Biomedical subjects

P Karran

Publications and source records attributed to P Karran.

At least 19 recordsLinked to original sources

PubMed2001

Photoactivation of DNA thiobases as a potential novel therapeutic option.

The thiopurines, 6-thioguanine and 6-mercaptopurine, are antileukemic agents that are incorporated into DNA following retrieval by the purine salvage pathway (see [1] for a review). Their toxicity requires active DNA mismatch repair (MMR), and thiopurine resistance is an acknowledged phenotype of MMR-defective cells [2, 3]. In addition to these direct cytotoxic effects, DNA thiobases have distinctive photochemical properties [4], the therapeutic potential of which has not been extensively evaluated. We report here that the thiopyrimidine nucleoside 4-thiothymidine is incorporated into DNA. It does not induce MMR-related toxicity, but it interacts synergistically with UVA light and dramatically sensitizes cultured human cells to very low, nonlethal UVA doses. 4-thiothymidine induced UVA dose enhancements of around 100-fold in DNA repair-proficient cells. Nucleotide excision repair-defective xeroderma pigmentosum cells were sensitized up to 1000-fold, implicating bulky DNA photoproducts in the lethal effect. The synergistic action of thiothymidine plus UVA required thymidine kinase, indicating a selective toxicity toward rapidly proliferating cells. Cooperative UVA cytotoxicity is a general property of DNA thiobases, and 6-thioguanine and 4-thiodeoxyuridine were also UVA sensitizers. Thiobase/UVA treatment may offer a novel therapeutic approach for the clinical management of nonmalignant conditions like psoriasis or for superficial tumors that are accessible to phototherapy.

Cell Line↗

PubMed2001

Mechanisms of tolerance to DNA damaging therapeutic drugs.

The cytotoxic effect of many anticancer drugs relies on their ability to damage DNA. Drug resistance can be associated with the ability to remove potentially lethal DNA lesions. DNA damage tolerance offers an alternative route to resistance. In a drug-tolerant cell, persistent DNA damage has become uncoupled from cell death. Tolerance to some DNA damaging drugs is accompanied by inactivation of the cell's DNA mismatch repair pathway. This is widely acknowledged as the mechanism underlying resistance to methylating agents and to 6-thioguanine which produce structurally similar types of DNA damage. Defects in mismatch repair are also associated with resistance to numerous drugs that produce a wide variety of structurally diverse DNA lesions. Here I consider possible mechanisms by which mismatch repair might influence drug resistance and the extent to which loss of mismatch repair might be considered to confer a multidrug resistance phenotype.

Animals↗

PubMed2000

Spontaneous development of drug resistance: mismatch repair and p53 defects in resistance to cisplatin in human tumor cells.

The contributions of defective mismatch repair and mutated p53 to cisplatin resistance of human tumor cells were analysed. Mismatch repair defects were not associated with a predictable degree of resistance among several tumor cell lines. Repair defective variants of the A2780 ovarian carcinoma cell line which were isolated by selection for a methylation tolerant phenotype and did not express the hMLH1 mismatch repair protein, were highly resistant to cisplatin. Their cisplatin resistance was not a simple consequence of the mismatch repair defect. They were members of a drug-naive subpopulation of A2780 in which a silent hMLH1 gene accompanies a mutated p53. Two complementary approaches indicated that each defect contributes to cisplatin resistance independently and to a different extent. Firstly, separate introduction of a p53 defect into A2780 cells significantly increased their cisplatin resistance; defective hMLH1 provided less extensive protection. Secondly, azadeoxycytidine reactivation of the silent hMLH1 gene or expression of a transfected hMLH1 cDNA sensitized the doubly hMLH1/p53 deficient cells only slightly to cisplatin. Both approaches indicate that defective p53 status is a major determinant of cisplatin resistance and defective mismatch repair is a minor, and independent, contributor. The data have implications for the development of intrinsic cisplatin resistance.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Alkylating Agents↗

PubMed2000

Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair.

A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5-fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of </=25 bases of DNA.

Adaptor Proteins, Signal Transducing↗

PubMed2000

DNA double strand break repair in mammalian cells.

Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.

Animals↗

PubMed2000

Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents.

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.

Alkylating Agents↗

PubMed2000

Mismatch repair and p53 independently affect sensitivity to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea.

