Evidence for a Connection Between the Mismatch Repair System and the G2 Cell Cycle Checkpoint1

[CANCER RESEARCH 55, 3721-3725. September l, 1W5] Advances in Brief

Evidence for a Connection between the Mismatch Repair System and the G2 Cell Cycle Checkpoint1

Mary T. Hawn, Asad Umar, John M. Carethers, Giancarlo Marra, Thomas A. Kunkel, C. Richard Boland,2 and Minoru Koi Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0586 IM. T. H., J. M. â‚¬.,G. M., C. R. B., M. K.I; Gastroenterology Section, Veteran's Affairs Medical Center, Ann Arbor, MI 48105-2399 [M. T. H., J. M. C., G. M., C. K. B., M. K.j; and Laboratories of Molecular Carcinogenesis JM. K.j and Molecular Genetics Â¡A.U., 7".A. K.j, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

Abstract instability has been reported to occur in approximately 10-15% of sporadic colonie neoplasias (2, 3), and has also been linked to a defect The human colon tumor cell line HCT116 is deficient in wild-type in mismatch repair in sporadic colon and endometrial tumor cell lines liMIJII. is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to JV-methylWV'-nitro-JV-nitrosoguanidine (7-9). Previous work with the human colon cancer cell line HCT116, (MNNG). Transferring a normal copy of li.MI.III on chromosome 3 into which has no normal hMLHl gene product, has shown that this line is the cell line restores MMR activity, stabilizes microsatellite loci, and deficient in MMR (8, 9), exhibits microsatellite instability, and is increases the sensitivity of the cell to MNNG. Previous studies in other cell tolerant to the methylating agent MNNG (7). These studies suggested lines tolerant to alkylating agents such as MNNG or JV-methylnitrosourea that inactivation of both copies of the hMLHl gene leads to complete have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypoth loss of mismatch repair activity, and that such a defect is responsible esis that tolerance to MNNG or 6TG may be the result of MMR deficiency. for the microsatellite instability found in HNPCC and some sporadic To test this hypothesis, we studied the effects of 6TG on the MNNG- tumors. Consistent with this hypothesis, we previously demonstrated tolerant, MMR-deficient IK I IK. cell line and its MNNG-sensitive, that the introduction of one copy of the wild-type hMLHl gene on MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Con chromosome 3 into HCT116 cells (HCT116+chr3) restored mismatch tinuous exposure to low doses of 6TG (0.31-1.25 ug/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient repair activity and lowered mutation frequency at a microsatellite cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dose- locus (7). Tolerance to alkylating agents such as MNNG has been dependent decrease in CFA. Growth kinetics and cell cycle analysis re observed in cell lines (10, 11) that have also been shown to be vealed that the growth of 6TG-treated HCT116+chr3 cells was arrested at deficient in MMR and demonstrate microsatellite instability (9, 12, G2 after exposure to low dose of 6TG. In contrast, the same exposure to 13). Previously, we demonstrated that transfer of chromosome 3 into 6TG did not induce G2 arrest but rather a G, delay in IK"l'I 16 and HCT116 cells diminished the tolerance of the cells to MNNG toxicity. HCT116+chr2. To obtain further evidence for the role of MMR on 6TG After MNNG treatment, these cells exhibit prolonged growth arrest at and MNNG toxicity, we isolated an MNNG-resistant revertan! clone, M2, G2, followed by eventual cell death (7), suggesting that the mismatch from the MNNG-sensitive, MMR-proficient HCT116+chr3 