Showing papers by "Marco Tafani published in 2006"

The course of etoposide‐induced apoptosis in Jurkat cells lacking p53 and Bax

Abstract: Jurkat T-lymphocytes lack p53 and Bax but contain p73 and Bid and are killed by etoposide (ETO). With ETO c-abl is phosphorylated and phosphorylated p73 increased. Translocation of full-length Bid to mitochondria follows, with induction of the mitochondrial permeability transition (MPT) and release of cytochrome c into the cytosol. Pronounced swelling of mitochondria was evident ultrastructurally, and the MPT inhibitor cyclosporin A prevented the release of cytochrome c. Overexpression of Bcl-2 prevented the translocation of Bid, the release of cytochrome c, and cell death. The pan-caspase inhibitor ZVAD-FMK prevented the cell killing, but not the initial release of cytochrome c. An accumulation of tBid occurred at later times in association with Bid degradation. A sequence is proposed that couples DNA damage to Bid translocation via activation of c-abl and p73. Bid translocation induces the MPT, the event that causes release of cytochrome c, activation of caspases, and cell death.

Re-evaluation of the distinction between type I and type II cells: the necessary role of the mitochondria in both the extrinsic and intrinsic signaling pathways upon Fas receptor activation.

Abstract: Cyclosporin A (CyA) and bongkrekic acid (BK) prevented Fas-induced apoptosis in two type I cell lines (H9 and SKW6.4) and two type II cell lines (Jurkat and CEM). CyA and BK inhibited the release of cytochrome c in all four cell lines. In type I cells and in CEM cells, CyA and BK did not prevent the translocation of Bax to the mitochondria. In these same cells, full-length Bid decreased in the mitochondria and cytosol. The cleavage product of Bid, tBid, appeared in the cytosol and to a lesser extent in the mitochondria. In Jurkat cells, Bid also decreased in the cytosol, but increased in the mitochondria. Similar to the other cells, tBid appeared in the mitochondria and cytosol. In the type I H9 and SKW6.4 cells and type II Jurkat cells, the caspase-8 inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD) prevented the cell killing. In the type I cells, IETD prevented the translocation of Bax, the degradation of Bid and the accumulation of tBid. By contrast, IETD only marginally protected the type II CEM cells. In these cells in the presence of IETD, Bax translocated to the mitochondria, in the absence of any degradation of Bid or accumulation of tBid. In the type I H9 cells, IETD produced a depletion of ATP, an effect that did not occur in the type II CEM cells. It is concluded that in type I cells the extrinsic signaling pathway is mitochondrial dependent to the same extent as is the intrinsic pathway in type II cells.

p27kip1 overexpression promotes paclitaxel-induced apoptosis in pRb-defective SaOs-2 cells.

Abstract: p27kip1 is a cyclin-dependent kinase (CDK) inhibitor, which controls several cellular processes in strict collaboration with pRb. We evaluated the role of p27kip1 in paclitaxel-induced apoptosis in the pRb-defective SaOs-2 cells. Following 48 h of exposure of SaOs-2 cells to 100 nM paclitaxel, we observed an increase in p27kip1 expression caused by the decrease of the ubiquitin-proteasome activity. Such increase was not observed in SaOs-2 cells treated with the caspase inhibitors Z-VAD-FMK, suggesting that p27kip1 enhancement at 48 h is strictly related to apoptosis. Finally, we demonstrated that SaOs-2 cells transiently overexpressing the p27kip1 protein are more susceptible to paclitaxel-induced apoptosis than SaOs-2 cells transiently transfected with the empty vector. Indeed, after 48 h of paclitaxel treatment, 41.8% of SaOs-2 cells transiently transfected with a pcDNA3-p27kip1 construct were Annexin V-positive compared to 30.6% of SaOs-2 cells transfected with the empty vector (P < 0.05). In conclusion, we demonstrated that transfection of the pRb-defective SaOs-2 cells with the p27kip1 gene via plasmid increases their susceptibility to paclitaxel-induced apoptosis. The promoting effect of p27kip1 overexpression on apoptosis makes p27kip1 and proteasomal inhibitors interesting tools for therapy in patients with pRb-defective cancers. J. Cell. Biochem. © 2006 Wiley-Liss, Inc.

Detection of oncogenic HPV and identification of 72Arg polymorphic p53 by in situ PCR for clinical routine purposes

Abstract: Background: Ano-genital carcinoma is a poly- factorial and polygenic disease Certain strains of human papillomavirus (HPV) have been detected in a high percentage of patients It has been suggested that p53 polymorphisms may be relevant for the interaction with viral proteins that inactivates p53 Materials and Methods: Patients were selected on the basis of HPV infection, clinical history, positive PAP test and type of lesion In situ PCR was performed on smear samples, in four steps: a) preparation on clean biobond-treated slides, b) permeabilisation and digestion; c) in situ PCR amplification; d) in situ hybridisation with a fluorescent probe Results: In situ PCR analysis of the smears confirmed the results obtained by classic PCR and by in situ PCR of frozen sections Conclusion: In situ PCR on smears could be used in targeted-screening for young and post-menopausal women, as well as in the development of large scale studies to establish the connection among the presence of HPV, p53 poly- morphisms and the risk of cervical cancer Human papillomavirus (HPV) is associated with proliferative disorders, including condylomata and carcinoma of the cervix, bladder, penis and anus and papilloma of the larynx (1-6) Oncogenic HPV can be detected in 98% of high-grade lesions (7), in most invasive carcinoma of the cervix (8, 9) and in 60-80% of head and neck carcinoma (10, 11) This association is not a simple cause-effect relationship, but is conditioned by a number of factors related to the host and/or virus A routine and balanced evaluation of such