The contributions of defective mismatch repair (MMR) and the p53-response to cell killing by N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) were evaluated. MMR defects were previously shown to be associated with CCNU sensitivity (G. Aquilina et al., Cancer Res., 58: 135-141, 1998). Unexpectedly, eight MMR-deficient variants of the A2780 human ovarian carcinoma cell line were 3-fold more resistant to CCNU than the MMR-proficient parental cells. The variants were members of a preexisting subpopulation of drug-resistant A2780 cells. In addition to deficient expression of the MMR protein hMLH1, an essential component of the hMutL alpha repair complex, the variants exhibited alterations in the expression of other genes that influence drug sensitivity. Although A2780 cells possess a wild-type p53 gene, all of the clones contained a heterozygous G to T tranversion at codon 172. This change resulted in a Val to Phe substitution and was associated with a constitutive production of high levels of p53, which was inactive as a transcriptional activator of bax and p21. The hMLH1/p53 defective variants displayed a less prominent cell cycle arrest and reduced apoptosis after CCNU treatment. In contrast, MMR-defective A2780 variants, which had a similar hMutL alpha defect but retained a wild-type p53, did exhibit the expected CCNU sensitivity. Expression of a dominant-negative p53val135 increased CCNU resistance of both MMR-proficient and MMR-deficient A2780 cells. Thus, defective MMR and p53 influence CCNU sensitivity in opposite directions. Their effects are independent, and sensitization by defective MMR does not require a functional p53 response.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.

The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage.

3T3 Cells↗

PubMed1999

Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes methylation but not UV- or ionizing radiation-induced DNA damage.

Interaction between long patch mismatch repair (MMR) and persistent DNA O6-methylguanine or 6-thioguanine (6-TG) is implicated in the cytotoxicity of methylating agents and 6-TG, respectively. Human cells with defective MMR tolerate DNA methylation damage and are cross-resistant to 6-TG. To determine whether MMR contributes to the lethal effects of persistent UV-induced DNA lesions, MMR deficiency was introduced into nucleotide excision repair (NER)-defective XP12RO cells. The doubly repair-defective cells, designated XP12ROB4, did not express detectable hMSH2 protein. They had the mutator phenotype, N-methyl-N-nitrosourea and 6-TG resistance typical of MMR-defective cells. Active MMR was not required for the cytotoxicity of UV light, and the hMSH2 defect did not detectably alter the survival of XP12ROB4. The level of spontaneous or UV-induced SCE was also similar in XP12RO and XP12ROB4, indicating that hMSH2 is not required for this recombination process. The combined deficiency in MMR and NER did not confer a significant degree of tolerance to ionizing radiation, and the survival of XP12RO and XP12ROB4 after gamma-radiation was similar. Although it recognizes and processes some persistent damaged or modified DNA base pairs, MMR is unlikely to serve as a general sensor of DNA damage.

Cell Line↗

PubMed1999

The cytotoxicity of DNA carboxymethylation and methylation by the model carboxymethylating agent azaserine in human cells.

Carboxymethylating agents are potential sources of endogenous DNA damage that have been proposed as possible contributors to gastrointestinal carcinogenesis. The cytotoxicity of the model DNA carboxymethylating agent azaserine was investigated in human cells. Expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) did not affect sensitivity to the drug in two related Raji Burkitt's lymphoma cell lines. DNA mismatch repair-defective variants of Raji cells which display increased tolerance to DNA methylation damage were not selectively resistant to azaserine. Complementary results were obtained with a second carboxymethylating agent, potassium diazoacetate. In contrast, lymphoblastoid cell lines representative of each of the xeroderma pigmentosum complementation groups, including the variant, were all significantly more sensitive to azaserine than nucleotide excision repair-proficient cells. The hypersensitivity of XP cells was not due to systematic differences in the concentrations of intracellular thiol compounds or related thiol metabolizing enzymes. The data indicate that of the two types of potentially lethal DNA damage which azaserine introduces, carboxymethylated bases and O(6)-methylguanine, the former are repaired by nucleotide excision repair and are a more significant contributor to azaserine lethality in human cells.

Adenine↗

PubMed1998

Frameshift mismatch recognition by the human MutS alpha complex.