cell line and repair system is involved in mediating MNNG toxicity. characterized the MMR activity, hMLHl status, and 6TG response. The Alkylating agent-resistant cells have been shown to be cross-toler results showed that M2 cells lost MMR activity as well as the previously ant to the guanine base analogue 6TG (11, 14, 15). O''-methylguanine, introduced normal hMLHl gene. Restoration of the CFA of M2 and an the major methylation product from MNNG adduci formation (12), absence of G2 arrest were observed after treatment with low doses of 6TG. has a similar molecular volume to 6TG (16). Both 6TG and 0"- These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The methylguanine are unable to form stable base pairs with either of the results further suggest that any agent that induces DNA mispairs will pyrimidines (16). Several investigators have hypothesized that toler cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells. ance to 6TG and O6-methylguanine may be the result of a defect in the MMR system (11, 13, 15). Introduction To test this hypothesis, we examined whether MMR-deficient, HNPCC3 tumors exhibit a high rate of mutation at microsatellite MNNG-tolerant HCT116 cells were also tolerant to low doses of 6TG and whether MMR-proficient, MNNG-sensitive HCT116 containing sequences (1-3). This phenotype has been linked to an inherited the transferred chromosome 3 (HCT116+chr3-6) were sensitive to the germline mutation in one of the MMR genes, hMLHl, hMSH2, same doses of 6TG. In addition, we isolated and characterized an hPMSl, or HPMS2, in which a subsequent somatic mutation in the MNNG-resistant revertant clone from HCT116+chr3-6 cells. Using wild-type alÃeleoccurs before tumor formation (4-6). Microsatellite these HCT116 cell line derivatives, we then addressed whether the MMR system mediates 6TG toxicity by examining the colony-form Received 5/31/95; accepted 7/21/95. The costs of publication of this article were defrayed in part by the payment of page ing ability and cell cycle progression after treatment with 6TG. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1This work was partly supported by NIH Grant CA39233, the University of Michigan Cancer Center Grant CA46592, the Johnson Family Fund for Familial Colorectal Cancer, Chemical Reagents. 6TG and SAG (Sigma Chemical Co., St. Louis. MO) National Cancer Institute Grant R25 CA57716, the James Lind Scholarship, and the were dissolved in 0.1 N NaOH and stored at -10Â°C. MNNG (Aldrich Chemical National Institute of Environmental Health Sciences. 2 To whom requests for reprints should be addressed, at University of Michigan Co., Milwaukee, WI) was dissolved in DMSO (Sigma) and stored at -10Â°C. Medical Center, Department of Internal Medicine, 4410 Keesge III, 200 Zina Pitcher Cell Culture. The human colon cancer cell line HCT116 was maintained in Place, Ann Arbor, MI 48109-0586. IMDM (GIBCO-BRL, Grand Island, NY) containing 10% FBS. HCT116 + hu 1The abbreviations used are: HNPCC, hereditary nonpolyposis colon cancer; MMR, mismatch repair; MNNG,-/V-methyl-N'-nitro-W-nitrosoguanidine; 6TG, 6-thioguanine; man chromosome 2, clone 1 (HCT116+chr2-l), HCT116 + human chromo SAG, 8-azaguanine; IMDM, Iscove's modified Dulbecco's medium; FBS, fetal bovine some 2, clone 3 (HCT116+chr2-3), HCT116 + human chromosome 3, clone 5 serum; hprl, hypoxanthine phosphoribosyl transferase; HAT, hypoxanthine aminopterin (HCT116+chr3-5), and HCT116 + human chromosome 3, clone 6 thymidine; SSCP, single-strand conformational polymorphism. (HCT116+chr3-6) represent cell lines modified by the transfer of a single 3721

