Using the yeast two-hybrid system we have identi®ed novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein speci®cally interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4 and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by aecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

Insulin treatment of adipocytes causes the rapid phosphorylation of the insulin receptor substrate-1 (IRS-1) on tyrosine. The phosphotyrosine [Tyr(P)] form of IRS-1 then complexes with the enzyme phosphatidylinositol (PI) 3-kinase. In this study, we have investigated the effect of this association on PI 3-kinase activity in 3T3-L1 adipocytes. Insulin stimulated cytosolic PI 3-kinase activity about sevenfold. This stimulation was maximal after 1 min of exposure of cells to insulin, persisted for at least 1 h, and occurred over the range of insulin concentrations that saturate its receptor. By means of immunoprecipitation of IRS-1, it was shown that virtually all of the enhanced activity was due to PI 3-kinase complexed with IRS-1. Moreover, the purified Tyr(P) form of IRS-1, either isolated from 3T3-L1 adipocytes or obtained by phosphorylation of the recombinant protein with the insulin receptor, markedly stimulated the activity of purified rat liver PI 3-kinase. These results show that the association of Tyr(P) IRS-1 with PI 3-kinase activates the enzyme and thereby can explain the elevation of PI 3,4-bisphosphate and PI 3,4,5-trisphosphate in vivo observed upon treatment of adipocytes with insulin.

The phosphotyrosine form of the major substrate for the insulin receptor tyrosine kinase, insulin receptor substrate 1 (IRS-1), associates with and activates the enzyme phosphatidylinositol 3'-kinase (PtdIns 3'-kinase). IRS-1 contains nine potential tyrosine phosphorylation sites within YMXM or YXXM sequences known to bind to the two SH2 domains on the 85-kDa regulatory subunit of PtdIns 3'-kinase. We used sequences within IRS-1 as a model for synthesizing phosphotyrosine and nonhydrolyzable phosphonotyrosine peptides containing two YMXM motifs and tested them for their ability to bind to the SH2 domains of PtdIns 3'-kinase and stimulate its activity. We demonstrated for the first time that IRS-1-derived peptides containing two tyrosine phosphorylated YMXM motifs are capable of stimulating PtdIns 3'-kinase activity in the cytosol of 3T3-L1 adipocytes at nanomolar concentrations, similar to that required by purified phosphoryl-IRS-1 [Lamphere, M., Carpenter, C. L., Sheng, Z., Kallen, R. G., & Lienhard, G. E. (1994) Am. J. Physiol. 266 (Endocrinol. Metab. 29), E486-E489] and the extent of activation by these peptides was similar to that seen by maximal stimulation of cells with insulin. In contrast, those phosphotyrosine peptides containing only a single YMXM motif were able to stimulate PtdIns 3'-kinase activity only at concentrations over 10 microM. We conclude from these results that the high-affinity activation of PtdIns 3'-kinase requires the simultaneous binding of two phosphorylated YMXM motifs on IRS-1 to the two SH2 domains of PtdIns 3'-kinase.

Cyclin-dependent kinase inhibitors (CDKi's) may be useful to treat hyperproliferative vascular disorders, such as restenosis induced following angioplasty or vein engraftment. We have shown that a novel fusion protein of the CDKi's p27 and p16, named W9, significantly reduces proliferation of human coronary smooth muscle cells in vitro, by blocking cell proliferation without inducing apoptosis. We have now evaluated the efficacy of adenovirus-mediated gene transfer of W9 (AV-W9) in a balloon-injury model, in the carotid arteries of cholesterol-fed rabbits. We observed that intravascular delivery of 2 x 10(11) viral particles of AV-W9 3 days following balloon injury inhibited intimal hyperplasia by 60% compared to a control virus (P > 0.001). PCNA expression in the AV-W9-treated vessels, a marker of injury-induced cell proliferation, was also reduced compared to the control virus-treated vessels. Direct comparison of the efficacy of AV-W9 and AV-p16 and AV-p27 in this model indicated that delivery of either of the parental genes was significantly less effective in inhibiting intimal thickening compared to the AV-W9 treatment. We conclude that combining the activities of multiple cell cycle regulatory proteins greatly increases the potency of cytostatic gene therapy in the treatment of balloon injury-induced intimal hyperplasia and represents a promising potential approach to preventing postangioplasty restenosis.