Tatsuhiko Furukawa

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells.

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. Int. J. Cancer 91:126–131, 2001.

Expression and sub-cellular localization of human ABH family molecules

AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1‐methyladenine and 3‐methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2‐oxoglutarate‐ and Fe(II)‐dependent (2OG‐Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG‐Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4–hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG‐Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG‐Fe(II) oxygenase domain but both an RNA‐binding motif and a methyl‐transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub‐cellular localization of EmGFP‐hABH8 was restricted to the cytoplasm. EmGFP‐hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP‐hABH2 splice variant localized in nucleoplasm with a dot‐like pattern. In some HeLa cells transfected with EmGFP‐hABH5, dot‐like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function

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.

Inhibition of Metastasis of Tumor Cells Overexpressing Thymidine Phosphorylase by 2-Deoxy-l-Ribose

Thymidine phosphorylase (TP) catalyzes the reversible conversion of thymidine to thymine, thereby generating 2-deoxy-d-ribose-1-phosphate, which upon dephosphorylation forms 2-deoxy-d-ribose (d-dRib), a degradation product of thymidine. We have previously shown that d-dRib promotes angiogenesis and chemotaxis of endothelial cells and also confers resistance to hypoxia-induced apoptosis in some cancer cell lines. 2-Deoxy-l-ribose (l-dRib), a stereoisomer of d-dRib, can inhibit d-dRib anti-apoptotic effects and suppressed the growth of KB cells overexpressing TP (KB/TP cells) transplanted into nude mice. In this study, we examined the ability of l-dRib to suppress metastasis of KB/TP cells using two different models of metastasis. The antimetastatic effect of l-dRib was first investigated in a liver-metastasis model in nude mice inoculated with KB/TP cells. Oral administration of l-dRib for 28 days at a dose of 20 mg/kg/day significantly reduced the number of metastatic nodules in the liver and suppressed angiogenesis and enhanced apoptosis in KB/TP metastatic nodules. Next, we compared the ability of l-dRib and tegafur alone or in combination to decrease the number of metastatic nodules in organs in the abdominal cavity in nude mice receiving s.c. of KB/TP cells into their backs. l-dRib (20 mg/kg/day) was significantly (P < 0.05) more efficient than tegafur (100 mg/kg/day) in decreasing the number of metastatic nodules in organs in the abdominal cavity. By in vitro invasion assay, l-dRib also reduced the number of invading KB/TP cells. l-dRib anti-invasive activity may be mediated by its ability to suppress the enhancing effect of TP and d-dRib on both mRNA and protein expression of vascular endothelial growth factor and interleukin-8 in cultured KB cells. These findings suggest that l-dRib may be useful in a clinical setting for the suppression of metastasis of tumor cells expressing TP.

Targeted Deletion of Both Thymidine Phosphorylase and Uridine Phosphorylase and Consequent Disorders in Mice

ABSTRACT Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP−/− UP−/−) mice. TP activities were inhibited in TP−/− UP−/− mice, and the level of thymidine in the plasma of TP−/− UP−/− mice was higher than for TP−/− mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP−/− UP−/− mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T2 maps in the brain and axonal edema by electron microscopic study of the brain in TP−/− UP−/− mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Differential Effects of Leukocyte Common Antigen-related Protein on Biochemical and Biological Activities of RET-MEN2A and RET-MEN2B Mutant Proteins

Protein-tyrosine-phosphatases (PTPs), in conjunction with protein-tyrosine kinases, play essential regulatory roles in diverse cellular activities by modulating the phosphorylation state of target proteins. Leukocyte common antigen-related (LAR) protein is a widely expressed receptor-type protein-tyrosine-phosphatase that is implicated in the regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. The gene for LAR is localized to human chromosome 1p32, a region frequently deleted in tumors of neuroectodermal origin, including neuroblastoma, pheochromocytoma, and medullary thyroid carcinoma. On the other hand, the RET gene codes for a transmembrane tyrosine kinase and is responsible for the development of multiple endocrine neoplasia (MEN) 2A and 2B. To explore the potential role of LAR in RET tyrosine kinase activity and RET-induced signal transduction, we cotransfected LAR and RET with a MEN2A or MEN2B mutation (designatedRET-MEN2A or RET-MEN2B) into the NIH 3T3 cell line. Here we show that LAR reduces the constitutive tyrosine autophosphorylation and kinase activity of RET-MEN2A but not RET-MEN2B, accompanying a significant decrease of phosphorylation of phospholipase Cγ, AKT, and ERK1/2. Interestingly, LAR expression significantly decreased the levels of disulfide-linked RET-MEN2A dimerization. Moreover, reduced oncogenic activity of RET-MEN2A by overexpression of LAR was observed both by an in vitro colony formation assay and by in vivo tumorigenicity in scid mice. These results thus suggest that LAR may contribute to deactivation of the RET-MEN2A mutant protein and reduction of its oncogenic activityin vivo.

