An RGD containing peptide from HIV-1 Tat-(65-80) modulates protooncogene expression in human bronchoalveolar...

Tat (transactivator of transcription) is essential for HIV-1 replication in vivo and in vitro. Tat-(65-80), an RGD containing domain, has been shown to regulate proliferative function of a variety of cell lines, including a human adenocarcinoma cell line, A549. The exact cellular and molecular mechanisms by which these effects are mediated, remain unknown. To evaluate the hypothesis that Tat-(65-80) modulates the expression of immediate early genes (IEG) c-jun, c-myc, c-fos and the tumor suppressor gene p53, serum starved A549 cells were incubated with Tat-(65-80) or heat-inactivated Tat-(65-80) at 10 ng/ml. Total cellular RNA was isolated from the cells at various time points (0-24 hours). In each case, 5 micrograms of RNA was reverse transcribed in 20 microliters of reaction volume. Equal amounts of cDNA were subjected to polymerase chain reaction (PCR) and analyzed by electrophoresis. The photographic negatives of the ethidium bromide stained gels were quantitated by densitometric scanning and normalized to corresponding beta-actin PCR products. Treatment with Tat-(65-80) showed a twofold induction of c-jun at 0.5 h. Peak expression occurred at 60 minutes and remained above baseline at 24 hours (h). c-myc was increased at 0.5 h, reached a twofold increase at 2 h and remained above baseline at 24 h. c-fos increased seven fold at 0.5 h and declined subsequently to baseline at 8 h. p-53 gene was reduced fivefold at 0.5 h and remained downregulated thereafter. These results show that Tat-(65-80) can modulate growth related genes in human lung epithelial cells.