The effects of telomere shortening on cancer cells: a network model of proteomic and microRNA analysis.

Previously, we have shown that shortening of telomeres by telomerase inhibition sensitized cancer cells to cisplatinum, slowed their migration, increased DNA damage and impaired DNA repair. The mechanism behind these effects is not fully characterized. Its clarification could facilitate novel therapeutics development and may obviate the time consuming process of telomere shortening achieved by telomerase inhibition. Here we aimed to decipher the microRNA and proteomic profiling of cancer cells with shortened telomeres and identify the key mediators in telomere shortening-induced damage to those cells. Of 870 identified proteins, 98 were differentially expressed in shortened-telomere cells. 47 microRNAs were differentially expressed in these cells; some are implicated in growth arrest or act as oncogene repressors. The obtained data was used for a network construction, which provided us with nodal candidates that may mediate the shortened-telomere dependent features. These proteins' expression was experimentally validated, supporting their potential central role in this system.

Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells-multiple regulatory pathways in vitro and ex vivo.

BACKGROUND: The importance of telomerase in multiple myeloma (MM) is well established; however, its response to bortezomib has not been addressed. METHODS: The effect of bortezomib on telomerase activity and cell proliferation was evaluated in four MM cell lines and in myeloma cells obtained from eight patients. The mechanism of telomerase regulation on epigenetic, transcriptional, and post-translational levels was further assessed in two selected cell lines: ARP-1 and CAG. Clinical data were correlated with the laboratory findings. RESULTS: Bortezomib downregulated telomerase activity and decreased proliferation in all cell lines and cells obtained from patients, albeit in two different patterns of kinetics. ARP-1 cells demonstrated higher and earlier sensitivity than CAG cells due to differential phosphorylation of hTERT by PKCα. Methylation of hTERT promoter was not affected. Transcription of hTERT was similarly inhibited in both lines by decreased binding of SP-1 and not of C-Myc and NFκB. The ex vivo results confirmed the in vitro findings and suggested existence of clinical relevance. CONCLUSION: Bortezomib downregulates telomerase activity in MM cells both transcriptionally and post-translationally. MM cells, both in vitro and in patients, exhibit different sensitivity to the drug due to different post-translational response. The effect of bortezomib on telomerase activity may correlate with resistance to bortezomib in patients, suggesting its potential utility as a pre-treatment assessment.

Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro.

UNLABELLED: Little is known about the interaction of novel anticancer drugs with other treatment modalities. THE AIM of this study was to examine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell lines - SKNMC cells derived from Ewing sarcoma and breast cancer MCF-7 cells. METHODS: Cell proliferation was determined using the sulphorodamine B cytotoxicity assay. Cell cycle analysis was performed with flow cytometry. Apoptosis was determined using a commercial cell death ELISA plus kit. Phosphorylated AKT, which has been suggested to be involved in radiation resistance, was detected by Western blot analysis. RESULTS: Exposure of SKNMC cells to STI-571 resulted in a dose-dependent antiproliferative effect and a decrease in phosphorylated AKT expression. There was no evidence of apoptosis. The combination of STI-571 with radiation or cisplatin had an additive antiproliferative effect in SKNMC cells (60% reduction in cell number). A similar effect was observed in human MCF-7 breast cancer cells. CONCLUSION: STI-571 improves the outcome of cisplatin or irradiation treatment in vitro. AKT pathway may play a role in the additive effect of STI-571 and irradiation.

Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomerase-expressing cell lines.

Imatinib mesylate (IM) is a tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. It has proved beneficial in treating patients with chronic myeloid leukaemia (CML). In addition, IM demonstrates activity against malignant cells expressing c-kit and platelet-derived growth factor receptor (PDGF-R). The activity of IM in the blastic crisis of CML and against various myeloma cell lines suggests that this drug may also target other cellular components. In the light of the important role of telomerase in malignant transformation, we evaluated the effect of IM on telomerase activity (TA) and regulation in various malignant cell lines. Imatinib mesylate caused a dose-dependent inhibition of TA (up to 90% at a concentration of 15 microM IM) in c-kit-expressing SK-N-MC (Ewing sarcoma), SK-MEL-28 (melanoma), RPMI 8226 (myeloma), MCF-7 (breast cancer) and HSC 536/N (Fanconi anaemia) cells as well as in ba/F3 (murine pro-B cells), which do not express c-kit, BCR-ABL or PDGF-R. Imatinib mesylate did not affect the activity of other DNA polymerases. Inhibition of TA was associated with 50% inhibition of proliferation. The inhibition of proliferation was associated with a decrease in the S-phase of the cell cycle and an accumulation of cells in the G2/M phase. No apoptosis was observed. Inhibition of TA was caused mainly by post-translational modifications: dephosphorylation of AKT and, to a smaller extent, by early downregulation of hTERT (the catalytic subunit of the enzyme) transcription. Other steps of telomerase regulation were not affected by IM. This study demonstrates an additional cellular target of IM, not necessarily mediated via known tyrosine kinases, that causes inhibition of TA and cell proliferation.

Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating Protein (TRAP).

Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.

Psychosocial versus biomedical risk factors in Kevorkian's first forty-seven physician-assisted deaths.

This article examines biomedical and psychosocial data on the first forty-seven cases of physician-assisted suicide (PAS) of Kevorkian as collected by means of both a physical autopsy and a preliminary psychological autopsy. The following patterns emerge: 1) The physical condition of these PAS patients was not typical of the conditions that lead to death in the United States. 2) Consistent with the above findings, our pilot data indicate that only 31.1 percent of these patients were terminal. While 73.9 percent were described as reporting pain, only 42.6 percent were revealed at autopsy to have a specific anatomical basis for their pain. However 36 percent were described as depressed, 66 percent as having some disability, and perhaps of key importance, 90 percent expressed a fear of dependency. Most important, our pilot data suggest the possibility of large gender differences, since 3) 68.1 percent of these forty-seven PAS's are women and only 31.9 percent are men. This represents the reverse of the gender pattern for completed suicides in the United States in 1995, resembling instead the approximate pattern for unsuccessful suicide attempts. 4) Approximately 75 percent of both men and women in the above sample were described as reporting pain. Men were almost twice as likely to have had an anatomical basis for the pain and three times as likely to be terminal. Our pilot data indicate PAS women are more likely to be described as depressed and twice as likely to have had a history of previous unsuccessful suicide attempts. 5) Kevorkian's patients were older than the typical unaided suicides in America. Reported pain decreases with age as does depression; however anatomical basis for pain increases slightly with age, and no age effect emerges for terminality. 6) Approximately two-thirds of those physician-assisted suicides were at middle SES levels. History of disability was the biggest risk factor for the low SES patients and fear of dependency for the high SES patients.