normal human chromosome as described previously (7). These cell cultures exclusion. A nuclear prep was obtained as follows: cells were washed in PBS, were maintained in IMDM containing 10% FBS and G418 (400 /J.g/ml; resuspended (10s cells/ml) in nuclear lysis buffer [10 mM Tris (pH 7.5) 0.32 M GIBCO-BRL). The MNNG-resistant revertan! cell line derived from sucrose-3 mM MgCl2-2 mM CaClr().2% (v/v) NP40] and incubated on ice for HCT116+chr3-6, HCT116+chr3-6-M2, was maintained in IMDM containing 10 min. The tubes were centrifuged at 3000 rpm for 10 min. The nuclei were 10% FBS and G418. HAT medium (Sigma) was dissolved in IMDM contain then washed with nuclear lysis buffer (4 X H)5 cells/ml) without NP40 and ing 10% FBS at a final concentration of KM) /AM hypoxanthine, 0.4 JAM centrifuged for 10 min. Nuclei were stained with propidium iodine (5 /Ag/ml) aminopterin, and In JAMthymidine. in resuspension buffer [O.I MTris (pH 7.5)-0.15 M NaCl-1 mM CaCM).5 HIM 6TG Cytotoxicity Assay. Exponentially growing cell lines were plated in MgClrO.()l% (v/v) NP40] for 2-6 h. Cell cycle analysis was performed, and duplicate at densities of \(i2 to IO4 on 6-cm plates and IO5 to 10* on 10-cm percent of cells in each phase was determined using a Coulter Epics C flow plates. After allowing for attachment to the plate for 18-24 h, the medium was cytometer (Hialeah, FL). replaced with fresh medium containing 6TG (0.31-10 ng/ml). Cells were Mismatch Repair Activity. The efficiency of cytoplasmic extracts in maintained in 6TG-containing medium for 10 days, changing the medium repairing DNA mismatches was performed as follows. Cell-free extracts were every 3 days. The plates were washed with PBS, fixed with methanol, and prepared as described previously (10). Procedures for mismatch repair have stained with 3% Giemsa. Colonies with greater than 50 growing cells were been described (9). Mismatched substrates, prepared as described, contained a counted and expressed as a ratio of the plating efficiency for untreated cells. nick in the minus strand at position -264, where position +1 is the first Treatment of cells with 8AG was carried out in a similar manner. At least two transcribed base of the lacZa gene. Repair reactions (25 /A!)contained 30 mM independent experiments were performed in duplicate for each point. HEPES (pH 7.8); 7 mM MgCl,; 4 mM ATP; 200 /AMconcentrations each of Incorporation of 6TG into DNA. Cells (2 X IO7) were plated on 15-cm CTP, GTP, and UTP; 100 /AMconcentrations each of dATP, dGTP, dTTP, and plates and treated with 6TG (1.25 /Ag/ml) for 24 and 48 h. The cells were dCTP; 40 mM creatine phosphate; 100 mg/ml of creatine phosphokinase; 15 Irypsinized and treated with proteinase K (GIBCO-BRL) at 55Â°Covernight. mM sodium phosphate (pH 7.5); 1 fmol of the indicated heteroduplex DNA; DNA was extracted with phenol-chloroform, precipitated with ethanol, dried, and 50 /Â¿gofcell extract protein. After incubating at 37Â°Cfor 30 min, reactions and resuspended in TE (10 mM Tris-HCl-l mM EDTA) buffer. DNA was were processed and introduced into Escherichiu coli NR9162 (miitS), which quantitutcd using a diphenylamine (Sigma) assay, which is highly specific for were plated to score plaque colors. Repair efficiency is expressed in percent as DNA (17). Briefly, equal volumes of sample DNA solutions, including a 100 x (1 - the ratio of the percentages of mixed bursts obtained from standard human Cot 1 DNA (GIBCO-BRL; 0-20 fig/ml) and 10% perchloric extract-treated and untreated samples) (Ref. 9). acid were incubated at 70Â°Cfor 30 min. Two volumes of a diphenylamine/ acetaldehyde solution were added. Samples were incubated in the dark over Results and Discussion night, and the absorbance at 600 nm was measured by spectrophotometry. The Correlation between MMR Activity and 6TG Toxicity. To test concentration of sample DNA was calculated from the standard curve con structed from known concentrations of Cot 1 DNA. DNA from 6TG-treated whether MNNG sensitivity and MMR activity are correlated with cells and known amount of 6TG (0.1-10 fig/ml) were dissolved in 0.1 N HC1 6TG sensitivity, we first compared colony-forming ability in the and heated to 70Â°Cfor 30 min. The absorbance at 346 nm (the peak absorbance presence of low concentrations of 6TG (0.31-1.25 /Â¿g/ml)among MNNG-resistant, MMR- cells (HCT116 and HCT116+chr2) and for 6TG; Ref. 14) was measured, and the amount of 6TG in cellular DNA was MNNG-sensitive, MMR+ cells (HCT116+chr3). As shown in Fig. L4, determined using the standard curve constructed from the known amount of 6TG. HCT116 and two independent clones from HCT116+chr2 showed no Selection of 6TG-resistant Clones. Cells lines were plated (IO2 to IO4/ significant decrease in colony-forming ability after continuous expo 10-cm plate, 10s to 10"/15-cm plate) in continuous exposure to 6TG (1.25 and sure to low doses of 6TG. In contrast, two clones from HCT116+chr3 10 /Ag/ml) for 10-14 days. The medium was changed every 3-4 days. showed a dose-dependent decrease in colony-forming efficiency. Ten Individual colonies were isolated and rcplated. The ability to grow in HAT growing subclones from HCT116 and HCT116+chr2 were isolated medium (Sigma) was tested on all 6TG-resistant subclones. Selection of MNNG-resistant Clones from HCT116+chr3-6 Cells. Cells after exposure to 1.25 ttg/ml of 6TG and cultivated in HAT medium, (10s) were treated with 5 /AMMNNG in serum-free media for 45 min at 37Â°C. which is selectively cytotoxic to hprt-deficient cells. All clones were Cells were pelleted by centrifugation, washed with PBS, and plated on a 15-cm resistant to HAT selection, indicating that tolerance to 6TG was not plate. Individual colonies were isolated, grown, and treated with MNNG under due to inactivation of hprt gene. the same conditions. Individual colonies were again isolated and grown. One Another guanine analogue, SAG, which is also phosphoribosylated subclone (clone 2, HCT116+chr3-6-M2) was chosen for further analysis. by hprt and selects for hprt-deficient cells, is know to exert its primary Colony formation after MNNG treatment was determined as follows: cell lines toxicity by purine starvation (20). Treatment with SAG at 1-5 fig/ml were treated with 5 /AMMNNG, washed with PBS, resuspended in growth had similar effects on colony-forming ability in MMR-proficient and media, and plated in duplicate. Colony-forming ability was expressed relative MMR-deficient cells (Fig. IÃŸ).These results indicate that MMR to the plating efficiency of untreated cells. deficiency specifically correlates with 6TG tolerance but not SAG SSCP Analysis of HCT116+chr3-6-MNNG-resistant Revertant Clone toxicity. M2. DNA was isolated from cell lines as described by Miller et al. (18). To determine whether tolerance to 6TG in MMR" cells was con Ã¬iMLHIDNA was amplified from codons 228 to 263 using a sense primer, 5'-TGATAGAAATTGGATGTGAGG-3', and an antisense primer, 5'-TGAT- ferred by differential 6TG incorporation into DNA, the content of GAAGAGTAAGAAGATGC-3' (4). End labeling of the sense primer with 6TG in high molecular weight DNA from 6TG-treated cells was [â€¢y-32P|ATP(6000 Ci/mmol; Amersham, Arlington Heights, IL) was per measured and compared. HCT116 and HCT116+chr3 had similar formed. The PCR reaction (5 /A!)contained 1 X PCR buffer (GIBCO-BRL), 10 incorporation of 6TG after 24 and 48 h of exposure (Fig. 2). hprt- pmol each of labeled and unlabeled primer, 60 ng template DNA, 0.25 unit Taq deficient clones derived from HCT116 served as negative control and DNA polymerase (GIBCO-BRL), and 250 /AMof each dNTP. Reaction tubes had no appreciable incorporation of 6TG under the same conditions of were heated to 94Â°Cfor 2.5 min and then cycled 35 times at 93Â°Cfor 1 min, culture. 54Â°Cfor 1 min, 72Â°Cfor 1 min, followed by 72Â°Cfor 10 min in the thermal cycler (Perkin Elmer Cetus, Emeryville, CA). Samples were denatured at 94Â°C 6TG is known to exert its toxicity by two different mechanisms. 6TG, as well as SAG, can cause purine starvation in the nucleic acid for 8 min, placed on ice. and loaded on a 0.5X HydroLink MDE Gel (J. T. base pool and directly leads to cell death (19). A second mechanism Baker Chemical Co., Phillipsburg, NJ) containing 5% glycerol, 0.054 M Tris-borate, and 0.0012 M EDTA at 4Â°C.After electrophoresis, the gel was is thought to involve incorporation of 6TG into cellular DNA, but this dried and exposed to X-ray film. process is not well understood (20). Because incorporated 6TG is Cell Cycle Analysis. Cells (l(f) were plated in 6-cm plates. After 24 h, thought to mimic DNA mispairs (16) and 8AG is not as readily 6TG was added. Every 24 h for 5 days, the cells were trypsinized and incorporated into DNA as 6TG (21), our results suggest that MMR is harvested. The number of viable cells was determined by Trypan blue (Sigma) involved in mediating the second mechanism for toxicity in which the 3722

