The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.

Dependent on the viral vector and the specific assay used, viral titers produced from commonly used retroviral packaging cell lines have an upper limit in the range of 10(5) to 10(7) infectious units/ml. We have developed a generally applicable method, using hollow-fiber filtration technology, which allows for the concentration of infectious virus derived from packaging lines. This method resulted in a reproducible 10- to 30-fold increase in viral titer and can readily be scaled to accommodate larger input volumes. Over 80% of the input virus is recovered in an infectious form in the concentrate. Concentrated virus containing media was seen to produce higher infection frequencies in Jurkat T cells as compared to unconcentrated virus containing media; however, this was not proportional to the differences in viral titer observed by limiting dilution analysis on NIH-3T3 cells. These results are discussed in relation to the importance of factors other than viral titer in determining transduction frequencies.

The evidence for the multistage nature of tumor promotion in vivo and for multiple subclasses of phorbol ester receptors in vitro argues that multiple mechanisms of tumor promotion exist. The existence of multiple mechanisms suggests that brute force assay for tumor promoters in vivo may be inadequate and that understanding of mechanisms may be essential. The interest in the phorbol esters is not primarily that they are environmental hazards for man, but rather that they provide a probe for phorbol ester receptors. These receptors are found in people, and modulation of their activity may play a role in tumor promotion in man.

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s(4)dU)(35)]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s(4)dU)(35) is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC(50): 0.8-25.4 microg/ml) in vitro. Besides its previously reported anti-RT activity, (s(4)dU)(35) mediated its antiviral action by preventing virus attachment (IC(50): 0.002-0.003 microg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s(4)dU)(35) preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s(4)dU)(35) did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s(4)dU)(35) as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.

No abstract