The human (h) and rat (r) equilibrative (Na ؉ -independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K m values 0.7-2.6 mM), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.Plasma membrane transport processes for nucleosides and nucleobases play key roles in many aspects of mammalian physiology and pharmacology (1-5). In particular, uptake of exogenous nucleosides and nucleobases is the first step of nucleotide synthesis in tissues such as bone marrow and intestinal epithelium (and certain parasitic organisms) that lack de novo pathways for purine biosynthesis (5-7). The same transport processes also mediate cellular uptake of many synthetic nucleoside and nucleobase analogs used in cancer, viral, and parasite chemotherapy (3)(4)(5)8). Independent transport processes specific for nucleosides or nucleobases as well as shared mechanisms of nucleoside and nucleobase transport have been described (2, 4).In human and other mammalian cells and tissues, uptake of nucleosides is brought about by members of the concentrative (Na ϩ -dependent) nucleoside transporter (CNT) 1 and equilibrative (Na ϩ -independent) nucleoside transporter (ENT) families (3, 5). CNTs have been described primarily in specialized epithelia, whereas ENTs occur in most, possibly all, cell types and tissues. Three CNT and two ENT isoforms have been identified. Human (h) and rat (r) CNT1 and CNT2 both transport uridine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (9 -14). In contrast, hCNT3 and its mous...

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met 33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile 33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.

Recently we reported that human dermal fibroblasts, or conditioned media obtained from such cells, affect the growth of human melanoma cells as a direct function of tumor progression: melanoma cells obtained from earlystage (metastatically incompetent) primary lesions were growth inhibited, whereas cells obtained from more advanced (metastaticaly competent) primary lesions, or metastases, were growth stimulated. Ion-exchange and gel-filtration chromatography of fibroblast conditioned medium revealed the inhibitor to be a protein of molecular mass between 20 and 30 kDa and distinct from the stimulator. This is the approximate molecular mass of interleukin 6 (IL-6), a ubiquitous multifunctional cytokine known to affect in particular many kinds of hemopoietic and lymphoid cells. Since this cytokine is known to be made by fibroblasts, we attempted to determine if the human fibroblast-derived growth inhibitor (hFDGI) was identical to IL-6. Neutralizing antibodies specific for IL-6 completely eliminated the inhibitory activity ofhFDGI. Moreover, exposure to human recombinant IL-6 was found to inhibit the growth of earlystage melanoma cells obtained from radial growth phase (RGP) or early vertical growth phase (VGP) primary lesions in three of four cases. In contrast, melanoma cells from a number of more advanced VGP primary lesions, or from distant metastases, were completely resistant to this IL-6-mediated growth inhibition. Acquisition of an "IL-6-resistant" phenotype by metastatically competent melanoma cell variants may provide such cells with a proliferative advantage within the dermal mesenchyme (a hallmark of melanoma cells that are malignant), helping them eventually to dominate advanced primary lesions and to establish secondary growths elsewhere.The growth and spread of cancers can be strongly influenced by surrounding normal tissues in a variety of ways. Tumor angiogenesis-i.e., the process satisfying the absolute requirement of new blood vessel capillaries for solid tumors to grow beyond 1-2 mm in diameter-is perhaps the most striking illustration of this interaction (1). Similarly, a variety of different hormones or locally produced growth factors and cytokines secreted by various normal cells can stimulate tumor growth (2, 3). In addition to stimulating the growth of tumors, surrounding normal cells can, in other circumstances, significantly suppress such growth (4); this is especially evident in the case of an interspersed minority subpopulation of tumor cells surrounded by an excess of normal cells, such as fibroblasts (5).With few exceptions the effects of a given normal cell population on the growth and behavior of a particular type of tumor have not been studied in the context of different stages of tumor progression. It is possible, for example, that the effect of normal adjacent cells on the growth of tumor cells from primary lesions early in tumor progression is quite different from the effects of more advanced (metastatically competent) primary lesions, or metastases. We recently uncover...

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