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 ␤-galactosidase activity. The shortterm 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.

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 p38␣ knock-out mice (p38␣ ؊/؊ MEF), whereas they occur within 12 h of serum withdrawal in p38␣ ؉/؉ 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.Bcl-2 family proteins play differential roles in the regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to the cytosol (1, 2). Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator, voltage-dependent anion channel, by ADP/ATP exchange, or by interfering with oxidative phosphorylation during apoptosis (2). The three-dimensional structure of Bcl-2 proteins suggests that they are also capable of forming channels in mitochondrial membranes (3-5).Recent evidence indicates that the antiapoptotic functions of Bcl-2 can be regulated by post-translational modifications, including phosphorylation (6), dimerization (7), and/or proteolytic cleavage (8). There is still controversy over the significance of Bcl-2 phosphorylation, and two opposing models were initially proposed, the "taxol-induced" and the "interleukin-3-induced" Bcl-2 phosphorylation (6). In the first model, Bcl-2 phosphorylation induces a strong inhibition of the antiapoptotic potential and is related to hyperphosphorylation of several kinases, including Raf-1 kinase, but is independent of protein kinase C (9). In the second model, stimulation with growth factors, such as interleukin-3, induces Bcl-2 phosphorylation, which increases the antiapoptotic potential of the protein (10), whereas, in contrast, interleukin-3 deprivation inhibits Bcl-2-phosphorylation and induces cell death. However, subsequent reports clearly show that hyperphosphorylated Bcl-2 cannot be considered a hallmark of the growth/survival mechanism of action and that triggering specific tyrosine kinase receptors decreases ...

Our aim in this work was to define the role of c-Myc in the susceptibility to cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] in human melanoma cells. Two M14 melanoma cell clones obtained by transfection and expressing six to ten times lower c-Myc protein levels than the parental cells and the control clone were employed. Analysis of survival curves demonstrates an increase in CDDP sensitivity in c-Myc low-expressing clones if compared with the control clone and the parental line. The enhanced sensitivity is unrelated to the impairment in enzymatic DNA repair activity. Cell cycle analysis demonstrates that although the control clone is able to completely recover from the CDDP-induced S-G(2)/M block, this arrest is prolonged in c-Myc low-expressing clones and a fraction of cells undergoes apoptosis. Although no changes in P53, Bax, Bcl-2, and Bcl-x(L/S) protein levels are observed, apoptosis is associated with the formation of reactive oxygen species (ROS), activation of caspase-1, caspase-3 and cleavage of the specific caspase substrate poly-ADP-ribose polymerase. The use of the antioxidant N-acetyl cysteine and caspase inhibitors prevents CDDP-induced apoptosis in c-Myc low-expressing clones, demonstrating that ROS, caspase-1, and caspase-3 are required for apoptotic cell death. Moreover, ROS generation depends on caspase-1-like activation because the Ac-YVAD-cho inhibitor abrogates CDDP-induced ROS in the c-Myc low-expressing clones.

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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 ␥-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 ␥-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.