Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

Biological activity of the G-quadruplex ligand RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate) is associated with telomere capping alteration.

This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G(2)/M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and beta-galactosidase activity. The short-term biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.

Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis.

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.

Telomere dysfunction increases cisplatin and ecteinascidin-743 sensitivity of melanoma cells.

The aim of this study was to investigate the role of telomerase function on the chemosensitivity of melanoma cells. To this end, ecteinascidin-743 (ET-743) and cisplatin [cis-diamminedichloroplatinum(II) (CDDP)], two DNA-interacting drugs that invariably cause an arrest in the G(2)/M phase, and 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (LND), a mitochondria-targeting drug inducing a G(1) block, were used. As experimental model, human melanoma clones showing reduced human telomerase reverse transcriptase (hTERT) expression and telomerase activity and characterized by telomere dysfunction were used. Reconstitution of telomerase activity by exogenous hTERT expression improved telomere function and reduced the sensitivity to CDDP and ET-743 without affecting LND susceptibility. The decreased sensitivity to CDDP and ET-743 was mainly caused by the ability of cells to recover from drug-induced damage, evaluated in terms of both chromosomal lesions and cell survival. The ability of hTERT-reconstituted cells to recover from drug-induced damage was attributable to the restoration of cell cycle progression. In fact, the cells without hTERT restoration remained for a prolonged time in the G(2)/M phase, and this cell cycle alteration made irreversible the drug-induced S-G(2)/M block and led to the activation of apoptotic program. On the contrary, the hTERT-reconstituted cells progressed quickly through the cell cycle, thus acquiring the capacity to recover from drug-induced block and to protect themselves from the G(2)/M phase-specific drug-triggered apoptosis.

Glutathione influences c-Myc-induced apoptosis in M14 human melanoma cells.

The objective of this article is to dissect the mechanisms by which the down-regulation of c-Myc induces programmed cell death in melanoma cells. In stable and doxycycline-inducible M14 melanoma cells, down-regulation of c-Myc induced apoptosis subsequent to a decrease in the intracellular reduced glutathione content and a concomitant accumulation of its oxidized form. This redox alteration was associated with a decrease of the enzyme activities of gamma-glutamyl-cysteine synthetase and NADPH-dependent GSSG reductase, as well as a consequent glutathione release in the extracellular medium. Cytochrome c was released into the cytosol at very early stages of apoptosis induction, long before detectable production of reactive oxygen species and activation of caspase-9 and -3. Macroarray analysis revealed that down-regulation of c-Myc produced striking changes in gene expression in the section related to metabolism, where the expression of gamma-glutamyl-cysteine synthetase and GSSG reductase was found to be significantly reduced. The addition of N-acetyl-l-cysteine or glutathione ethyl ester inhibited the apoptotic process, thus confirming the key role of glutathione in programmed cell death induced by c-Myc.

Reconstitution of hTERT restores tumorigenicity in melanoma-derived c-Myc low-expressing clones.

c-Myc is involved in the control of telomerase activity through its ability to induce the expression of the catalytic subunit of the enzyme, the human telomerase reverse transcriptase (hTERT). Our aim was to study whether telomerase plays a critical role in c-Myc-dependent tumorigenicity of melanoma cells. By using M14-derived clones, expressing low levels of c-Myc, we demonstrated that the down-regulation of c-Myc reduced cell proliferation rate, cloning efficiency and tumorigenicity and increased the apoptotic rate. Decreased tumorigenic potential correlated with reduced hTERT gene expression, telomerase activity and telomere shortening. Introduction of wild-type hTERT into these cells increased their proliferation rate and partially re-established their tumorigenic potential, at early passages, even though the apoptotic rate of the population remained unaltered. After several in vitro passages, hTERT-mediated cell proliferation made the tumorigenic potential of the c-Myc low-expressing clones comparable to that of the M14 parental line. Over-expression of the mutant biologically inactive hTERT did not drive cells to proliferate. In conclusion, our results demonstrate that the reconstitution of high levels of telomerase activity reverses the low tumorigenicity due to low c-Myc expression.