We previously reported that the rat organic cation transporter rOCT1 could transport the nucleoside analog deoxytubercidin (dTub) (Chen R, Nelson JA. Biochem Pharmacol 2000;60:215-9). The cationic form of dTub (dTub(+)) appeared to be the true substrate of rOCT1. We also reported that although rOCT2 is similar to rOCT1, it does not transport dTub at pH 7.4. In this study, we measured the K(m) and V(max) values of dTub(+) uptake at a reduced pH (pH 5.4) for both rOCT1 and rOCT2. The difference in substrate activity appears due, in large part, to a poor affinity of rOCT2 for dTub(+). The transport efficiency estimated by V(max)/K(m) values for rOCT2 was only 6% that of rOCT1. Chimeras constructed between rOCT1 and rOCT2 revealed that the difference in dTub binding lies within transmembrane domains 2-7. To evaluate the potential of OCT1 in the renal secretion of dTub, tissue distribution and urinary excretion of dTub in OCT1 knockout mice were measured. No significant difference was observed in renal elimination, plasma level, and tissue distribution of dTub between the knockout and the wild-type mice. Therefore, dTub is a good substrate for OCT1; however, OCT1 does not appear to be necessary for its renal secretion.

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.

The failure of available anticancer drugs to cure the common human solid tumors is thought to be caused by their poor selective toxicity. As demonstrated herein, suicide gene therapy offers the potential to improve selective toxicity.

The bystander effect, in which unmodified cells are killed as the result of enzyme-prodrug activation in genetically modified neighboring cells, amplifies the suicide response in a tumor in which only a fraction of the cells are targeted. The drug verapamil (VRP), a calcium channel antagonist that is also used to counteract the multidrug resistance of tumor cells, is shown to inhibit the bystander effect by herpes simplex virus thymidine kinase (HSVtk) enzyme-prodrug therapy with ganciclovir by protecting beta geo marked bystander cells in both in vitro coculture assays and in an in vivo animal tumor model. VRP had no stimulatory or inhibitory effect on the proliferation of CT 26 cells, their tumorigenicity, or prodrug-activated cell death produced by the action of the HSVtk gene. The kinetics of the protection afforded by VRP was time dependent with respect to the time of addition of the prodrug, and protection was ineffective when added two or more days after prodrug administration.

Cysteine conjugate beta-lyase, an enzyme that converts cysteine S-conjugates to free thiols, pyruvate and ammonia, is normally expressed primarily in the liver and kidney. In theory, this selective distribution affords the opportunity to target thiol-containing drugs to these organs and, perhaps, to tumors derived from them. To assess the potential for delivery of such drugs to kidney-derived tissue, we have used a typical beta-lyase substrate, S-(2-benzothiazolyl)-L-cysteine, to measure the beta-lyase activity in normal and tumor tissue of kidneys removed from patients with renal carcinoma. Although considerable heterogeneity in enzyme activity levels was observed in normal and tumor-derived samples, a high proportion of tumor samples had enzyme activity that was at least 50% of that observed in adjacent normal tissue. Frequently, hypoxanthine-guanine phosphoribosyltransferase activity was observed to be greater in the tumor than in normal tissue. These results may aid in the development of therapy for renal carcinomas.

Cimetidine has been used as a relatively selective inhibitor of renal organic cation secretion, analogous to the use of probenecid to inhibit organic anion secretion. Many of the substrates for the multidrug transporter P-glycoprotein, which is overexpressed in multidrug-resistant tumor cells, are organic cations. Furthermore, the protein is normally expressed on the apical membranes of proximal tubule cells, the postulated site for active organic cation secretion. To test directly whether P-glycoprotein might serve as a carrier for cimetidine, we measured cimetidine transepithelial movement across Madin-Darby canine kidney cells grown as monolayers on membrane filters. A retrovirally transduced Madin-Darby canine kidney cell line (Madin-Darby canine kidney cells transfected with the human multiple drug resistance 1 cDNA for P-glycoprotein), that expresses the human form of P-glycoprotein on its apical membrane, had an increased capacity to transport cimetidine from the basolateral to apical medium (b-->a) but not in the reverse direction (i.e., a-->b). Qualitatively similar results were observed with daunomycin, a well established substrate for P-glycoprotein. Cellular uptake and energy-dependent efflux experiments further established cimetidine to be a substrate for the human P-glycoprotein. Thus, P-glycoprotein may play a role in the renal secretion of cimetidine and perhaps other organic cations.

2'-Deoxytubercidin is secreted by the organic cation secretory system of mouse kidney. The presented data indicate that this secretion occurs by a transport system other than the MDR1 P-glycoprotein.

6-Thioguanine (TG) incorporation into DNA has been associated with cytotoxicity and DNA damage in Chinese hamster ovary (CHO) and murine leukemia L1210 cells. According to alkaline elution analysis, single-strand breaks (SSB) occur in both cell types. DNA-protein and interstrand crosslinks are prominent features of TG effects in L1210, CEM, and HL-60 but not CHO cells. To assess which DNA strand experiences SSB in CHO cells, the cells were synchronized by growth to confluence (late g1, S). The cells were then diluted into fresh medium so that they underwent a round of division during a subsequent 16-hr interval. They were treated with TG during this first cell cycle, and mitotic cells were harvested at the end of the first cycle using colcemid. SSB were determined in parental DNA (radiolabeled with thymidine during growth to confluence), TG-containing DNA (radiolabeled with [14C]TG during drug exposure), and daughter DNA (labeled with thymidine during the second cell cycle). SSB occurred in TG-containing DNA late in the second cell cycle after drug exposure and in the DNA synthesized from a TG-DNA template (daughter DNA). This observation is consistent with the known delayed cytotoxicity and chromosomal aberrations seen in CHO cells. The SSB suggest relatively normal elongation of DNA containing TG but altered synthesis and/or ligation from a TG-DNA template. This premise was tested in synchronized CHO cells. The DNA strand incorporating TG elongated naturally; however, DNA elongation was impaired in the cell cycle following TG treatment. The results are consistent with SSB in daughter DNA synthesized from a TG-DNA template due to inability to elongate the newly-synthesized strand.

Inherited human immunodeficiences associated with loss of adenosine deaminase or purine nucleoside phosphorylase are thought to result from accumulation of the nucleoside substrates. This work attempts to identify lymphocyte subpopulations that are uniquely-sensitive to the endogenous substrates or their analogs.