Brian J. Druker

Oncogenes, growth factors, and signal transduction

HOW external growth signals are transmitted from the surface of the cell to the nucleus is one of the fundamental problems of cell biology. Research in the field of signal transduction has been mot...

VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo‐oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control.

Characterization of pp85, a target of oncogenes and growth factor receptors.

An 85,000-molecular-weight polypeptide (85K polypeptide) has previously been identified as a common substrate for tyrosine phosphorylation upon polyomavirus middle T transformation or upon platelet-derived growth factor stimulation of 3T3 cells. In each case, pp85 has an associated phosphatidylinositol kinase activity. The tissue distribution of pp85 was determined by middle T blotting experiments; the highest levels were found in brain, lung, and spleen tissues. High-resolution examination of 85K by isoelectric focusing demonstrated that there are at least 10 different forms. These were resolved into two families, 85K and 86K; the ratio of the two families changed in different cells. Similar forms were found for pp85 associated with pp60v-src. Individual species within each family differed by phosphorylation. Analysis of pp85 and pp86 by immunoprecipitation with anti-phosphotyrosine antibody showed increasing phosphorylation in response to middle T or pp60v-src transformation. The association of middle T with pp85 and pp60c-src was examined in pulse-chase experiments. Association of middle T with pp60c-src was slow and was accompanied by progressive modification of middle T. pp85 formed a dissociable complex with middle T within 2.5 min.

Absence of constitutive EGF receptor activation in ovarian cancer cell lines.

Previous investigators have noted that certain ovarian cancer cell lines secrete and respond to transforming growth factor-alpha (TGF-alpha), suggesting that endogenous activation of the epidermal growth factor (EGF) receptor through autocrine or paracrine mechanisms might contribute to the proliferative response. In order to determine whether autocrine stimulation was partly responsible for the proliferative response in ovarian cancer, we investigated whether the EGF receptor expressed by ovarian cancer cell lines was constitutively activated as assessed by the presence of tyrosine phosphorylation. A specific anti-phosphotyrosine antibody was used in conjunction with an immunoblotting technique in order to detect EGF receptor phosphorylation in ovarian cancer cell lines in the absence and presence of exogenous EGF. The effects of neutralising anti-EGF receptor antibody on the proliferation of ovarian cancer cell lines was also examined. We found no evidence for constitutive tyrosine phosphorylation of the p170 EGF receptor in eight epithelial ovarian cancer cell lines tested, although each line demonstrated inducible phosphorylation in response to exogenous EGF. The absence of constitutive EGF receptor activation was also noted when cells were grown under high density conditions, thus excluding a role for membrane-bound EGF or TGF-alpha in this process. Media conditioned by five ovarian cancer cell lines, as well as malignant ascites obtained from 12 different ovarian cancer patients, were not capable of stimulating EGF receptor phosphorylation. Finally, the proliferation of ovarian cancer cell lines was not significantly inhibited in the presence of neutralising anti-EGF receptor antibody. These data suggest that EGF receptor activation through autocrine pathways is not a major mechanism for the growth of many ovarian cancer cell lines. Other pathways of signal transduction which bypass the requirement for EGF receptor activation may be important in the proliferation for ovarian cancer cells. Such EGF receptor-independent pathways may limit the effectiveness of strategies designed to inhibit ovarian cancer cell growth through disruption of EGF receptor function.

Chlorambucil, vinblastine, procarbazine, and prednisone. An effective but less toxic regimen than MOPP for advanced‐stage Hodgkin's disease

The toxicity of MOPP chemotherapy, including nausea, vomiting, hair loss, and neuropathy, can limit patient compliance. Alternative regimens employing oral alkylating agents and vinblastine have been designed to ameliorate these toxicities. The authors reviewed their experience in 24 patients with advanced‐stage Hodgkins disease who were treated with chlorambucil, vinblastine, procarbazine, and prednisone (ChlVPP). Complete responses were obtained in 92% (11/12) of previously untreated patients and in 92% (11/12) of patients who relapsed after radiation (10/10) or chemotherapy (one of two). Overall, relapse‐free survival is 82% with a median duration of follow‐up of 5.5 years. Toxicity was minimal with myelo‐suppression being the dose‐limiting toxicity. Severe nausea and vomiting occurred in only two patients and was considered secondary to procarbazine. Mild nausea occurred in six other patients. Minimal alopecia was seen in three patients and only two patients developed a mild peripheral neuropathy. The authors conclude that ChlVPP appears as effective as MOPP chemotherapy for Hodgkins disease in comparable presentations but is a less toxic regimen. Thus, it may be useful in situations where poor compliance and patient acceptance may compromise optimal dose and frequency of drug administration.