A combination of MSH2 DNA mismatch repair deficiency and expression of the SV40 large T antigen results in cisplatin resistance of mouse embryonic fibroblasts

Mutations in the human mismatch repair (MMR) genes are associated with hereditary non-polyposis colorectal cancer as well as other sporadic cancers. MMR gene mutations have been implicated in the resistance of human tumours to cisplatin and several tumour-derived MMR-deficient cells show cisplatin resistance in vitro. In addition, hypoxia, a common feature of the tumour microenvironment, has been shown to influence tumour responses to conventional cancer treatments. We have examined the role of the mMSH2 MMR protein on repair of cisplatin-damaged DNA and cisplatin sensitivity in mMSH2-deficient murine fibroblasts and mMSH2-proficient controls under conditions of normoxia and hypoxia. Sensitivity to cisplatin was measured using the MTT assay and clonogenic survival. Repair of cisplatin-damaged DNA was measured using a host cell reactivation (HCR) assay employing a non-replicating recombinant virus expressing the β-galactosidase reporter gene. Sensitivity to cisplatin was significantly less and HCR of the cisplatin-damaged reporter gene was significantly greater in SV40-transformed mMSH2-deficient cells (MS5-7) compared to mMSH2-proficient controls (BC1-6) under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts under both normoxic and hypoxic conditions. Sensitivity to cisplatin was also significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a control plasmid under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was less and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a plasmid expressing SV40 large T antigen under both normoxic and hypoxic conditions. These results suggest that loss of MMR alone does not result in increased resistance to cisplatin in murine fibroblasts and that additional concomitant alterations in cells expressing the SV40 large T antigen are responsible for cisplatin resistance through a modulation of DNA repair capacity and/or apoptosis.     

Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis

DNA damage recognition plays an important role in DNA repair and mutagenesis. Failure to recognize DNA damage may lead to DNA replication without damage repair as well as mutation accumulation. Mutations can lead to many disease conditions. XPC is a DNA damage recognition protein that binds to damaged DNA templates at a very early stage during the DNA repair process. We have studied the role of the XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. When psoralen and cisplatin-damaged plasmid DNA was transfected into xeroderma pigmentosum group C (XPC) cells, which were defective in the XPC gene, very distinct mutation frequency and spectrum was observed: a decreased mutation frequency for psoralen-damaged plasmid and an increased mutation frequency for cisplatin-damaged plasmid; in contrast, most mutations generated by psoralen in XPC cells were T-to-G transversions and most mutations generated by cisplatin in XPC cells were large deletions. We also determined the DNA repair ability of XPC cells by both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed greatly reduced host cell reactivation of a luciferase reporter for both psoralen and cisplatin-damaged plasmid DNA in XPC cells. The in vitro DNA repair results revealed a defective repair capacity for both psoralen and cisplatin-damaged plasmid DNA in nuclear extract prepared from XPC cells. However, this defective DNA repair activity was partially restored when a functional XPC protein was supplemented into the XPC nuclear extract prior to the reaction. These results suggest that the XPC protein DNA damage recognition function plays a crucial role in DNA repair initiation and mutation avoidance and XPC defects may lead to increased mutations and high risk for disease progression.     

Analysis of microsatellite instability in acquired drug-resistance human tumor cell lines

