Thiol redox modulation of tumor necrosis factor-α responsiveness in cultured AIDS-related Kaposi's sarcoma cells

Abstract

Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). While virtually every cell responds to TNF-alpha with gene activation, the extent of TNF-alpha-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-alpha membrane interaction, are part of this TNF-alpha-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-alpha. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-alpha challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV normal fibroblasts. In contrast, following TNF-alpha challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-alpha-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-alpha challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-alpha, suggest a biochemical rationale for the recognized TNF-alpha AIDS-KS clinical correlation.

Thiol redox modulation of doxorubicin mediated cytotoxicity in cultured AIDS-related Kaposi's sarcoma cells

“…Consistent with our previous findings [Mallery et al, 1994[Mallery et al, , 1995[Mallery et al, , 1998], GSH levels were determined to be significantly higher in healthy HIV Ϫ donor fibroblasts relative to both KS lesional or nonlesional cells (Fig. 4A,B).…”

“…The AIDS-KS cells were isolated from the tissue specimen as previously described. [Mallery et al, 1994[Mallery et al, , 1995[Mallery et al, , 1998]. Prior to the doxorubicin challenge assays, the AIDS-KS cells were cultured in ''COMPLETE'' medium, which consisted of: M-199 (GIBCO, Grand Island, NY), supplemented with 15 mM HEPES, 0.23 mg/ml l-glutamine, 11 µg/ml Na pyruvate, sodium heparin (Sigma, St. Louis, MO; 90 µg/ml) endothelial cell growth supplement (prepared inhouse, from bovine brain, 150 µg/ml), 15% heat inactivated fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated pooled, male human serum (Sigma).…”

“…Cellular levels of nucleotides and nucleosides were determined by high performance liquid chromatography (HPLC), by a modification of the method of Geisbuhler et al [1984], as reported in our publications [Mallery et al, 1993[Mallery et al, , 1994[Mallery et al, , 1995[Mallery et al, , 1998]. Nucleotide and nucleoside results are expressed as nmol/mg protein.…”

“…[Mallery et al, 1994[Mallery et al, , 1995 Further, our laboratory has recently shown that as a consequence of an inherent perturbation in their cellular thiol redox status, AIDS-KS cells show an accentuated response to tumor necrosis factor ␣, a cytokine which employs reactive oxygen intermediates (ROI) in its signal transduction pathway [Mallery et al, 1998]. Collectively our data, which show that ROI persist longer in AIDS-KS cells, suggests that AIDS-KS cells should therefore demonstrate enhanced susceptibility to redox active agents such as doxorubicin [Mallery et al, 1994[Mallery et al, , 1995[Mallery et al, , 1998]. This current study was conducted to investigate the effects of doxorubicin on the cellular thiol redox bioenergetic status of three defined cellular populations, i.e., KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV Ϫ aged matched men.…”

Selenium and Interleukins in Persons Infected with Human Immunodeficiency Virus Type 1

“…In HIV-infected patients, TNF appears to play a major role in the pathogenesis of Kaposi's sarcoma. Several clinical studies have found a positive correlation between high levels of TNF and the presence of Kaposi's sarcoma [24,25]. Recent studies suggest that the effect of TNF on Kaposi's disease is mediated through the TNF receptor [25].…”