Project #1. Interferon and Cytokine Activation of Early Response Genes: We have developed a cell free system to study Both IFN alpha and IFN gamma activation of the assembly of several transcription complexes. Using these systems we have been able to demonstrate that activation of these complexes required a combination of a membrane associated tyrosine phosphatase(s) and tyrosine kinase(s). In monocyte membranes as well as in intact cells it is possible to directly activate the formation of the IFN alpha induced transcription complex FcRF gamma by the use of the tyrosine phosphatase inhibitor vanadate. This is the first demonstration of any agent which can mimic some of the actions of interferons. We are presently using a combination of antibodies to known components needed for either IFN alpha or IFN gamma induced gene expression to define their biochemical and biophysical interactions. In addition, we have recently obtained evidence that several other cytokines such as GM-CSF, IL-3, IL-5, IL-10, EPO prolactin and growth hormone are functioning by similar mechanisms as the IFNs to activate the expression of early response genes. Recently we have ontained evidence that tyrosine phosphorylated DNA-binding proteins in Drosophila. Efforts are now underway to clone this protein from Drosophila to proceed with developmental and gentic studies. Project #2: Modulation of IFN Signaling by Growth Factors and transformation. Phorbol Esters, expression of the viral of adenovirus E1A or human papilloma virus E6/7 inhibit IFN formation of the ISGF3 transcription complex. We have now characterized several cell lines which are defective in IFN activation of ISGF3 and contain within their cytoplasm a factor actually disrupts the formation of ISGF3. This factor (TKO) is present in human cell lines derived from cervical, hematopoetic, ovarian, colon and small cell lung cancers. TKO has been enriched about 5000 fold and we have purified the protein to near homogeneity. The purified protein is now being sequenced and we hope to have it cloned in the near future.