Genomic instability characterized as microsatellite instability (MIN) is associated with loss of DNA mismatch repair (MMR) protein. Several studies have shown that loss of DNA MMR protein confers resistance to some interacting DNA chemotherapeutic drugs, but also that exposure of MMR-proficient cells to these drugs can result in loss of MMR protein accompanied by induction of MIN. Such associations were mainly reported for cisplatin, but scarce data are available for doxorubicin (a DNA interacting agent), and nothing is known about vinblastine (an antitubulin agent). Thus, in this study we have analyzed MIN frequency in different type of human tumor cell lines characterized by their MMR protein status and resistant to doxorubicin or to vinblastine. Relationship between MIN occurrence and drug resistance was firstly verified in cisplatin resistant cells, and showed a MIN enrichment (33%) only in the MMR-deficient cells. In order to determine whether treatment of MMR-proficient cells with doxorubicin might lead to induction of MIN, we analyzed two different MMR-proficient cell lines. Variations of MIN frequency were found with either high levels of MIN (66%) or no MIN at all (0%). Effect of vinblastine was analyzed according to the MMR status in two different MMR-proficient and -deficient cells. No major change in MIN frequency was found either in the MMR-proficient (0%) or -deficient (9%) cells. Our results demonstrate that MIN occurs only in tumor cells resistant to cisplatin or doxorubicin, thus supporting earlier findings reporting such associations only with drugs interacting with DNA. Moreover, the data show that MIN does not appear in all tumor cell lines, suggesting that induction of MIN in relation to MMR status is a complex phenomenon which does not only depend on the drug considered (interacting or not with DNA), but also on the tumor cell variant.     

Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.     

Host-cell reactivation of cisplatin-damaged pRSVcat in a human lymphoid cell line

A method has been developed for studying host-cell reactivation of cisplatin-damaged plasmid DNA in human T lymphocytes. Parameters of electroporation were established for transfection of the shuttle vector pRSV cat into H9 cells, and a rapid single-vial assay was used for measurement of chloramphenicol acetyltransferase (CAT) activity in extracts of transfected cells. pRSVcat was modified with cisplatin to defined levels ranging from 5 to 40 platinum molecules per plasmid, and then transfected into H9 cells. Graded reductions in CAT activity were observed with increasing levels of platination of the plasmid. At 40 platinum molecules per plasmid, CAT activity was reduced to levels of the negative controls. The efficiency of electroporation-mediated transfer of plasmid into H9 cells was not reduced by high levels of cisplatin modification. The level of modification effecting a 63% reduction in CAT gene expression (the B0), was found to be 17 cisplatin molecules per plasmid. This assay system offers a rapid and sensitive method for assessing the capability of human lymphoid cells to reactivate cisplatin-damaged plasmid DNA.     

Targeting DNA mismatch repair for radiosensitization

Postreplicational mismatch repair (MMR) proteins are capable of recognizing and processing not only single base-pair mismatches and insertion-deletion loops (IDLs) that occur during DNA replication, but also adducts in DNA resulting from treatment with cancer chemotherapy agents. These include widely varying types of DNA adducts resulting from methylating agents such as MNNG, MNU, temozolomide, and procarbazine; CpG crosslinks resulting from cisplatin and carboplatin; and S(6)-thioguanine and S(6)-methylthioguanine residues in DNA. Although MMR proteins can recognize both replicational errors and chemotherapy-induced adducts in DNA, the end results of this recognition are very different. Base-base mismatches and IDLs can be repaired by MMR, restoring genomic integrity, whereas MMR-mediated recognition and processing of chemotherapy-induced adducts in DNA results in apoptosis. After the loss of MMR, the inability of cells to recognize and correct single base-pair mismatches and insertion-deletion loops can lead to secondary mutations in proto-oncogenes and tumor-suppressor genes, thereby contributing to the development of cancer. In addition, the inability of MMR-deficient cells to recognize chemotherapy-induced adducts in DNA can result in a damage-tolerant phenotype that translates to clinically significant resistance by allowing for selection of MMR-deficient cancer cells. We have shown recently that these MMR-deficient, drug-resistant cells can be targeted for radiosensitization by the halogenated thymidine analogs iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd). These thymidine (dThd) analogs become incorporated into DNA and form reactive uracil radicals after ionizing radiation (IR), increasing strand breaks. IdUrd and BrdUrd appear to be removed from DNA in MMR-proficient cells with limited toxicity or disruption of the cell cycle, while accumulating at much higher levels in MMR-deficient cells. As a result, it is possible to effectively increase the radiosensitization of MMR-deficient cells at levels of halogenated dThd analog that demonstrate limited toxicity to MMR-proficient cells. This indicates that a combined approach of IdUrd or BrdUrd with IR may be effective in killing MMR-deficient tumors in patients, which are resistant to many cancer chemotherapy agents commonly used in the clinic.     

