In the present study, effect of Na-Bu on the pRb phosphorylation was analysed in the primary cultures of 12 VS tumors. Primary cultures of VS tumors were established from the fresh tumor tissues removed surgically and were treated with Na-Bu. Na-Bu treatment for 48 h led to morphological changes and apoptotic cell death in VS tumor cells. Na-Bu treatment decreased level of total pRb and phosphorylated form of pRb and caused specific dephosphorylation at Ser 249/Thr 252 and Ser 567. In the untreated and Na-Bu treated cells (when present), pRb was localised in the nucleus. Moreover, in Na-Bu treated cells the nucleus appeared highly condensed as compared to untreated cells. Results of the present study indicated that Na-Bu treatment modulated pRb phosphorylation status and caused apoptotic cell death in VS tumors.

Cell death by apoptosis mediates several important physiologic and pathologic processes and appears to be intrinsically programmed. Its characteristic features are distinctive morphologic changes of nucleus and cytoplasm, along with cleavage of chromatin at regularly spaced sites. Here we study DNA organization and nuclear structure in apoptotic thymocytes to define the cleavage event and, by implication, the role of the responsible endonuclease. We show that in apoptosis, double-stranded cleavage of DNA generates two classes of chromatin fragments: 70% of DNA exists as long, H1-rich oligonucleosomes bound to the nucleus, and 30% comprises short oligonucleosomes and mononucleosomes, which are depleted in H1, enriched in HMG1 and 2, and not attached to the nucleus. This minority class probably derives from chromatin in a transcriptionally active configuration, which would allow better access to enzymes in the nucleoplasm, producing more complete digestion. The characteristic nucleolar morphology in apoptosis can also be explained in terms of cleavage of the transcriptionally active ribosomal genes, with conservation of the nucleolin-rich fibrillar center. The chromatin cleavage, nucleolar morphologic changes, and chromatin condensation were closely mimicked by micrococcal nuclease digestion of normal thymocyte nuclei in the presence of protease inhibitors. Thus, in apoptosis, selective activation of an endogenous endonuclease appears to be responsible not only for widespread chromatin cleavage but also for the major nuclear morphologic changes.

Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.

In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.

Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

Treatment of human HL-60 or KG1A leukemia cells with the topoisomerase II inhibitor etoposide resulted in extensive DNA degradation. When DNA integrity was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident 1.5-2 h after addition of etoposide to cells, increased in intensity over 6 h, and persisted at 24 h. Six h after addition of the drug, 94 +/- 4% of the cells excluded trypan blue even though as much as 90% of the DNA had been degraded to oligosomal fragments. Exposure of cells to 10 micrograms/ml (17 microM) etoposide for as little as 45 min was sufficient to induce this DNA damage 4 h later. Preincubation with dinitrophenol abolished the effect of etoposide, suggesting that an energy-requiring step occurred prior to or during the endonucleolytic cleavage. In contrast, the effect of etoposide was not prevented by preincubation of HL-60 cells with the RNA synthesis inhibitor 5,6-dichloro-1-beta-ribofuranosylbenzimidazole or the protein synthesis inhibitors cycloheximide or puromycin. On the contrary, high concentrations of 5,6-dichloro-1-beta-ribofuranosylbenzimidazole, cycloheximide, or puromycin by themselves induced the same endonucleolytic cleavage, as did a variety of diverse cytotoxic agents, including camptothecin (0.1 microM), colcemid (0.1 microgram/ml), cis-platinum (20 microM), methotrexate (1 microM), and 1-beta-D-arabinofuranosylcytosine (3 microM). These results suggest that endonucleolytic DNA damage by a preexisting cellular enzyme occurs soon after treatment of HL-60 cells with any of a variety of cytotoxic agents. The observation that a variety of nuclear proteins [including poly(ADP-ribose) polymerase, lamin B, topoisomerase I, topoisomerase II, and histone H1] are degraded concomitant with the DNA fragmentation calls into question the selectivity of the degradative process for DNA. The implications of these results for (a) current theories which focus upon endonucleolytic damage of DNA as a critical early event during cell death, and (b) use of topoisomerase-directed drugs to map topoisomerase-binding sites in active chromatin are discussed.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.

The antiapoptosis potential of Bcl-2 protein is well established, but the mechanism of Bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid or the chemotherapeutic drug taxol, we found that Bcl-2 was phosphorylated in lymphoid cells. Phospho amino acid analysis revealed that Bcl-2 was phosphorylated on serine. Under similar conditions, okadaic acid or taxol treatment led to the induction of apoptosis in these cells. Thus, phosphorylation of Bcl-2 seems to inhibit its ability to interfere with apoptosis. In addition, phosphorylated Bcl-2 can no longer prevent lipid peroxidation as required to protect cells from apoptosis.

Sodium butyrate, at millimolar concentrations, when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins, including enzymes, hormones, hemoglobin, inhibition of cell differentiation, reversion of transformed characteristics of cells to normal morphological and biochemical pattern, increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation, from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components.