P53 plays an important role in cell fate determination after exposure to microcystin-LR.

Background Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear. Objective We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure. Methods We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-α, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3β. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-α and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR. Conclusions This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.

Multidrug resistance-associated protein MRP-1 regulates dauer diapause by its export activity in Caenorhabditis elegans

Multidrug resistance-associated proteins (MRPs), when overexpressed, confer drug resistance to cancer cells by exporting anti-cancer agents through the cell membrane, but their role in animal development has not been elucidated. Here we show that an MRP homolog regulates larval development in the nematode Caenorhabditis elegans. C. elegans forms a special third-stage larva called a dauer larva under conditions inappropriate for growth. By contrast, we found that mutants in mrp-1, an MRP homolog gene, form dauer larvae even under conditions appropriate for growth, in the background of certain mutations that partially block the insulin signaling pathway. A functional mrp-1::GFP gene was shown to be expressed in many tissues, and the wild-type mrp-1 gene must be expressed in multiple tissues for a wild-type phenotype. Human MRP1 could substitute for C. elegans MRP-1 in dauer larva regulation, and an inhibitor of the human MRP1 transport activity impaired this function, showing that export activity is required for normal dauer larva regulation. Epistasis studies revealed that MRP-1 acts in neither the TGF-β nor the cGMP signaling pathway. mrp-1 mutations enhanced the dauer-constitutive phenotype of mutants in the insulin signaling pathway more strongly than that in other pathways. Thus, MRP-1, through its export activity, supports the induction of the normal (non-dauer) life cycle by the insulin signaling pathway.

Copper-Transporting P-Type ATPase, ATP7A, Confers Multidrug Resistance and Its Expression Is Related to Resistance to SN-38 in Clinical Colon Cancer

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cis-diaminedichloroplatinum (II) (CDDP). In this study, we found that ATP7A transfection of Chinese hamster ovary cells (CHO-K1) and fibroblasts isolated from Menkes disease patients enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10-hydroxy-camptothecin (SN-38), etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more resistant to SN-38 than ATP7A-negative cells. Thus, ATP7A confers resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Copper Transport Systems are Involved in Multidrug Resistance and Drug Transport

Copper is an essential trace element and several copper containing proteins are indispensable for such processes as oxidative respiration, neural development and collagen remodeling. Copper metabolism is precisely regulated by several transporters and chaperone proteins. Copper Transport Protein 1 (CTR1) selectively uptakes copper into cells. Subsequently three chaperone proteins, HAH1 (human atx1 homologue 1), Cox17p and CCS (copper chaperone for superoxide dismutase) transport copper to the Golgi apparatus, mitochondria and copper/zinc superoxide dismutase respectively. Defects in the copper transporters ATP7A and ATP7B are responsible for Menkes disease and Wilsons disease respectively. These proteins transport copper via HAH1 to the Golgi apparatus to deliver copper to cuproenzymes. They also prevent cellular damage from an excess accumulation of copper by mediating the efflux of copper from the cell. There is increasing evidence that copper transport mechanisms may play a role in drug resistance. We, and others, found that ATP7A and ATP7B are involved in drug resistance against the anti-tumor drug cis-diamminedichloroplatinum (II) (CDDP). A relationship between the expression of ATP7A or ATP7B in tumors and CDDP resistance is supported by clinical studies. In addition, the copper uptake transporter CTR1 has also been reported to play a role in CDDP sensitivity. Furthermore, we have recently found that the effect of ATP7A on drug resistance is not limited to CDDP. Using an ex vivo drug sensitivity assay, the histoculture drug response assay (HDRA), the expression of ATP7A in human surgically resected colon cancer cells correlated with sensitivity to 7-ethyl-10-hydroxy-camptothecin (SN-38). ATP7A-overexpressing cells are resistant to many anticancer drugs including SN-38, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), vincristine, paclitaxel, etoposide, doxorubicin (Dox), and mitoxantron. The mechanism by which ATP7A and copper metabolism modulate drug transport appears to involve modulation of drug cellular localization via modulation of the vesicle transport system. In ATP7A overexpressing cells, Dox accumulates in the Golgi apparatus. In contrast, in the parental cells, Dox is localized in the nuclei, where the target molecules of Dox, topoisomerase II and DNA, are found. Disruption of the intracellular vesicle transport system with monensin, a Na+/H+ ionophore, induced the relocalization of Dox from the Golgi apparatus to the nuclei in the ATP7A overexpressing cells. These data suggested that ATP7A-related drug transport is dependent on the vesicle transport system. Thus copper transport systems play important roles in drug transport as well as in copper metabolism. Components of copper metabolism are therefore likely to include target molecules for the modulation of drug potency of not only anti-cancer agents but also of other drugs.