DNA mismatch binding by an extensively purified hMutS alpha mismatch recognition complex was investigated using a bandshift assay. The complex bound to G. T mispairs and to looped structures containing an unpaired single or two adjacent bases. A CA loop was preferentially recognised if the unpaired bases formed part of a repeated sequence. In general, single base loops were also more favourably recognised by hMutS alpha when present in monotonic runs of two to five. In one series of substrates, based on a known hotspot for frameshift mutations in the hypoxanthine-guanine phosphoribosyltransferase gene, in which different length G, A, C or T tracts were flanked by closely similar sequences, an A loop was bound preferentially in the absence of adjacent As and a C loop in the absence of adjacent Cs. This preferential binding was influenced by the base immediately 5' to the loop. Thus, while repeated regions generally favour recognition of single base loops by hMutS alpha, other factors related to local sequence may influence this interaction.

Binding Sites↗

PubMed1997

Mismatch repair defects and O6-methylguanine-DNA methyltransferase expression in acquired resistance to methylating agents in human cells.

Fifteen variants with >/=30-fold resistance to N-methyl-N-nitrosourea were isolated from the Burkitt's lymphoma Raji cell line. Eight had received a single treatment with a highly cytotoxic dose. The remainder, including the previously described RajiF12 cell line, arose following multiple exposures to initially moderate but escalating doses. Surprisingly, methylation resistance arose in three clones by reactivation of a previously silent O6-methylguanine-DNA methyltransferase gene. Five clones, including RajiF12, displayed the microsatellite instability and increased spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus, consistent with deficiencies in mismatch repair. Defects in either the hMutSalpha or hMutLalpha mismatch repair complexes were identified in extracts of these resistant clones by in vitro complementation using extracts from colorectal carcinoma cell lines. Defects in hMutLalpha were confirmed by Western blot analysis. Remarkably, five methylation-resistant clones in which mismatch repair defects were demonstrated by biochemical assays did not exhibit significant microsatellite instability.

Blotting, Western↗

PubMed1997

DNA mismatch repair in lymphoblastoid cells from hereditary non-polyposis colorectal cancer (HNPCC) patients is normal under conditions of rapid cell division and increased mutational load.

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have germ-line mutations in one of four DNA mismatch repair genes (hMSH2, hMLH1, hPMS1 and hPMS2). It is supposed that a single functional copy of these genes is sufficient for normal mismatch repair, but it is not certain that this is the case under abnormal conditions such as rapid cell division or an increased tendency to DNA replication errors (RERs). We have analysed mismatch repair by examining replication errors in immortalised lymphoblastoid cells derived from two HNPCC patients heterozygous for mismatch repair defects (one hMSH2 mutant and one hMLH1 mutant), and from control individuals. Three conditions of cell culture have been used: (i) relatively slow cell growth and division; (ii) relatively fast growth and division; and (iii) chronic perturbation of the intracellular dNTP pool to promote a increased frequency of replication errors. No significant differences in microsatellite instability were found between HNPCC patients and controls in any of these environments. Lymphoblastoid cells from hMSH2 and hMLH1 mutant/wild-type heterozygotes appear, therefore, to have normal levels of mismatch repair, even under conditions that increase the requirement for repair. The pool bias cultures from the HNPCC patients and controls did, however, show similar, increased frequencies of RERs, suggesting that the mismatch repair capacity of the cells had been overloaded, but that the number of normal HNPCC alleles was not the limiting factor.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.

Animals↗

PubMed1997

Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins.

The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines. Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink. Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand. Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly. The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation. Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch. It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.

Adenosine Triphosphatases↗

PubMed1997

DNA excision repair pathways.

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair proteins in knockout mice provides information on the roles of such factors in vivo and recent three-dimensional structures of several repair enzymes explain their detailed modes of action.

Animals↗

PubMed1996

Selection for beta 2-microglobulin mutation in mismatch repair-defective colorectal carcinomas.

Novel peptide antigens complexed with human leukocyte antigen (HLA) and beta 2-microglobulin (beta 2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumor cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and beta 2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3,4] and loss of beta 2M (or HLA) expression [5,6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of beta 2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating beta 2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring beta 2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.

Colorectal Neoplasms↗