used, containing a covalently closed (+) strand and a (-) strand with a nick (to direct repair to this strand) located several hundred base pairs away from the G-G mispair at position 88 in the lacZ a-comple- mentation coding sequence. The (+) strand encodes a colorless plaque phenotype, whereas the (-) strand encodes a blue plaque phenotype. â€”Oâ€” HCTl 16 --A-- HCTl 16+2-1 If the unrepaired heteroduplex is introduced into an E. coli strain â€”-B.... HCTl 16+2-3 deficient in methyl-directed heteroduplex repair, plaques will have a â€”Mâ€” M2 -â€”oâ€”â€¢HCTl16+3-6 mixed plaque phenotype on selective plates due to expression of both -.-â€¢-.- HCT116+3-5 strands of the heteroduplex. However, repair occurring during incu bation of the substrate in a repair-proficient human cell extract will reduce the percentage of mixed plaques and increase the ratio of the (+) strand phenotype (colorless with this substrate) relative to that of the (-) strand phenotype (blue) because the nick directs repair to the 0.00 0.25 0.50 0.75 1.00 1.25 (-) strand. 6TG (ng/ml) Mismatch repair is readily detected in a HeLa cell extract (22), as indicated by the reduction in mixed plaque phenotypes and the change in the blue:white plaques ratio when compared to an unrepaired control heteroduplex (Fig. 3). Most important, an extract of M2 cells is defective in mismatch repair, as indicated by both a high percentage â€”aâ€” HCTl 16 â€”Â«â€” HCT116+chr2-l â€”oâ€”- HCT116+chr3-i