DNA polymerase zeta accounts for the reduced cytotoxicity and enhanced mutagenicity of cisplatin in human colon carcinoma cells that have lost DNA mismatch repair.

The mutagenicity of cis-diamminedichloroplatinum(II) (DDP; cisplatin) and the rate at which resistance develops with repeated exposure to DDP are dependent on mutagenic translesional replication across DDP DNA adducts, mediated in part by DNA polymerase zeta, and on the integrity of the DNA mismatch repair (MMR) system. The aim of this study was to determine whether disabling Pol zeta by suppressing expression of its hREV3 subunit in human cancer cells can reduce the mutagenicity of DDP and whether loss of MMR facilitates mutagenic Pol zeta-dependent translesional bypass. The HCT116+ch3 (MMR(+)/REV3(+)) and HCT116 (MMR(-)/REV3(+)) human colon carcinoma cell lines were engineered to suppress hREV3 mRNA by stable expression of a short hairpin interfering RNA targeted to hREV3. The effect of knocking down REV3 expression was to completely offset the DDP resistance mediated by loss of MMR. Knockdown of REV3 also reduced the mutagenicity of DDP and eliminated the enhanced mutagenicity of DDP observed in the MMR(-)/REV3(+) cells. Similar results were obtained when the ability of the cells to express luciferase from a platinated plasmid was measured. We conclude that Pol zeta plays a central role in the mutagenic bypass of DDP adducts and that the DDP resistance, enhanced mutagenicity, and the increased capacity of MMR(-)/REV3(+) cells to express a gene burdened by DDP adducts are all dependent on the Pol zeta pathway.     

Selective sensitivity to carboxyamidotriazole by human tumor cell lines with DNA mismatch repair deficiency

We have previously reported that high-dose nifedipine had a selective antiproliferative effect on colon cancer cell lines deficient in DNA mismatch repair (MMR). We hypothesized that carboxyamidotriazole (CAI), a calcium channel blocker, would also have a selective inhibitory effect on colon cancer cell lines with DNA MMR deficiency. In addition, we speculated that this effect may also be seen in cell lines deficient in DNA MMR derived from other tumor types. Fourteen human cancer cell lines with and without DNA MMR derived from carcinomas of the colon, bladder, ovary and prostate were treated with CAI, vehicle or control drugs (nifedipine and 5-flurouracil). The effect of treatment on growth inhibition, invasion, apoptosis and cell cycle progression was assessed. Selective sensitivity to CAI was observed in all cancer cell lines deficient in MMR. Compared with the MMR-proficient cells, the matched deficient cells were significantly more sensitive to the growth inhibitory effect of CAI and nifedipine, but less sensitive to 5-flurouracil. CAI significantly inhibited the invasive ability of MMR-deficient cancer cells compared to 5-flurouracil. CAI induced more apoptosis but similar level of G(2)/M arrest in MMR (hMLH1- or hMSH6-)-deficient colon cancer cells than MMR-proficient counterparts. CAI selectively inhibits proliferation and invasion in MMR-deficient human cancer cell lines. The antitumor effect is at least partly explained by G2/M cell cycle arrest and induction of apoptosis. These findings may have clinical implications directing clinical trials in selectively targeted patients with DNA MMR tumors.     

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.     

Host cell reactivation of cisplatin-treated adenovirus is reduced in nucleotide excision repair deficient mammalian cells and several human tumour cells

Cisplatin is widely used for chemotherapy of a variety of human cancers. Cisplatin exerts its toxic effect by covalently binding to DNA, resulting in monofunctional adducts, intrastrand crosslinks, and interstrand crosslinks. Several recent reports suggest that the cellular capacity for DNA repair, especially nucleotide excision repair (NER), is an important determinant in the sensitivity of cells to cisplatin. We have used a sensitive host cell reactivation (HCR) technique to examine the repair capacity for cisplatin-damaged DNA in several different mammalian cell types. HCR of cisplatin-damaged adenovirus (Ad) was reduced in all UV-sensitive NER deficient Chinese hamster ovary (CHO) cells examined (complementation groups 1 to 6) compared to NER proficient CHO cells. HCR of cisplatin-damaged Ad was also reduced in fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, B, C, D, F, and G compared to that in normal human fibroblasts. Differences in the HCR of cisplatin-treated Ad were also detected among human cancer cell lines, suggesting some tumour cells may be deficient in the NER of cisplatin-DNA adducts.     

