No abstract

The human immunodeficiency virus type 1 (HIV-1) Tat protein is a key regulatory protein in the HIV-1 replication cycle. Tat interacts with cellular transcriptional factors and cytokines, such as tumor necrosis factor (TNF-alpha), and alters the expression of a variety of genes in HIV-1-infected and noninfected cells. To further elucidate the mechanisms by which HIV-1 Tat amplifies the activity of TNF-alpha, we transfected the HIV-1 tat gene into an epithelial (HeLa) cell line. We observed that Tat-expressing cells had increased NF-kappa B-dependent trans-activational activity due to enhanced NF-kappa B--DNA binding in response to TNF-alpha treatment. Tumor necrosis factor receptor (TNFR) p55 was the prominent receptor, as neutralizing antibodies to TNFR p55, but not to TNFR p75, blocked TNF-alpha-mediated NF-kappa B activation. Furthermore, tat-transfected cells were more sensitive to TNF-alpha-induced cytotoxicity and only the neutralizing antibodies to TNFR p55 completely protected the cells. To determine whether TNFR p55 was involved in amplification of cellular response to TNF-alpha by HIV-1 Tat, we investigated the effect of TNF-alpha on TNFR p55 expression in the tat-transfected cells. TNF-alpha treatment resulted in a reduction in both TNFR p55 mRNA and protein levels in the control cells but not in the tat-transfected cells as determined with Northern blot and Western blot analyses, respectively. Our results indicate that HIV-1 Tat may inhibit TNF-alpha-induced repression of TNFR p55 and thereby amplify TNF-alpha activity in these stably transfected cells.

An immortal line of chemically altered rat hepatocytes was used to study the effects of the liver tumor promoter, phenobarbital (PB), on hepatocyte growth and viability in vitro. When the serum concentration in medium was changed from 10% to 0.5%, cell proliferation decreased and hepatocytes died. Death of the hepatocytes occurred after 2 days in low-serum medium. PB appeared to control the type of cell death that occurred. In the absence of PB in low-serum medium, most dead cells had morphological changes that are characteristic of necrosis as determined by both light and electron microscopy. In the presence of PB, the dead cells had alterations typical of apoptosis. Biochemical features of cell death in low-serum medium were also analyzed. DNA isolated from cells in low serum with PB showed nucleosome-length fragments after gel electrophoresis, whereas DNA from cells in low serum without PB appeared as randomly degraded fragments. Although proliferation of hepatocytes in low-serum decreased by 75%, the appearance of apoptosis in the presence of PB was associated with increased expression of the c-myc gene. Based on these observations, we conclude that PB can modulate the type of cell death that occurs after serum deprivation in this line of immortal rat hepatocytes. PB seemed to prevent necrotic cell death in low serum, and cells died through a gene-directed pathway of apoptosis.

An immortal line of chemically altered rat hepatocytes was used to study the effects of the liver tumor promoter, phenobarbital (PB), on hepatocyte growth and viability in vitro. When the serum concentration in medium was changed from 10% to 0.5%, cell proliferation decreased and hepatocytes died. Death of the hepatocytes occurred after 2 days in low-serum medium. PB appeared to control the type of cell death that occurred. In the absence of PB in low-serum medium, most dead cells had morphological changes that are characteristic of necrosis as determined by both light and electron microscopy. In the presence of PB, the dead cells had alterations typical of apoptosis. Biochemical features of cell death in low-serum medium were also analyzed. DNA isolated from cells in low serum with PB showed nucleosome-length fragments after gel electrophoresis, whereas DNA from cells in low serum without PB appeared as randomly degraded fragments. Although proliferation of hepatocytes in low-serum decreased by 75%, the appearance of apoptosis in the presence of PB was associated with increased expression of the c-myc gene. Based on these observations, we conclude that PB can modulate the type of cell death that occurs after serum deprivation in this line of immortal rat hepatocytes. PB seemed to prevent necrotic cell death in low serum, and cells died through a gene-directed pathway of apoptosis.