Mismatch Repair Deficient Cells Research Articles

Cellular Physiology of Mismatch Repair

The DNA mismatch repair system maintains genomic stability by correcting DNA sequence errors generated during DNA replication, during genetic exchanges between chromosomes i.e., recombination, and by correcting DNA lesions caused by mutagenic agents such as cis-platinum. Post-synthesis mismatch repair improves almost 1000-fold the fidelity of DNA replication; however, the functions of mismatch repair proteins extend well beyond DNA repair. Recent studies suggest that mismatch repair is part of the machinery that couples DNA damage and repair to cell cycle regulation and apoptosis. These studies indicate that tolerance to certain DNA lesions (such as methylation and cis-platinum adducts) is associated with inefficient activation of cell cycle checkpoints and inefficient activation of apoptosis in mismatch repair deficient cells. Hence, mismatch repair proteins regulate the survival threshold to DNA damage, and this function provides a novel platform for understanding the role of mismatch repair in B cells, in tumor formation, as well as in resistance to chemotherapy. In this communication, we review how mismatch repair may contribute to the physiology of cells and may be regulated by the intracellular trafficking of mismatch repair proteins.

DNA methyltransferase Dnmt1 and mismatch repair.

We have examined the relationship between DNA mismatch repair and deficiency of DNA methylation in a mouse embryonic cell line, Dnmt1-/- ES, with homologous deletion of the gene coding for the maintenance DNA methyltransferase Dnmt1. With the use of a sensitive PCR for the assay of two mononucleotide- and three dinucleotide microsatellite markers that exhibited instabilities in mismatch repair-deficient cells, significantly higher frequencies of instability were detected at three of the five markers in Dnmt1-/- ES than the wild-type ES. The data suggest that Dnmt1 enzyme plays an integrating role in DNA replication and/or repair. The implication that Dnmt1 enzyme and/or cytosine methylation may participate in the strand discrimination of mismatch repair during eukaryotic DNA replication is discussed.

234 Induction of cell death by imidazoacridinone derivative C-1311 in T lymphoblastoid leukemia MOLT4 cells

Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) consist of a conventional NSAID to which an NO-releasing moiety is attached covalently, often via a spacer molecule. NO-NSAIDs represent an emerging class of compounds with chemopreventive properties against a variety of cancers, demonstrated in preclinical models including cell culture systems and animal tumor models; their potential efficacy in humans has not been assessed. Their mechanism of action appears complex and involves the generation of reactive oxygen species, suppression of microsatellite instability in mismatch repair-deficient cells, and modulation of several signaling cascades that culminate in inhibited cell renewal and enhanced apoptosis. NO, long appreciated to be able to protect from and also promote cancer, is released form NO-NSAIDs and constitutes their defining property. Existing data are consistent with the notion that NO may mediate their anticancer effect. In addition there is evidence that long-term administration of NO-donating compounds is not associated with increased incidence of colon cancer. Whether NO release is required for the anticancer effect of NO-NSAIDs has being questioned by recent data indicating that, at least in the case of NO-aspirin, the NO-releasing moiety may serve as a leaving group while the spacer actually being the moiety responsible for its pharmacological action. Regardless of mechanistic issues, these compounds promise to contribute to the control of cancer.

Phase II trial of oxaliplatin (OXA) combined with paclitaxel (P) inplatinum + taxanes sensitive advanced ovarian cancer (AOC) patients: Final results