Hypoxia-induced enrichment and mutagenesis of cells that have lost DNA mismatch repair

Loss of DNA mismatch repair (MMR) increases the risk of spontaneous mutations. We sought to determine whether there was an interaction between hypoxia and MMR deficiency that might contribute to the phenomenon of tumor progression. Human colon carcinoma HCT116+ch2 (MMR-deficient) and HCT116+ch3 (MMR-proficient) sublines were exposed for varying periods of time to an environment of <0.1% O2 and pH as low as 6.1. When a population containing 5% MMR-deficient cells and 95% MMR-proficient cells was subjected to hypoxia for 72 h, the MMR-deficient cells were enriched by a factor of 2-fold in the surviving population, whereas no enrichment was detected in cells maintained under aerobic conditions. The potential of hypoxia to destabilize the genome was determined by measuring the frequency of clones in the surviving population resistant to very high concentrations of 6-thioguanine or cisplatin. A 72-h exposure to hypoxia did not increase the frequency of resistant clones in the MMR-proficient cells but produced a 7.8-fold increase in 6-thioguanine-resistant clones and a 2.5-fold increase in cisplatin-resistant clones in the MMR-deficient cells. Loss of MMR increased the frequency of mutations in a reporter vector sensitive to frameshift mutations in a microsatellite sequence. Exposure to hypoxia for a time period as short as 48 h further increased the number of mutations in both cell types, but the absolute number of mutants was higher in the MMR-deficient cells. These results indicate that hypoxia and its accompanying low pH enrich for MMR-deficient cells and that loss of MMR renders human colon carcinoma cells hypersensitive to the ability of hypoxia to induce microsatellite instability and generate highly drug-resistant clones in the surviving population.     

DNA mismatch repair proficiency executing 5-fluorouracil cytotoxicity in colorectal cancer cells

Background

5-fluorouracil (5-FU)-based chemotherapy is the standard treatment for advanced stage colorectal cancer (CRC) patients. Several groups including ours have reported that stage II-III colorectal cancer patients whose tumors retain DNA Mismatch repair (MMR) function derive a benefit from 5-FU, but patients with tumors that lost MMR function do not. Although, MMR recognition of 5-FU incorporated in DNA has been demonstrated biochemically, it has not been demonstrated within cells to execute 5-FU cytotoxicity.

Aim

To establish an efficient construction model for 5-FU within DNA and demonstrate that 5-FU incorporated into DNA can trigger cellular cytotoxicity executed by the DNA MMR system.

Methods

We constructed a 5FdU-containing heteroduplex plasmid (5FdU plasmid) and 5FdU-containing linear ds-DNA (5FdU linear DNA), and transfected these into MMR-proficient, hMLH1^-/- and hMSH6^-/- cells. We observed cell growth characteristics of both transfectants for 5-FU-induced cytotoxicity.

Results

MMR-proficient cells transfected with the 5FdU plasmid but not the 5FdU linear DNA showed reduced cell proliferation by MTS and clonogenic assays, and demonstrated cell morphological change consistent with apoptosis. In MMR-deficient cells, neither the 5FdU plasmid nor 5FdU linear DNA induced cell growth or morphological changes different from controls.

Conclusion

5FdU as heteroduplex DNA in plasmid but not linear form triggered cytotoxicity in a MMR-dependent manner. Thus 5-FU incorporated into DNA, separated from its effects on RNA, can be recognized by DNA MMR to trigger cell death.Key words: 5-FU, colorectal cancer, mismatch repair system, heteroduplex plasmid     

DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells.