6- HCTl 16

HCT116+chr3-6 4- 2345 Â¡I ED M2 SAG (ug/ml) 2- HCTl 16 hpn" Fig. 1. A, percent colony-forming ability after 10 days of continuous exposure Io 6TG O (0.31, 0.625, and 1.25 ng/ml) is depicted in MMR-deficient cell lines (HCTl 16, HCT116+chr2-l, HCT116+chr2-3, and M2) and MMR-proficient cell lines (HCT116+chr3-5 and HCT116+chr3-6). HCT116+chr3, clones 5 and 6, demonstrates significantly decreased colony-forming ability when compared to HCTl 16 MMR-defi cient cells at 0.625 and 1.25 |Â¿g/mlof 6TG (*, P < 0.01; **, P < 0.005). B, percent colony-forming ability after 10 days of continuous exposure to SAG (0.31-5 fig/ml) in HCTl 16, HCT116+chr2-l, and HCT116+chr3-6. No statistically significant difference was observed among the groups for concentrations up to 2.5 (Â¿g/ml.The difference seen at 5 jÂ¿g/mlis not significant but most likely represents the difference in mutation rate at Fig. 2. A representative 6TG incorporation experiment is shown. HCTl 16, the hpn locus. HCTl 16+chr3-6, and M2 all have similar incorporation of 6TG into DNA after 24 and 48 h of 6TG exposure, hprt-deficient cells are unable to use 6TG in DNA synthesis. An hprt-deficicnt clone derived from HCTl 16 (HCTl 16 hprt") was assayed as a negative control and has no appreciable incorporation of 6TG after 24 and 48 h of exposure. induction of mispairs and their recognition by the MMR system is crucial. MNNG Resistance Correlates with 6TG Tolerance, Loss of MMR Activity, and Wild-type hMLHl. To obtain further evi dence for the role of MMR in MNNG and 6TG toxicity, we isolated MNNG-resistant clones from MNNG-sensitive, MMR+ HCT116+chr3 cells and then examined whether MNNG resistance - Ã¶ correlated with loss of MMR activity and the reacquisition of 6TG tolerance. HCT116+chr3, clone 6 cells were treated with 5 JU.M HE MNNG for 45 min at 37Â°C. Most of the treated cells showed fa growth arrest and gradual cell death. Two weeks after treatment, growing colonies were obtained at a frequency of about 10~4 P* a treated cells. Ten such clones were independently isolated and SÃ subjected to MNNG treatment (5 /XM45 min at 37Â°C). Eight of m these clones showed no growth arrest after MNNG treatment. One such clone, M2, was chosen for further characterization. Col ony-forming ability in MNNG and 6TG was determined. M2 exhib ited tolerance to both 6TG (Fig. L4) and MNNG (not shown) that was similar to HCTl 16 cells. M2 had similar 6TG incorporation into its DNA, as did the parental HCT116+chr3 cells (Fig. 2), eliminating the Fig. 3. Repair efficiencies in extracts of the HCTl 16 cell lines are shown above. Mock, possibility that tolerance was due to a lack of 6TG incorporation. negative control; HeLu cell extract, positive control. As illustrated, HCTl 16 is repair deficient, HCTl 16+chr3 (clone 6) is repair proficient, and M2 is repair deficient. HEC59 We next compared MMR activity in extracts from M2 cells to that cell extract, which is known to be hMSH2 deficient, complements M2 cell extract, of extracts of control cells. A circular M13mp2 DNA substrate was indicating that the loss of repair for M2 is not due to hMSH2 inactivation. 3723