5023 Background: OXA is active in mismatch repair (MMR) deficient cells, a condition prevalent in pretreated AOC. OXA/P shows high activity in investigator initiated studies (Faivre et al, Ann Oncol 10:1125, 1999). We evaluated efficacy and safety of OXA/P in AOC pts with clinically evaluable disease pretreated with 1 platinum based regimen ± taxanes and a progression-platinum-free interval (PPFI) ≥6 months (mo). Methods: From Dec 99 to Apr 02, 105 pts, 14 centres received P 175 mg/m2, 3h iv followed by OXA 130 mg/m2, 2h iv, every 3 weeks, for up to 9 cycles (cy) without G-CSF. Median (m) age: 58 years (range 37–83), 96% PS 0/1. PPFI: 69 pts >12 mo, 34 pts 6–12 mo; 80 pts (76%) taxane-pretreated. Results: 708 cy administered; m 7 cy/pt (1–9). Relative dose intensity: OXA 0.95, P 0.97. Efficacy: 98 pts eligible, 7 non-eligible (3 bowel occlusion, 3 >1 prior platinum-based chemotherapy, 2 PPFI not determinable). 98 pts evaluable for response (see table) ORR: 81% (95%CI 71–88), all but 3 responses third party reviewed; 90 pts evaluable for biological response (baseline CA125 ≥50 UI/ml): 89% response (decrease >75% or CA125 normalization >1 mo). Combined RECIST /biological response: 88% (95%CI 80–94). M follow-up: 27 mo (14–48); m PFS: 10.2 mo; projected OS >28 mo (44 events). Safety (NCI-CTC); % pts with grade (gr) 3/4: neutropenia 21/46; thrombocytopenia 15/1; nausea-vomiting 9/2; asthenia 7/1; allergic reaction: 1/1; cumulative reversible neurotoxicity gr 2/3 (OXA specific scale): 50/25 after a m 7 cy (3–9); febrile neutropenia: 9 episodes/8 pts; 4% cy required G-CSF; no treatment-related deaths. Conclusion: The OXA/P combination shows the highest level of efficacy in platinum-sensitive third party reviewed large phase II studies in platinum/taxane pretreated AOC pts with PPFI >6 mo, and can be administered in an outpatient setting without special safety measures. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MGI Pharma, Inc.

Phase II trial of oxaliplatin (OXA) combined with paclitaxel (P) inplatinum + taxanes sensitive advanced ovarian cancer (AOC) patients: Final results

5023 Background: OXA is active in mismatch repair (MMR) deficient cells, a condition prevalent in pretreated AOC. OXA/P shows high activity in investigator initiated studies (Faivre et al, Ann Oncol 10:1125, 1999). We evaluated efficacy and safety of OXA/P in AOC pts with clinically evaluable disease pretreated with 1 platinum based regimen ± taxanes and a progression-platinum-free interval (PPFI) ≥6 months (mo). Methods: From Dec 99 to Apr 02, 105 pts, 14 centres received P 175 mg/m2, 3h iv followed by OXA 130 mg/m2, 2h iv, every 3 weeks, for up to 9 cycles (cy) without G-CSF. Median (m) age: 58 years (range 37–83), 96% PS 0/1. PPFI: 69 pts >12 mo, 34 pts 6–12 mo; 80 pts (76%) taxane-pretreated. Results: 708 cy administered; m 7 cy/pt (1–9). Relative dose intensity: OXA 0.95, P 0.97. Efficacy: 98 pts eligible, 7 non-eligible (3 bowel occlusion, 3 >1 prior platinum-based chemotherapy, 2 PPFI not determinable). 98 pts evaluable for response (see table) ORR: 81% (95%CI 71–88), all but 3 responses third party reviewed; 90 pts evaluable for biological response (baseline CA125 ≥50 UI/ml): 89% response (decrease >75% or CA125 normalization >1 mo). Combined RECIST /biological response: 88% (95%CI 80–94). M follow-up: 27 mo (14–48); m PFS: 10.2 mo; projected OS >28 mo (44 events). Safety (NCI-CTC); % pts with grade (gr) 3/4: neutropenia 21/46; thrombocytopenia 15/1; nausea-vomiting 9/2; asthenia 7/1; allergic reaction: 1/1; cumulative reversible neurotoxicity gr 2/3 (OXA specific scale): 50/25 after a m 7 cy (3–9); febrile neutropenia: 9 episodes/8 pts; 4% cy required G-CSF; no treatment-related deaths. Conclusion: The OXA/P combination shows the highest level of efficacy in platinum-sensitive third party reviewed large phase II studies in platinum/taxane pretreated AOC pts with PPFI >6 mo, and can be administered in an outpatient setting without special safety measures. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MGI Pharma, Inc.

