with ICR191. As shown in Fig. 4, mutations in
the [+1] frameshift-sensitive vector increased in a dose-dependent
manner in both the mismatch repair-proficient and mismatch
repair-deficient cell lines. However, at all doses of ICR191, the
mismatch repair-defective HCT116 cell line demonstrated a fivefold to
10-fold greater induction of mutations than did its mismatch
repair-proficient counterpart, the HCT116+C3 cells. Moreover, in HCT116
cells, 8 µg/mL ICR191 induced a 45-fold increase in vector mutation
frequency above the spontaneous vector mutation frequency of 1.8 x
10-3. Thus, repair-deficient cells may carry a markedly
elevated total mutational burden arising from both an increased basal
mutation rate and an increased sensitivity of these cells to induced
mutations generated by specific chemical or environmental agents.
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DISCUSSION |
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Our observation that human cells deficient in mismatch repair are susceptible to induced mutations caused by an intercalating agent is consistent with findings that mismatch repair-deficient bacteria show an increased susceptibility to mutations induced by ICR191 (34), by the chemically related noncovalently binding acridine derivatives 9-aminoacridine and proflavin (34), and by iPr-OPC, an intercalating oxazolopyridocarbazole (35). The mismatch repair system recognizes single-strand loops that arise by strand slippage during replication (36), and a mechanistic basis for involvement of mismatch repair in the prevention of ICR191-induced mutation is suggested by models in which aminoacridines stabilize stem loop structures (37-39) that give rise to strand slippage events that lead to frameshift mutations (40,41). Consistent with the previously demonstrated specificity of ICR191 to induce mutations in homopolymeric G : C repeat sequences, we did not observe a substantial increase in ICR191-induced mutation rates in studies using the pCAR reporter vector that bears a CA repeat (30) or employing a pCAR-derived reporter vector containing a poly A : T repeat (30).
Mismatch repair-defective colon cancers account for an estimated 12% of colon cancer cases annually (3). These cancers can arise from either inheritance of germ line defects in mismatch repair genes or somatic inactivation of mismatch repair genes (1-4) that most commonly is due to methylation and silencing of the hMLH1 gene (42-44). An unexplained conundrum has been that both inherited and sporadic repair-deficient colon cancers demonstrate a marked predilection for the proximal colon. We and other investigators (5-9,45) have previously demonstrated that mismatch repair deficiency elevates the spontaneous mutation rate of expressed homopolymeric sequences by several orders of magnitude. We now find in human colon cancer cells that mismatch repair deficiency also dramatically elevates the susceptibility of these sequences to induced frameshift mutations. In this context, our data are consistent with the hypothesis that susceptibility to an environmental carcinogen may target repair-deficient cancers to the proximal colon over other sites, even in individuals who bear germ line mutations in mismatch repair genes throughout their bodies. Importantly, such exposures would represent a risk factor in these cancer-prone individuals, which could be reduced by intervention. Moreover, even a twofold increase in mutation rate due to environmental agents present in the colon could theoretically lead to a marked preference for tumor formation in the colon. Since five or more mutations are required to transform a normal epithelial cell (3), a twofold increase in the rate of each mutation would increase by 25, or 32-fold, the overall rate of cancer formation. Thus, while we have used a single high-dose exposure to a mutagen, ICR191, chronic low-level exposures could also substantially alter the risk of cancer arising in mutagen-exposed versus unexposed tissues. This effect would be particularly pronounced for mutagens that target key sequence motifs, such as the RII polyA tract.
In conclusion, this study shows that mismatch repair-defective cells exhibit increased sensitivity to both the toxicity and mutagenicity of specific DNA-interactive agents. These observations imply that some environmental agents could have an impact on tumor development from mismatch repair-defective progenitor cells. This study also proves in principle that a mismatch repair defect can lead to selective sensitivity to the cytotoxic effects of certain agents. Therefore, drugs similar to the frameshift mutagen ICR191 may provide strategies for selective targeting of mismatch repair-defective tumors. We note with interest that amsacrine, an investigational chemotherapeutic agent used in human clinical trials, is an acridine derivative with frameshift-inducing activity (46). In addition, the bisimidazoacridones, which are acridine-related compounds, have been noted to be active against HCT116 cells in both cell line and xenograft models (47). Moreover, a number of new acridine-derived compounds are currently being evaluated for anticancer activity in human trials (48,49). Further elucidation of environmental agents that potentially promote malignant progression of repair-deficient colon cells and of therapeutic agents that exploit the vulnerability of these cells to killing by frameshift mutagens should have clear implications for the care of individuals at risk for or affected by this class of malignancies.
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NOTES |
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Supported by Public Health Service grants R01CA67409 (to S. D. Markowitz), R01CA70788 (to M. L.Veigl), and K08CA66628 (to J. R. Eschleman) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by grant 96B084 (to W. D. Sedwick) from the American Institute for Cancer Research. S. D. Markowitz is an Investigator of the Howard Hughes Medical Institute.
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Manuscript received June 23, 1999; revised December 2, 1999; accepted December 14, 1999.
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