were analyzed using SSCP analysis. As shown in Fig. 4, HCT116 v

CONTROL CONTROL 48 HOURS 96 HOURS % CELL CYCLE PHASE .625 ug/ml

Gl S G2 ED CONTROL DNA CONTENT D 48 HOURS @ 96 HOURS

Fig. 5. A, growth curves in the presence of 6TG (0.625 (ig/ml) are shown for HCT116, HCT116+chr2-l. HCT116+chr3-6, and M2. All cells are growth inhibited by 6TG, however, HCTI lh+chr.3-6 does not double in number. B. representative flow cytometrograms and percent of cells in each phase of the cell cycle after 48- and %-h exposures to 6TG (0.625 . HCTI 16+chr3-6 cells accumulate in G:. HCTI 16, HCTI l6+chr2-l. and M2 cells demonstrate a G, arrest in response lo 6TG. 3724

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1995 American Association for Cancer Research. MISMATCH REPAIR AND G, CELL CYCLE CHECKPOINT effect on cell cycle kinetics (20, 23). Our results demonstrate that the 3. lonov. Y.. Peinado, M. A., Malkhosyan, S., Shihata. D.. and Perucho, M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonie G2 effect is MMR dependent, and the G, arrest is independent of the carcinogenesis. Nature (Lond.), ibi: 558-561, 1993. MMR system. The absence of the G, effect in HCT116+chr3-6 cells 4. Papadopoulos. N., Nicolaides, N. C, Wei, Y. R. Ruben, S. M., Carter, K. C, Rosen, after 6TG treatment may be due to the fact that these cells are arrested C. A., Haselline, W. A., Fleischmann, R. D., Fraser. C. M., Adams. M. D., Venter, J. C., Hamilton, S. R.. Kinzlcr. K. W., and Vogelstein, B. Mutation of a mutL at the first G2 after 6TG incorporation. homolog in hereditary colon cancer. Science (Washington DC), 163: 1625-1629, Recently, it has been shown that hMSH2 specifically binds to 1994. mispairs (24). Since O6-methylguanine, a methylation product of 5. Fishel, R., Lescoe, M. K., Rao, M. R. S., Copeland, N. G., Jenkins, N. A.. Garbcr. J.. Kane. M.. and Kolodner. R. The human mutator gene homolog imli2 and its MNNG, and 6TG are known to form unstable base pairs with other association with hereditary nonpolyposis colon cancer. Cell, 75: 1027-1038. 1993. purines or pyrimidines when incorporated into cellular DNA, it may 6. Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y. F., Carter, K. C., Ruben, S. M., be that hMSH2 binds to environmentally induced mispairs and me Rosen. C. A., Haseltine. W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, J. C., Dunlop. M. C.. Hamilton. S. R., Petersen, G. M., de la Chapelle, A., diates its repair function in collaboration with the other MMR pro Vogclstein, B., and Kinzler. K. W. Mutations of two PMS homologues in hereditary teins, including hMLHl (25). Our results suggest that MMR not only nonpolyposis colon cancer. Nature (Lond.), 371: 75-80. 1994. repairs DNA damage but also recognizes DNA damage and is in 7. Koi, M., Umar. A.. Chauhan, D. P., Cherian. S. P.. Carethers, J. M., Kunkel, T. A., and Boland. C. R. Human chromosome 3 corrects mismatch repair deficiency and volved in a process that signals G2 arrest. The MMR protein complex microsatellite instability and reduces W-methyl-W-nitro-A'-nitrosoguanidine tolerance might interact directly or indirectly with other proteins or protein in colon tumor cells with homozygous hMLHl mutation. Cancer Res., 54: 4308- complexes that are responsible for the G2 checkpoint. Alternatively, 4312. 1994. 8. Parsons, R.. Li. G. M.. Longlcy. M. J.. Fang, W.. Papadopoulos. M., Jen, J., de la G2 arrest may be signaled by DNA strand breaks generated by Chapelle, A.. Kinzler, K. W.. Vogelstein. B., and Modrich. P. Hypermulability and mismatch repair activity (26). Further studies are needed to identify mismatch repair deficiency in RER+ tumor cells. Cell, 75: 1227-1236, 1993. the cellular mechanisms that result in G2 arrest in response to the 9. Umar, A.. Boyer. J. C.. Thomas, D. C., Nguyen, D. C., Risinger. J. !.. Boyd. J.. lonov. Y.. Perucho. M., and Kunkel. T. A. Defective mismatch repair in extracts of colorectal presence of inappropriate base pairing in DNA. and cndometrial cancer cells lines exhibiting microsatellite instability. J. Biol. Chem., We used HCT116+chr3 cells containing one copy of wild-type 269.- 14367-14370, 1994. liMLHI as MMR+ cells. The heterozygous state of the hMLHl locus 10. Karran. P.. and Bignami. M. Self-destruction and tolerance in resistance of mamma lian cells to alleviation damage. Nucleic Acids Res.. 12: 2933-2940, 1992. in HCT116+chr3 cells is similar to that of the cells from HNPCC 11. Green. M. H. L.. Lowe, J. E.. Petit-FrÃ¨re, C.. Karran. P.. Hall, J., and Kalaoka. H. patients. As shown in this study, a single treatment with MNNG Properties of Ar-methyl-/V-nitrosourca-resistant. Mex- derivatives of an SV4()-immor- selected MMR~ cells (i.e., M2 cells) from MMR+ HCT116+chr3 talized human fibroblast cell line. Carcinogenesis (Lond.), 10: 893-898, 1989. 