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells.

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.

Effect of H 2O 2 on cell cycle and survival in DNA mismatch repair-deficient and -proficient cell lines

Patients who develop tumors with Lynch syndrome, which is caused by mutational inactivation of the DNA mismatch repair (MMR) system, have a relatively favorable prognosis compared to patients who develop sporadic tumors. Paradoxically, DNA MMR-deficient cells are resistant to many chemotherapeutic agents, and are capable of bypassing the G2/M checkpoint in vitro. Colon cancers that develop in the setting of Lynch syndrome show an abundant recruitment of immune cells into tumor tissues, which might be expected to increase oxyradical formation, and make the tumor cells more vulnerable to cell death. We examined the chemosensitivity and cell cycle response to oxidative stress in several MMR-deficient (HCT116, SW48, and DLD1) and -proficient (CaCo2, SW480, and HT29) colorectal cancer cell lines. H 2O 2 induced a G2/M cell cycle arrest in both MMR deficient and proficient cell lines, however MMR-deficient cell lines were more sensitive to H 2O 2 toxicity, and the response was more prolonged in MMR-deficient cells. Interestingly, human MutL-homologue ( hMLH1-)defective HCT116 and hMLH1-restored HCT116+ch3 cell lines responded to H 2O 2 with the same degree of G2/M arrest. The survival response of HCT116+ch3 was nearly identical to that of hMLH1-defective HCT116+ch2, although better than the response observed in HCT116 cells. In conclusion, greater cellular sensitivity and G2/M arrest in response to oxidative stress in MMR-deficient colorectal cancer cells could be one of the reasons for the more favorable prognosis seen in patients with Lynch syndrome. However, this sensitivity appears not to be a direct result of a deficient MMR function, but is more likely attributable to spectrum of target gene mutations that occurs in MMR-deficient tumors.

Photodynamic therapy of DNA mismatch repair-deficient and -proficient tumour cells.

Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumours. DNA mismatch repair-deficient cells have been reported to be resistant to many chemotherapeutic agents and to radiotherapy, and to have the potential of rapidly acquiring additional mutations leading to tumour progression. Photodynamic therapy is a new treatment modality using light to activate a photosensitiser that preferentially localises in tumour cells. An oxygen dependent photochemical reaction ensues, resulting in selective tumour necrosis. The effect of loss of DNA mismatch repair activity on the sensitivity to photodynamic therapy was tested using pairs of cell lines proficient or deficient in mismatch repair due to loss of either MLH1 or MSH2 protein function. Cells were incubated with the photosensitiser 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin and exposed to laser light at 652 nm with various optical doses ranging from 0–1 J cm−2. Cell survival was assessed using the clonogenic assay. Loss of MLH1 or MSH2 function was not associated with resistance to photodynamic therapy. MCF-7 cells repeatedly treated with photodynamic therapy expressed parental levels of MLH1, MSH2, MSH6, and PMS2. DNA mismatch repair-deficient and -proficient cells showed similar subcellular distributions of meta-tetra(hydroxyphenyl)chlorin as analysed by laser scanning and fluorescence microscopy. Therefore, repeated exposure of tumour cells to photodynamic therapy does not seem to result in loss of DNA mismatch repair, and loss of mismatch repair, in turn, does not seem to contribute to resistance to photodynamic therapy. Our results suggest recommending photodynamic therapy as a strategy for circumventing resistance due to loss of DNA mismatch repair.British Journal of Cancer (2002) 86, 1130–1135. DOI: 10.1038/sj/bjc/6600218 www.bjcancer.com© 2002 Cancer Research UK

Multiple mutations are common at mouse Aprt in genotoxin-exposed mismatch repair deficient cells.

