BACKGROUND/AIM: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a guanine acyclic nucleotide analog whose targeted prodrugs are being investigated for chemotherapy of lymphomas. Its antiproliferative effects have been attributed to cell cycle arrest and induction of apoptosis, however, the underlying mechanisms remain poorly understood. The objective of this study was to determine the requirements for caspase and CD95/Fas activation in PMEG-induced apoptosis. Additionally, the influence of PMEG on cell cycle regulatory proteins was explored. MATERIALS AND METHODS: CCRF-CEM cells were exposed to PMEG with/without caspase inhibitor or anti-Fas blocking antibody and assayed for phosphatidyl serine externalization, mitochondrial depolarization and the cleavage of procaspase 3 and the nuclear protein poly (ADP-ribose) polymerase (PARP). RESULTS: Despite an observed increase of caspase 3, 8 and 9 proteolytic activity, neither pretreatment of the cells with cell-permeable caspase inhibitors nor blocking the death receptor with anti-Fas antibody did prevent apoptosis induced by PMEG. CONCLUSION: PMEG-induced apoptosis is caspase- and CD95/Fas-independent.
- MeSH
- aktivace enzymů MeSH
- antigeny CD95 metabolismus MeSH
- apoptóza účinky léků fyziologie MeSH
- buněčný cyklus účinky léků fyziologie MeSH
- cyklin E biosyntéza genetika MeSH
- cyklin-dependentní kinasy biosyntéza metabolismus MeSH
- guanin analogy a deriváty farmakologie MeSH
- inhibiční proteiny cyklin-dependentních kinas biosyntéza metabolismus MeSH
- kaspasy metabolismus MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie účinky léků fyziologie MeSH
- nádorové buněčné linie MeSH
- onkogenní proteiny biosyntéza genetika MeSH
- organofosforové sloučeniny farmakologie MeSH
- S fáze účinky léků MeSH
- signální transdukce MeSH
- T-lymfocyty cytologie účinky léků enzymologie metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- imunohistochemie metody MeSH
- lidé MeSH
- membránové proteiny analýza biosyntéza imunologie MeSH
- morfogeneze imunologie MeSH
- onkogenní proteiny analýza biosyntéza imunologie MeSH
- vývoj plodu imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- kongresy MeSH
Activation of conditional alleles of Myc can induce proliferation in quiescent cells. We now report that induction of Myc in density-arrested fibroblasts triggers rapid hyperphosphorylation of the retinoblastoma protein and activation of both cyclin D1- and cyclin E-associated kinase activities in the absence of significant changes in the amounts of cyclin-cdk complexes. Kinase activation by Myc is blocked by inhibitors of transcription and requires intact DNA binding and heterodimerization domains of Myc. Activation of cyclin E-cdk2 kinase in serum-starved cells occurs in two steps. The first is induced by Myc and involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa inactive complex that is present in starved cells. This is necessary, but not sufficient, to generate full kinase activity, as cdc25 phosphatase activity is limiting in the absence of external growth factors. In vivo cdc25 activity can be supplied by the addition of growth factors. In vitro recombinant cdc25a strongly activates the 120 kDa, but only poorly activates the 250 kDa cyclin E-cdk2 complex. Our data show that two distinct signals, one of which is supplied by Myc, are necessary for consecutive steps during growth factor-induced formation of active cyclin E-cdk2 complexes in G(o)-arrested rodent fibroblasts.
- MeSH
- aktivace enzymů MeSH
- buněčné linie MeSH
- cyklin D1 MeSH
- cyklin-dependentní kinasa 2 MeSH
- cyklin-dependentní kinasy biosyntéza metabolismus MeSH
- cykliny biosyntéza metabolismus MeSH
- fibroblasty MeSH
- fosfatasy cdc25 MeSH
- fosforylace MeSH
- geny myc MeSH
- kinasy CDC2-CDC28 * MeSH
- krysa rodu rattus MeSH
- messenger RNA biosyntéza MeSH
- onkogenní proteiny biosyntéza metabolismus MeSH
- protein-serin-threoninkinasy biosyntéza metabolismus MeSH
- proteinfosfatasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- protoonkogenní proteiny c-myc fyziologie MeSH
- retinoblastomový protein metabolismus MeSH
- růstové látky krev fyziologie MeSH
- S fáze MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Immunohistochemical staining with a monoclonal antibody against human cyclin D1 can be used to identify breast cancers that have an amplification of the q13 region of chromosome 11. In general, the intensity of staining is directly proportional to the degree of DNA amplification. In two unusual tumors, in which the CCND1 locus is highly amplified but staining is relatively weak, it appears that the DNA has undergone rearrangement and that the amplified/rearranged CCND1 allele may have reduced transcriptional activity. More significantly, the immunohistochemical technique identifies additional tumors in which the cyclin D1 gene is overexpressed with only marginal or undetectable increases in copy number, implying that other mechanisms can lead to deregulated expression. These results suggest that the frequency of overexpression is much higher than previously concluded from DNA-based analyses and that more than one-third of human breast cancers may contain excessive levels of cyclin D1. The technique we describe should facilitate the detection of this abnormality in a clinical setting and clarify its prognostic significance.
- MeSH
- alely MeSH
- amplifikace genu * MeSH
- cyklin D1 MeSH
- cykliny analýza biosyntéza genetika MeSH
- DNA nádorová analýza metabolismus MeSH
- exprese genu MeSH
- genetická transkripce MeSH
- imunohistochemie MeSH
- lidé MeSH
- lidské chromozomy, pár 11 * MeSH
- mapování chromozomů MeSH
- monoklonální protilátky MeSH
- nádorové buňky kultivované MeSH
- nádory prsu metabolismus patologie MeSH
- onkogenní proteiny analýza biosyntéza genetika MeSH
- prognóza MeSH
- RNA nádorová analýza biosyntéza MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