PURPOSE: The DNA mismatch repair (MMR) system plays an important role in mediating cell death after treatment with various types of chemotherapeutic agents, although the molecular mechanisms are not well understood. In this study, we sought to determine what signal is introduced by MMR after 6-thioguanine (6-TG) treatment to signal a G(2)-M arrest leading to cell death. EXPERIMENTAL DESIGN: A comparison study was carried out using an isogenic MMR(+) and MMR(-) human colorectal cancer RKO cell system, which we established for this study. Cells were exposed to 6-TG (3 micro M x 24 h) and then harvested daily for the next 3-6 days for growth inhibition assays. Cell cycle effects were determined by flow cytometry, and DNA strand breaks were measured using pulsed-field gel electrophoresis and alkaline Comet assays. RESULTS: We first established MMR(+) RKO cell lines by transfection of human MutL homologue 1 (hMLH1) cDNA into the hMLH1-deficient (MMR(-)) RKO cell line. The ectopically expressed hMLH1 protein restored a MMR-proficient phenotype in the hMLH1(+) transfectants, showing a significantly increased and prolonged G(2)-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G(2)-M arrest started on day 1, peaked on day 3, and persisted to day 6 after 6-TG removal. We found that DNA double-strand breaks were comparably produced in both our MMR(+) and MMR(-) cells, peaking within 1 day of 6-TG treatment. In contrast, single-strand breaks (SSBs) were more frequent and longer lived in MMR(+) cells, and the duration of SSB formation was temporally correlated with the time course of 6-TG-induced G(2)-M arrest. CONCLUSIONS: Our data suggest that MMR mediates 6-TG-induced G(2)-M arrest by introducing SSBs to signal a persistent G(2)-M arrest leading to enhanced cell death.     

Mutations in DNA mismatch repair genes: implications for DNA damage signaling and drug sensitivity (review)

The DNA mismatch repair (MMR) system functions in the elimination of biosynthetic errors that arise during DNA replication, in DNA damage surveillance, and in the prevention of recombination between non-identical sequences. It therefore substantially contributes to the maintenance of genome integrity. It is thus not surprising that loss of MMR function may lead to cancer. Inherited defects due to germline mutations in the MMR genes underlie the hereditary non-polyposis colon cancer (HNPCC) syndrome in humans, and epigenetic silencing of the hMLH1 gene accounts for sporadic cancers, including those of the endometrium and ovaries. Another hallmark of MMR is its capacity to elicit DNA damage-induced cell death. Although this might seem to make MMR a useful target for anticancer agents, it has become clear that tumor cells with defective MMR display reduced sensitivity to the cytotoxic effect of DNA damaging agents such as alkylating agents and cisplatin. This is of clinical relevance, as these agents provide MMR-deficient tumor cells a growth advantage. This effect is exacerbated by the potential of some agents to cause the de novo generation of MMR-resistant variants. Thus, the discovery of antitumor agents that retain sensitivity against or specifically target MMR-deficient tumor cells, and the development of strategies to overcome MMR-related drug resistance assumes clinical importance. Efforts have been taken in the past years to come to a better understanding of the molecular mechanisms of MMR, including those underlying MMR-related tumorigenesis, those determining the substrate-specificity of MMR-dependent damage recognition, and those linking damage recognition to cell death responses. This information is also expected to contribute to the establishment of diagnostic methods to screen for MMR gene mutations in tumors and to the development of strategies which enable the oncologists to apply appropriate and specific regimens for tumor treatment.     

Hypersensitivity in DNA mismatch repair-deficient colon carcinoma cells to DNA polymerase reaction inhibitors

We studied the cytotoxic effects of various DNA replication inhibitors on MMR-deficient and -proficient colon carcinoma cell lines. DNA polymerase (pol) inhibitors including aphidicolin and gemcitabine, and hydroxyurea were more toxic (1.7 to 2.8-fold) to hMLH1-deficient HCT116 than to hMLH1-proficient HCT116+ch3. Similarly, pol inhibitors were more toxic to hMSH2-deficient LoVo than to hMSH2-proficient LoVo+ch2. In contrast, DNA topoisomerase I inhibitors, such as CPT-11, SN-38, and topotecan, were more toxic to MMR-proficient cells. Our results suggest that MMR-deficient colon carcinoma cells are hypersensitive to inhibitors of the pol reaction.     