Mismatch repair deficiency is known to contribute to elevated rates of mutations, particularly at mono- and dinucleotide repeat sequences. However, such repeats are often missing from the coding regions of endogenous genes. To determine the types of mutations that can occur within an endogenous gene lacking highly susceptible repeat sequences, we examined mutagenic events at the 2.3 kb mouse Aprt gene in kidney cell lines derived from mice deficient for the PMS2 and MLH1 mismatch repair proteins. The Aprt mutation rate was increased 33-fold and 3.6-20-fold for Mlh1 and Pms2 null cell lines, respectively, when compared with a wild-type kidney cell line. For the Pms2 null cells this increase resulted from both intragenic events, which were predominantly base-pairs substitutions, and loss of heterozygosity events. Almost all mutations in the Mlh1 null cells were due to base-pair substitutions. A:T-->G:C transitions (54% of small events) were predominant in the Pms2 null cells whereas G:C-->A:T transitions (36%) were the most common base-pair change in the Mlh1 null cells. Interestingly, 4-9% of the spontaneous mutant alleles in the mismatch repair deficient cells exhibited two well-separated base-pair substitution events. The percentage of mutant alleles with two and occasionally three base-pair substitutions increased when the Pms2 and Mlh1 null cells were treated with ultraviolet radiation (15-21%) and when the Mlh1 null cells were treated with hydrogen peroxide (35%). In most cases the distance separating the multiple base-pair substitutions on a given allele was in excess of 100 base-pairs, suggesting that the two mutational events were not linked directly to a single DNA lesion. The significance of these results is discussed with regards to the roles for the PMS2 and MLH1 proteins in preventing spontaneous and genotoxin-related mutations.

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells.

We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.

MutS Preferentially Recognizes Cisplatin- over Oxaliplatin-modified DNA

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.

Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells

Microsatellites are DNA elements composed of short tandem repeats of 1–5 bp. These sequences are particularly prone to frameshift mutation by insertion–deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA) n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number ( n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene ( neo), disrupted the reading frame of the gene in the (−1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA) 17 and (CA) 30 tracts than for (CA) 8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA) 17 and (CA) 30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (≥8 bp) were observed in these tracts, where as deletions this large were not found in the (CA) 8 tract. Single-repeat mutations of both types were made at similar rates in (CA) 8 tracts in human mismatch repair deficient (MMR −) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR +) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.

Mutation frequency in coding and non-coding repeat sequences in mismatch repair deficient cells derived from normal human tissue.

Repetitive tracts within the coding regions of TGFBR2 and BAX are frequently mutated in mismatch repair deficient tumours and are implicated in tumour progression. However, there has been little study of the balance between selection pressure and inherent instability at sequences within these genes. To determine whether TGFBR2 and BAX are inherently prone to mutations in the presence of MMR defects, we studied MMR deficient cells derived from B-lymphocytes. By analysing cells derived from normal tissue we aimed to minimize the effects of selection pressures that bias the apparent frequency of mutation. We definitively show that certain sequences, usually repaired by MMR, are inherently unstable. Using a small pool PCR technique we confirmed these cells exhibit microsatellite instability. Additionally, we demonstrate that MMR deficiency results in an excess of mutations, specifically at the poly(A)(10) tract compared to other regions of the TGFBR2 gene (P<0.001). Conversely, an excess of mutations does not appear to arise at the poly(G)(8) tract of the BAX gene. These studies provide insight into the mechanism by which TGFBR2 and BAX genes become mutated during tumorigenesis. These findings invoke the notion of "unmasking" specific hypermutable sequences in particular genes adding further complexity to the concept of the mutator phenotype.

