For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by gamma-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.

• MeSH
• apoptóza účinky záření   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin účinky záření   MeSH
• chromatinová imunoprecipitace MeSH
• DNA účinky záření   MeSH
• fibroblasty cytologie   metabolismus   účinky záření   MeSH
• fluorescenční protilátková technika MeSH
• G1 fáze fyziologie   účinky záření   MeSH
• histondeacetylasy metabolismus   MeSH
• hybridizace in situ fluorescenční MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory histondeacetylas MeSH
• kůže cytologie   metabolismus   účinky záření   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• oprava DNA účinky záření   MeSH
• poškození DNA účinky záření   MeSH
• radioizotopy kobaltu MeSH
• regulace genové exprese účinky záření   MeSH
• S fáze fyziologie   účinky záření   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas the abundance of most of the CKIs, including p27Kip1, p57Kip2, p15ink4b and p18ink4c, was relatively maintained in the migrating epithelial tongue. These data indicate that downmodulation of several G(1)/S-phase cyclins and a relative excess of CKIs may cooperate to ensure the quiescent state of migrating keratinocytes during wound healing.

• MeSH
• cykliny metabolismus   MeSH
• dospělí MeSH
• epitelové buňky fyziologie   metabolismus   MeSH
• G1 fáze fyziologie   MeSH
• hojení ran * fyziologie   MeSH
• lidé MeSH
• pohyb buněk MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• S fáze fyziologie   MeSH
• ústní sliznice metabolismus   zranění   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH

The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130.

• MeSH
• buněčné linie MeSH
• cyklin D MeSH
• cyklin E metabolismus   MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• cykliny metabolismus   MeSH
• DNA vazebné proteiny * MeSH
• fosfoproteiny * genetika   chemie   metabolismus   MeSH
• fosforylace MeSH
• G1 fáze * fyziologie   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• peptidové mapování MeSH
• protein p130 podobný retinoblastomu MeSH
• proteiny buněčného cyklu * MeSH
• proteiny * MeSH
• retinoblastomový protein * genetika   chemie   metabolismus   MeSH
• transkripční faktor DP1 MeSH
• transkripční faktory E2F MeSH
• transkripční faktory metabolismus   MeSH
• transportní proteiny * MeSH
• vazebná místa genetika   MeSH
• vazebný protein 1 retinoblastomu MeSH
• Check Tag
• lidé MeSH

The Chk2 kinase is a tumor suppressor and key transducer of DNA-damage checkpoints. We show that the human Chk2 protein is relatively stable, nuclear, and responding to gamma-radiation throughout the cell cycle. Contrary to the retinoblastoma protein-regulated, labile Chk1 kinase restricted to S-G(2) phases, Chk2 remains activatable even in quiescent and differentiating cells. In human tissues, Chk2 is homogeneously expressed in renewing cell populations such as epidermis or intestine, heterogeneous in conditionally renewing tissues, and absent or cytoplasmic in static tissues such as muscle or brain. These data highlight striking differences between Chk2 and Chk1 and show unexpected correlation of Chk2 expression with tissue biology.

• MeSH
• aktivace enzymů MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné dělení fyziologie   MeSH
• buněčné linie MeSH
• buněčný cyklus fyziologie   MeSH
• checkpoint kinasa 2 MeSH
• fibroblasty cytologie   enzymologie   MeSH
• G1 fáze fyziologie   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• nádorové buňky kultivované MeSH
• osteosarkom enzymologie   patologie   MeSH
• poškození DNA * fyziologie   MeSH
• protein-serin-threoninkinasy * MeSH
• proteinkinasy fyziologie   imunologie   metabolismus   MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH

• MeSH
• G1 fáze fyziologie   MeSH
• myši MeSH
• oocyty fyziologie   MeSH
• proteiny buněčného cyklu fyziologie   MeSH
• S fáze genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

• MeSH
• CDC geny fyziologie   MeSH
• cykliny fyziologie   MeSH
• G1 fáze fyziologie   MeSH
• nádorová transformace buněk fyziologie   účinky léků   účinky záření   MeSH
• nádorový supresorový protein p53 fyziologie   MeSH
• poškození DNA MeSH
• S fáze genetika   MeSH

The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.

