Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.