Reduction of spontaneous mutagenesis in mismatch repair-deficient and proficient cells by dietary antioxidants

Cells lacking mismatch repair (MMR) exhibit elevated levels of spontaneous mutagenesis. Evidence exists that MMR is involved in repair of some DNA lesions besides mismatches. If some oxidative DNA lesions are substrates for MMR, then the excess mutagenesis in MMR − cells might be blocked by dietary antioxidants. Effects of the dietary antioxidants ascorbate, α-tocopherol, (−)-epigallocatechin gallate (EGCG) and lycopene on spontaneous mutagenesis were studied using mismatch repair-deficient (hMLH1 −) human colon carcinoma HCT116 cells and HCT116/ch3 cells, in which normal human chromosome 3 has been added to restore mismatch repair. HCT116 cells have a 22-fold higher spontaneous mutation rate compared with HCT116/ch3 cells. HCT116 cells cultured in 1% fetal bovine serum (FBS) have twice the spontaneous mutation rate of those cultured in 10% FBS, most likely due to reduction in serum antioxidants in the low serum medium. As expected, α-tocopherol (50 μM) and ascorbate (284 μM) reduced spontaneous mutagenesis in HCT116 cells growing in 1% serum more dramatically than in cells cultured in 10% serum. The strongest antimutagenic compound was lycopene (5 μM), which reduced spontaneous mutagenesis equally (about 70%) in HCT116 cells growing in 10 and 1% FBS and in HCT116/ch3 cells. Since lycopene was equally antimutagenic in cells growing in low and high serum, it may have another antimutagenic mechanism in addition to its antioxidant effect. Surprisingly, EGCG (10 μM) was toxic to cells growing in low serum. It also reduced spontaneous mutagenesis equally (nearly 40%) in HCT116 and HCT116/ch3 cells. The large proportion of spontaneous mutagenesis that can be blocked by antioxidants in mismatch repair-deficient cells support the hypothesis that a major cause of their excess mutagenesis is endogenous oxidants. Blocking spontaneous mutagenesis, perhaps with a cocktail of antioxidants, should reduce the risk of cancer in people with a genetic defect in mismatch repair as well as other individuals.

Acyl derivatives of demethylpenclomedine, an antitumor-active, non-neurotoxic metabolites of penclomedine.

The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

Mutagenesis by O6-Methyl-, O6-Ethyl-, and O6-Benzylguanine and O4-Methylthymine in Human Cells: Effects of O6-Alkylguanine-DNA Alkyltransferase and Mismatch Repair

Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ' gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent with m(6)G > b(6)G. The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions. However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.

Heterocyclic amine induced apoptotic response in the human lymphoblastoid cell line TK6 is linked to mismatch repair status

The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Augmentation of the p53 protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1 DNA or condensed nuclei) revealed that only 10–20% of the PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5μg/ml PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5μg/ml PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient colorectal carcinoma cell line, also exhibited lower PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following PhIP treatment. These results show that PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair protein(s) are involved downstream of p53 or that PhIP-induced apoptosis is p53-independent.

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase β were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.

Combined effects of adenovirus-mediated wild-type p53 transduction, temozolomide and poly (ADP-ribose) polymerase inhibitor in mismatch repair deficient and non-proliferating tumor cells.

Lack of p53 or mismatch repair (MR) function and scarce cell proliferation are commonly associated with tumor cell resistance to antineoplastic agents. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) has been considered as a tool to overcome resistance of MR-deficient tumors to methylating agents. In the present study we demonstrated that infection with p53 expressing adenovirus (Ad-p53), enhances chemosensitivity of MR-deficient tumor cell lines to the methylating agent temozolomide (TZM), either used as single agent or, more efficiently, when combined with PARP inhibitor. Moreover, the association of Ad-p53 with drug treatment induced a more pronounced growth inhibitory effect than that provoked by Ad-p53 infection only. Cells, growth arrested by p53 transduction, and then subsequently exposed to the drugs, were still highly susceptible to cytotoxicity induced by TZM and PARP inhibitor. The results suggested that this drug combination might be effective even in non-proliferating tumor cells. It is conceivable to envisage future possible strategies to enhance cytostatic or cytotoxic effects induced by Ad-p53, based on the use of TZM, alone or combined with PARP inhibitor for the therapy of resistant tumors.