12. Aquilina. G.. Hess, P.. Branch. P.. MacGcoch, C., Casciano. I.. Karran. P.. and cells. M2 lost the normal hMLHl that had been introduced. It is not Bignami. M. A mismatch recognition defect in colon carcinoma confers DNA clear whether MNNG treatment is the cause of hMLHl loss; however, microsatcllite instability and a mutator phenotype. Proc. Nati. Acad. Sci. USA. 91: 8905-89(19, 1994. these results indicate that MNNG allowed selective clonal expansion of the subpopulation of MMR" cells present in HCT116+chr3. This 13. Kat. A., Thilly, W. G., Fang, W-H.. Longley. M. J.. Li, G-M. and Modrich. P. An alkylation-tolerant. mutator human cell line is deficient in strand-specific mismatch implies that agents that induce DNA damage that mimics mispairs repair. Proc. Nati. Acad. Sci. USA, 90: 6424-6428, 1993. 14. Aquilina. G.. Giammarioli, A. M.. Zijno. A.. DiMuccio, A., Dogliotti, E. and may present a risk to cells that are heterozygous for MMR gene loci, such Bignami. M. Tolerance to i/'-methylguanine and 6-thioguanine cytotoxic effects: a as all cells in HNPCC patients. These agents might not only induce cross-resistant phenotype in Â¿V-methylnitrosourea-resistant Chinese hamster ovary mutations in the wild-type alÃele,butmay simultaneously select for clonal cells. Cancer Res., 50: 4248-4253, 1990. 15. Aquilina, G., Zijno. A.. Moscufo. N., Dogliotti. E.. and Bignami. M. Tolerance to expansion of those cells which incurred inactivation of that alÃele. methylnilrosourea-induced DNA damage is associated with 6-thioguanine resistance In summary, these data provide evidence for a connection between in CHO cells. Carcinogenesis (Lond.). 10: 1219-1223. 1989. the MMR system and the G2 checkpoint. Our results also suggest that 16. Rappaport, H. P. The 6-thioguanine/5-mcthyl-2-pyrimidinonc base pair. Nucleic Acids Res., 16: 7253-7267, 1988. agents that cause mispairs in cellular DNA may induce G2 growth 17. Brunk. C. F., Jones, K. C., and James. T. W. Assay for nanogram quantities of DNA arrest in MMR-proficient but not in MMR-deficient cells. The link in cellular homogenales. Anal. Biochem., 92: 497-500, 1979. between the MMR system and G-, arrest suggests that the MMR 18. Miller, S. A., Dykes, D. D., and Polesky, H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16: 1215. 1988. system is critical not only for DNA repair, but has additional functions 19. Tidd. D. M.. and Peterson. A. R. P. Distinction between inhibition of purine synthesis that limit the replication of cells with drug-induced mispairs that can and delayed cytotoxic reaction of 6-MP. Cancer Res., 34: 733-737. 1974. 20. Maybaum, J., Morgans. C. W., and Hink. L. A. Comparison of in w'wÂ»andin vitro not be repaired. effects of continuous exposure of L1210 cells to 6-thioguanine. Cancer Res., 47: 3083-3087, 1987. Acknowledgments 21. vanDiggelen, O. P.. Donahue. T. F., and Shin, S-I. Basis for differential cellular sensitivity to 8-azaguaninc and 6-thioguaninc. J. Cell Physiol.. Wf: 59-72. 1979. The authors thank Drs. Cynthia Afshari and Richard S. Paules for their 22. Thomas. D. C.. Roberts. J. D.. and Kunkel. T. A. Hcteroduplex repair in extracts of critical review of the manuscript. human HeLa cells. J. Biol. Chem., 266: 3744-3751, 1991. 23. Wotring, L. L.. and Roti Roti. J. L. Thioguanine-induced S and G, blocks and their significance to the mechanism of cytotoxicity. C'ancer Res., 40: 1458-1462. 1980. References 24. Fishel, R., Ewel, A., and Lescoe. M. K. Purified human MSH2 protein hinds to DNA 1. Aaltoncn, L. A., Pellomaki, P., Leach, F. S.. Sislonen, P.. Pylkkanen, L., Mecklin, containing mismatched nucleotides. Cancer Res., 54: 5539-5542. 1994. J-P., Jarvinen, H., Powell, S. M., Jen, J., Hamilton, S. R., Peterscn, G. M., Kinzler, 25. Prolla, T. A., Pang, O., Alani, E.. Kolodner. R. D.. and Liskay. R. M. MLHI. PMS1, K. W., Vogelstein, B., and de la Chapelle, A. Clues to the pathogenesis of familial and MSH2 interactions during the initiation of DNA mismatch repair in yeast. colorectal cancer. Science (Washington DC). 260: 812-816. 199.1. Science (Washington DC), 265: 1091-1093, 1994. 2. Thihodeau. S. N.. Bren, G., and Schaid, D. Microsatellite instability in cancer of the 26. Modrich, P. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1995 American Association for Cancer Research. Evidence for a Connection between the Mismatch Repair System and the G 2 Cell Cycle Checkpoint

Mary T. Hawn, Asad Umar, John M. Carethers, et al.

Cancer Res 1995;55:3721-3725.

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