Xinjian Lin

DNA mismatch repair and p53 function are major determinants of the rate of development of cisplatin resistance

As opposed to factors that control sensitivity to the acute cytotoxic effect of cisplatin, little is known about the factors that determine the rate at which resistance develops. This study examined how loss of p53 or DNA mismatch repair (MMR) function affected the rate of development of resistance to cisplatin in human colon carcinoma cells during sequential cycles of cisplatin exposure that mimic the way the drug is used in the clinic. We used a panel of sublines molecularly engineered to express either the MMR- and p53-proficient phenotype or singly or doubly deficient phenotypes. Loss of either MMR or p53 alone increased the rate of development of resistance to cisplatin by 1.8- and 2.4-fold, respectively; however, loss of both MMR and p53 increased the rate by 4.8-fold. Inhibition of DNA polymerase ζ by suppression of the expression of its REV3 subunit eliminated the increased rate of development of resistance observed in the MMR-deficient cells. Loss of p53 or MMR increased the steady-state level of REV3 and of REV1 mRNA; loss of both functions increased these levels much further by a factor of 20.2-fold for REV3 and 10.3-fold for REV1. The basal level of homologous recombination measured using a reporter vector was 1.3- to 1.7-fold higher in cells that had lost either p53 or MMR function, and 2.6-fold higher in cells that had lost both. In the p53- and MMR-proficient cells, cisplatin induced a 17-fold increase in homologous recombination even when the recombining sequences that did not contain cisplatin adducts; the magnitude of induction was even greater in cells that had lost either one or both functions. We conclude that separate from effects on sensitivity to the acute cytotoxic effect of cisplatin, loss of MMR, especially when combined with loss of p53, results in rapid evolution of cisplatin resistance during sequential rounds of drug exposure that is likely mediated by enhanced mutagenic translesion synthesis. The DNA damage response activated by cisplatin is accompanied by a p53- and MMR-dependent increase in homologous recombination even between adduct-free sequences. [Mol Cancer Ther 2006;5(5):1239–47]

Identification of genes whose expression is associated with cisplatin resistance in human ovarian carcinoma cells

The goal of this study was to identify genes consistently differentially expressed in multiple pairs of isogenic cisplatin (DDP)-sensitive and resistant human ovarian carcinoma cell lines using microarray-based expression profiling. Expression profiling was carried out on six pairs of ovarian carcinoma cells lines growing under identical conditions; each cell expression profile was independently replicated six times. No genes were differentially expressed in all six pairs of cells or even in even in any five of the six pairs. Eighteen genes and 1 EST were upregulated, and four genes and 1 EST were downregulated, in at least four cell pairs. Of these, only metallothionein 2A has previously been implicated in DDP resistance. Among the genes identified on the basis of six replicates, an average of 24.8% would have been missed if only five replicates had been performed, and 38.3% would have been missed with only four replicates. The genes did not identify a dominant biochemical pathway or ontology category as being linked to DDP resistance; however, hierarchical clustering provided evidence for two classes DDP-resistant phenotypes within which there are additional cell pair-specific alterations. Many of the genes identified in this study play important roles in cell surface interactions and trafficking pathways not previously linked to DDP resistance. The genes discovered by this extensively replicated analysis are candidates for prediction of DDP responsiveness in ovarian cancer patients.

DNA Polymerase ζ 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 ζ, and on the integrity of the DNA mismatch repair (MMR) system. The aim of this study was to determine whether disabling Pol ζ 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 ζ-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 ζ 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 ζ pathway.

Integrin αV control of Cu homeostasis and cisplatin sensitivity

Resistance to chemotherapy is a major factor responsible for cancer recurrence and failure of therapy. Recent studies have disclosed that the nature of the extracellular matrix and the crosstalk between the stroma and the malignant cells have substantial effects on the sensitivity to standard chemotherapeutic agents. The αV-containing integrins play a central role in the interaction between the matrix and tumor cells, and together with its β integrin partners participates in the regulation of numerous signal transduction pathways through the activation of FAK, Src and related kinases. However, little is known about how αV integrin modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum-containing drugs. Reduced drug accumulation due to impaired influx is a common feature of cells that have acquired resistance to cisplatin (cDDP) and carboplatin. Both are quite polar and do not cross lipid membranes readily. There is now a substantial body of evidence, derived from multiple cell models, that the copper (Cu) influx transporter CTR1 regulates the cellular pharmacology of these drugs.1 The discovery that CTR1 accounts for a significant fraction of the cDDP uptake introduced the concept that cDDP exploits its mimicry of the chemical characteristics of Cu+1 to get into the cell where it is now known to form complexes with several Cu-binding proteins containing a CXXC motif.2 We recently discovered that the loss of the cell-ECM interaction mediated by αV is associated with an increase in CTR1 expression, enhanced cDDP accumulation and DNA adduct formation, and greater sensitivity to the cytotoxic effect of cDDP.3 This suggests that the signals generated by the αV-containing integrins normally serve to repress CTR1 levels. The linkage between αV and CTR1 expression is of potential clinical significance because elevated hCTR1 expression, as determined by immunohistochemical and mRNA expression profiling analysis, is associated with favorable treatment outcome in several diseases including ovarian, non-small cell lung and endometrial cancers in which cDDP is used as part of primary therapy.