Enhanced host cell reactivation of damaged plasmid DNA in HeLa cells resistant to cis-diamminedichloroplatinum(II)

Human HeLa cells resistant to cisplatin were established by stepwise selection. The selected cells showed a 15- to 20-fold cisplatin resistance (CPR) at the dose level resulting in 50% inhibition. These cells were cross-resistant to mitomycin C, melphalan, and ethyl methanesulfonate but not to Adriamycin, colchicine, or vinblastine. The expression of cisplatin-damaged plasmid DNA carrying the bacterial chloramphenicol acetyltransferase (CAT) gene after its transfection into CPR cells was enhanced by approximately 3-fold. This did not correlate with the degree of CPR. However, the development of the CPR phenotype paralleled the enhanced CAT activity. The addition of aphidicolin (an inhibitor of DNA alpha-polymerase) to CPR cells effectively diminished the enhanced CAT activity and CPR. These studies have identified an enhanced host cell reactivation of the damaged plasmid in the acquisition of CPR, suggesting that DNA repair is a potential mechanism for the development of CPR phenotype in human cells.     

Cyclin E and histone H3 levels are regulated by 5-fluorouracil in a DNA mismatch repair-dependent manner

Several studies indicate that the DNA mismatch repair (MMR) system may trigger cytotoxicity upon 5-fluorouracil (5-FU) recognition, but signaling pathways regulated by MMR in response to 5-FU are unknown. We hypothesize that recognition of 5-FU in DNA by MMR proteins trigger specific signaling cascades that results in slowing of the cell cycle and cell death. Whole human genome cDNA microarrays were used to examine relative signaling responses induced in MMR-proficient cells after 5-FU (5 μM) treatment for 24 hours. Analysis revealed 43 pathways differentially affected by 5-FU compared to control (P 1.4-fold) and downregulated cdc25C, cyclins B1 and B2, histone H2A, H2B, and H3 (< -1.4-fold) over control. Cell cycle analysis revealed a G1/S arrest by 5-FU that was congruent with increased cyclin E and decreased cdc25C protein expression. Importantly, with knockdown of hMLH1 and hMSH2, we observed that decreased histone H3 expression by 5-FU was dependent on hMLH1. Additionally, 5-FU treatment dramatically decreased levels of several histone H3 modifications. Our data suggest that 5-FU induces a G1/S arrest by regulating cyclin E and cdc25C expression, and MMR recognition of 5-FU in DNA may modulate cyclin E to affect the cell cycle. Furthermore, MMR recognition of 5-FU reduces histone H3 levels that could be related to DNA access by proteins and/or cell death during the G1/S phase of the cell cycle.     

DNA Repair capacity and cisplatin sensitivity of human testis tumour cells

Metastatic testicular germ cell tumours can be cured using cisplatin-based combination chemotherapy. To investigate the role of DNA repair in cisplatin sensitivity, we measured the formation and removal of cisplatin adducts in the whole genome and in specific genomic regions in 3 testis and 3 bladder cancer cell lines. Following a 1 hr exposure to 50 μM cisplatin, the mean level of DNA platination was lower in the testis tumour cell lines. During a 72 hr post-treatment incubation period, the 3 bladder cell lines removed platinum from the overall genome, whereas 2 of the testis tumour cell lines showed relatively little reduction of DNA platination. The third testis tumour cell line, SuSa, showed an intermediate capacity to remove cisplatin. Cisplatin-induced damage and repair in selected regions of the actively transcribed N-ras gene and the inactive CD3δ gene were measured using quantitative PCR. The data were in agreement with those obtained with atomic absorption spectroscopy for the whole genome, showing that the bladder lines were repair-proficient: 2 of the testis tumour cell lines showed no repair, and the third testis line, SuSa, showed an intermediate level of repair in these 2 genes. Our findings confirm that reduced capacity to repair cisplatin-damaged DNA may contribute to the hypersensitivity of testis tumour cells to DNA-damaging agents. Int. J. Cancer 70:551–555. © 1997 Wiley-Liss Inc.