• MeSH
• biologické modely MeSH
• buněčné klony MeSH
• cyklin E genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• DNA vazebné proteiny * MeSH
• fosfatasy cdc25 metabolismus   MeSH
• G1 fáze fyziologie   MeSH
• interfáze * fyziologie   MeSH
• kinasy CDC2-CDC28 * MeSH
• nádorové buňky kultivované MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• replikace DNA MeSH
• retinoblastomový protein * metabolismus   MeSH
• S fáze fyziologie   MeSH
• transgeny MeSH
• transkripční faktor DP1 MeSH
• transkripční faktor E2F1 MeSH
• transkripční faktory E2F MeSH
• transkripční faktory * metabolismus   MeSH
• transportní proteiny * MeSH
• vazebný protein 1 retinoblastomu MeSH

p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.

• MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy biosyntéza   metabolismus   MeSH
• G1 fáze genetika   fyziologie   MeSH
• inhibitor p16 cyklin-dependentní kinasy biosyntéza   genetika   fyziologie   MeSH
• inhibitory růstu genetika   fyziologie   MeSH
• kinasy CDC2-CDC28 * MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• osteosarkom MeSH
• protein-serin-threoninkinasy biosyntéza   metabolismus   MeSH
• retinoblastomový protein biosyntéza   genetika   fyziologie   MeSH
• S fáze genetika   MeSH
• technika přenosu genů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor-dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3-cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.

• MeSH
• AMP cyklický metabolismus   MeSH
• buněčné dělení účinky léků   fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• cyklin D3 MeSH
• cyklin-dependentní kinasa 4 MeSH
• cyklin-dependentní kinasy biosyntéza   metabolismus   MeSH
• cykliny metabolismus   MeSH
• epidermální růstový faktor farmakologie   MeSH
• epitopy analýza   MeSH
• fluorescenční protilátková technika MeSH
• G1 fáze fyziologie   MeSH
• hypoglykemika farmakologie   MeSH
• inzulin farmakologie   MeSH
• krevní proteiny farmakologie   MeSH
• kultivované buňky MeSH
• mitogeny farmakologie   MeSH
• protoonkogenní proteiny * MeSH
• psi MeSH
• štítná žláza cytologie   enzymologie   MeSH
• thyreotropin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mammalian D-type cyclins D1, D2, and D3 activate the cyclin-dependent kinases CDK4 and CDK6 in G1 and thereby promote the cell's commitment to enter S phase. To elucidate the extent of functional overlap among the D-type cyclins, we have examined several aspects of the least characterized member of this subfamily of G cyclin proteins, cyclin D3. Microinjection of cyclin D3-neutralizing antibody inhibited G1/S transition in human (IMR-90) and rat (R12) diploid fibroblasts, indicating that analogous to cyclins D1 and D2, cyclin D3 is essential for timely progression through G1. In contrast to cyclins D1 and D2, cyclin D3 was (i) ubiquitously expressed among a panel of 70 human cultured cell types; (ii) strongly upregulated upon induction of HL-60 leukaemia cells to differentiate; and (iii) accumulated to high levels in a wide range of quiescent cell types in mouse and human differentiated tissues. Complementary analyses of human biopsies and mouse tissues at different stages of foetal and postnatal development revealed lineage-dependent transient or long-term accumulation of the cyclin D3 protein, correlating with initiation/establishment or maintenance of the mature phenotypes, respectively. Our data support the notion that the biological roles of the individual D-type cyclins are not fully redundant, and suggest a possible dual role for cyclin D3 in cell proliferation and induction and/or maintenance of terminal differentiation.

• MeSH
• akutní promyelocytární leukemie patologie   MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné dělení fyziologie   MeSH
• buněčné linie MeSH
• cyklin D3 MeSH
• cykliny biosyntéza   imunologie   fyziologie   MeSH
• G1 fáze fyziologie   MeSH
• HL-60 buňky MeSH
• interfáze fyziologie   MeSH
• kosterní svaly cytologie   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky chemie   MeSH
• nádorové buňky kultivované MeSH
• orgánová specificita MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH