Yvan Canitrot

Multidrug resistance mediated by the multidrug resistance protein (MRP) gene

Inherent or acquired resistance to multiple natural product drugs is a major obstacle to the success of chemotherapy. Two proteins have been shown to cause this type of multidrug resistance in human tumour cells, the 170 kDa P-glycoprotein and the 190 kDa multidrug resistance protein (MRP). Overexpression of these N-glycosylated phosphoproteins in mammalian cells is associated with reduced drug accumulation. Both MRP and p-glycoprotein belong to the ATP-binding cassette superfamily of transmembrane transport proteins, but they share only 15% amino acid identity. Furthermore, their predicted membrane topologies differ considerably, with MRP containing three multispanning transmembrane domains compared with the two that are present in P-glycoprotein. The drug cross-resistance profiles of cells that overexpress MRP or P-glycoprotein are similar but not identical. For example, lower levels of taxol resistance are associated with overexpression of MRP than with overexpression of P-glycoprotein. There also appear to be fundamental differences in the mechanisms by which the two proteins transport chemotherapeutic drugs. P-glycoprotein-enriched membrane vesicles have been shown to directly transport several chemotherapeutic drugs, whereas vincristine transport by MRP-enriched membrane vesicles is demonstrable only in the presence of reduced glutathione. Several potential physiologic substrates of MRP including leukotriene C4 and 17 beta-estradiol-17-(beta-D-glucuronide) have been identified. In contrast, these conjugated organic anions are transported poorly, if at all, by P-glycoprotein. Finally, agents that reverse P-glycoprotein-associated resistance are usually much less effective in MRP-associated resistance. Antisense oligonucleotide-mediated suppression of MRP synthesis offers a highly specific alternative approach to circumventing resistance mediated by this novel drug resistance protein.

The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides

Multidrug resistance protein (MRP) is a member of the ATP-binding cassette superfamily of transport proteins which has been demonstrated to cause multidrug resistance when transfected into previously sensitive cells. Sixteen eicosomeric oligonucleotides complementary to different regions along the entire length of the MRP mRNA reduced MRP mRNA and protein levels in drug-resistant small cell lung cancer cells that highly overexpress this protein. In MRP-transfected HeLa cells that express intermediate levels of MRP, one oligonucleotide, ISIS 7597, targeted to the coding region of the MRP mRNA, decreased the levels of MRP mRNA to < 10% of control levels in a concentration-dependent manner. This effect was rapid but transient with a return to control levels of MRP mRNA 72 hr after treatment. A double treatment with ISIS 7597 produced a sustained inhibition, resulting in a greater than 90% reduction in MRP mRNA for 72 hr and a comparable decrease in protein levels. Increased sensitivity to doxorubicin was observed under these conditions. Northern blotting analyses using two DNA probes corresponding to sequences 5 and 3 of the ISIS 7597 target sequence, respectively, revealed the presence of low levels of two smaller sized RNA fragments as expected from an RNase H-mediated mechanism of action of the antisense oligonucleotide. These studies indicate that a specific reduction in MRP expression can be achieved with antisense oligonucleotides, a finding that has potential implications for the treatment of drug-resistant tumors.

Multidrug resistance genes (MRP) and MDR1 expression in small cell lung cancer xenografts: relationship with response to chemotherapy.

Intrinsic or acquired drug resistance is a major limiting factor of the effectiveness of chemotherapy. Increased expression of either the MRP gene or the MDR1 gene has been demonstrated to confer drug resistance in vitro. In this study, we examined MRP and MDR1 gene expression in a panel of 17 small cell lung cancers (SCLC) xenografted into nude mice from treated and untreated patients using an RT-PCR technique. For some of them, the outcome of the corresponding patients was known and we related MDR1/MRP expression with the xenograft response to CCAV (cyclophosphamide, cisplatin, adriamycin and etoposide) combined chemotherapy. Fifteen (88%) of the 17 cases of SCLC were found to be positive for either MDR1 or MRP. MRP gene expression was present in 12 (71%) of 17 cases, whereas MDR1 gene expression was detected in eight (50%) of 16 cases. For six SCLC, the survival duration of patients differed, with three patients surviving for more than 30 months after therapy. Among these six turnours, five expressed MRP and/or MDR1. These six xenografts responded to the CCAV treatment but a significant rate of cure was obtained in only three cases. No obvious relationship was observed between the response to this treatment and MRP or MDR1 expression. However, the remarkably high levels and frequency of MRP expression in some SCLC samples indicate that future developments in chemotherapy of this tumour type should anticipate that drugs which are substrates of MRP may be of limited effectiveness.

Functional study of multidrug resistance with fluorescent dyes. Limits of the assay for low levels of resistance and application in clinical samples

Fluorescent dyes such as rhodamine 123 (R123) and Hoechst 33342 (Ho342) have been widely used to characterize multidrug-resistance (MDR) phenotype cells in cell populations, on the basis of their reduced accumulation in resistant cells. Taking advantage of the high fluorescence quantum yield of R123 and Ho342 compared with that of anthracyclines, we investigated the limits of fluorescence image cytometry in detecting MDR by the level of R123 and Ho342 accumulation and efflux. We were able to separate with this technique a cell line with a level of resistance as low as 3. We then studied the presence of MDR cells in lymphocytes isolated from patients with hematological malignancies.

Regulation of DNA Polymerase β by the LMP1 Oncoprotein of EBV through the Nuclear Factor-κB Pathway

The repair DNA polymerase beta (Polbeta), when overexpressed, plays a critical role in generating genetic instability via its interference with the genomic replication program. Up-regulation of Polbeta has been reported in many tumor types that exhibit genetic aberrations, including EBV-related B-cell lymphomas. However, the mechanisms responsible for its overexpression have never been examined. Here, we report that both expression and activity of Polbeta, in EBV-immortalized B cells, are induced by several natural genetic variants of LMP1, an oncoprotein associated with the vast majority of EBV-related tumors. Conversely, we found that the expression of Polbeta decreased when LMP1 signaling was down-regulated by a dominant negative of LMP1 or an inhibitor of the nuclear factor-kappaB (NF-kappaB) pathway, the main transduction pathway activated by LMP1, strongly supporting a role of NF-kappaB in the LMP1-mediated Polbeta regulation. Using electrophoretic mobility shift assay experiments from several EBV-immortalized B-cell nuclear extracts, we identified an LMP1-dependent p50/c-Rel heterodimer on a proximal kappaB binding site (-211 to -199nt) of the Polbeta promoter. This result was correlated with a specific Polbeta kappaB transcriptional activity. Taken together, our data enlighten a new mechanism responsible for Polbeta overexpression in EBV-infected cells, mediated by LMP1 and dependent on NF-kappaB activation.