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Thymidine phosphorylase (TP), an enzyme involved in pyrimidine metabolism, is identical with an angiogenic factor, platelet-derived endothelial cell growth factor. 2-Deoxy-D-ribose (D-dRib), the degradation product of thymidine generated by TP activity, has been suggested to be a downstream mediator of TP function. 2-Deoxy-L-ribose (L-dRib), a stereoisomer of D-dRib, inhibited the promotion of angiogenesis, tumor growth and metastasis by TP. In our study, we have shown that nude mice inoculated with TP-overexpressing KB/TP cells had shorter survival times than those injected with control KB/CV cells. KB/TP tumors were also more highly invasive than KB/CV tumors in mice. The expression levels of matrix metalloproteinase (MMP)-9 in KB/TP tumors were significantly higher than those in KB/CV tumors. L-dRib and a TP inhibitior, TPI, extended the survival period of KB/TP tumor-bearing mice. L-dRib also reduced MMP-9 mRNA levels in KB/TP tumors. Furthermore, L-dRib suppressed the mRNA level of MMP-9 in cultured KB/TP cells, and the invasive activity of the cells. L-dRib may be useful for the suppression of invasion of TP-expressing tumor cells.

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer. © 1996 Wiley-Liss, Inc.

V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine- or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

We previously isolated agosterol A (AG-A) from a marine Spongia sp. and found that it completely reversed colchicine resistance in P-glycoprotein (Pgp)-over-expressing KB-C2 cells and vincristine resistance in multidrug-resistance protein (MRP)1-over-expressing CV60 cells. However, a tri-deacetylated derivative of AG-A (IAG-A) showed almost no activity in reversing Pgp- or MRP1-mediated drug resistance. In this study, we examined the mechanisms by which AG-A reverses MRP1-mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1-over-expressing human KB carcinoma (KB/MRP) cells. [3H]-Leukotriene C4 (LTC4), [3H]-2,4-dinitrophenyl-S-glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [3H]-vincristine accumulation and efflux in KB/MRP cells were measured with or without AG-A and/or inactive IAG-A. AG-A reduced MRP1-mediated [3H]-LTC4 transport in a dose-dependent manner, but IAG-A did not. Inhibition by AG-A was competitive, with a Ki value of 31 μM. AG-A at 10 μM enhanced the accumulation of [3H]-vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent. Likewise, ATP-dependent efflux of [3H]-vincristine from KB/MRP cells was enhanced compared with KB-3-1 cells and inhibited by AG-A. In addition, AG-A reduced intracellular levels of glutathione, a compound required for MRP1-mediated transport of some anti-cancer drugs. These findings suggest that AG-A reverses MRP1-mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs. In addition, the capacity of AG-A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1. © 2001 Wiley-Liss, Inc.

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.

MRP has been identified as another multidrug-resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug-resistance KB sublines (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug-sensitive human KB cells. MRP gene expression was elevated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous-cell carcinomas of the lung.

The expression of multidrug-resistance-associated protein (MRP) was assessed in various types of untreated lung cancer using an immunohistochemical technique. MRP was abundantly expressed in 28 of 59 adenocarcinoma specimens (47%) and its expression was associated with the degree of glandular differentiation of the tumor. MRP expression in well-differentiated adenocarcinomas (56%) was higher than in poorly differentiated adenocarcinomas (22%) (p < 0.01). lower--20% in squamous-cell carcinomas, 20% in large-cell carcinomas and 0% in small-cell carcinomas and carcinoids. RT-PCR showed that the MRP-positive adenocarcinomas and squamous-cell carcinomas expressed mrp mRNA significantly. Immunoelectron microscopically, MRP was localized in the plasma membrane and rough endoplasmic reticulum. It is thus important to take MRP into account when considering chemotherapy for lung cancers because levels of mdr I gene product, another multidrug-resistance gene family, are low in untreated lung cancers.

One thousand fungi and Actinomycetes were investigated to see whether they produced compounds that reverse multi-drug resistance in KB cells. Only one Aspergillus strain M39 produced agents with resistance-reversing activity and these compounds were identified to be rubrofusarin B and dianhydro-aurasperone C. Rubrofusarin B only slightly reversed the resistance of KB-C2 cells to Adriamycin and daunomycin, partially reversed the resistance to chromomycin A3, and almost completely reversed the resistance to vincristine and mitomycin C. Purified dianhydro-aurasperone C and rubrofusarin B had similar effects on drug resistance in KB-8-5 cells. Dianhydro-aurasperone C enhanced the accumulation of vinblastine in KB-8-5 cells and inhibited the efflux of vinblastine from the cells. Dianhydro-aurasperone C and rubrofusarin B at 10 microM completely inhibited 3H-azidopine photolabelling of P-glycoprotein. The two products of Aspergillus strain M39 appear to reverse multi-drug resistance by interacting with P-glycoprotein and inhibiting its role as an active efflux pump.

The original article to which this Erratum refers was published in International Journal of Cancer; 2001; 93(2) 212–217.

After nitrosoguanidine mutagenesis we isolated from mouse leukemia L5178Y cells a mutant cell (Bla-R) resistant to blasticidin S, an inhibitor of protein synthesis. Neither growth nor leucine incorporation into hot-acid insoluble fraction of Bla-R cell was inhibited by 5 to 20 microgram/ml blasticidin S, which almost completely blocked protein synthesis as well as growth of the parental L5178Y cells. However, other inhibitors such as fusidic acid, cycloheximide, ricin D or L-asparaginase blocked protein synthesis in Bla-R cells to the same extent as in L5178Y cells. Protein synthesis in vitro using S-30 extracts from the parental cell line L5178Y was almost completely blocked in the presence of the antibiotic, while no inhibition by blasticidin S occurred when S-30 extracts -rom Bla-R mutant cells were used. Protein synthesis assays were made by using the S100 fraction from rat liver together with ribosomes from either L5178Y cells or Bla-R cells. Blasticidin S inhibited protein synthesis when ribosomes were derived from L5178Y cells, but not from Bla-R mutant.