"IZ1919"
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Cyclin D1 is a cell cycle regulator essential for G1 phase progression and a candidate proto-oncogene whose deregulated expression has been implicated in pathogenesis of several types of cancer. We have examined expression of cyclin D1 in 212 primary tumours of five histogenetically distinct types by immunohistochemistry and found strong aberrant accumulation of the protein in 21%, and a moderate overabundance in further 25% of cases. While the abnormalities were more frequent in carcinomas of the breast, i.e. the cancer type known for cyclin D1 gene amplification, aberrant expression was also seen in significant subsets of colorectal cancers, soft tissue sarcomas, uterine carcinomas and malignant melanomas. Comparison of distinct stages of tumour progression showed concordant cyclin D1 patterns in the in situ vs invasive breast carcinoma components (n = 37) and between primary and metastatic lesions (n = 51) of several tumour types. The specificity of the immunohistochemical data was supported by immunoblotting analysis of tissue and tumour lysates, and the tumour-specific over-expression was confirmed by computer-assisted image analysis. These observations suggest that alterations of cyclin D1 expression represent a common feature of malignancies of diverse histogenesis and indicate that both the spectrum of tumour types and the frequency of cyclin D1 aberrations significantly exceed previous estimations based on genetic analyses.
The D-type cyclins are positive regulators of the G1 phase of the mammalian cell cycle. Cyclins D1 or D2 are over-expressed in several types of cancer, transform rodent cells in culture and therefore harbor hallmarks of cellular proto-oncogenes. In contrast, no data on expression of cyclin D3 in tissues and tumours are presently available. We have raised monoclonal antibodies (MAbs) specific for cyclin D3 and examined abundance and subcellular localisation of this G1 cyclin in a series of human cultured cell types and in 180 primary tumours of diverse histogenesis. Cyclin D3 localised predominantly in nuclei of normal and tumour cells both in culture and in situ, and a pronounced cell-to-cell variation of its abundance was reminiscent of cyclins D1 and D2. Immunohistochemical analysis of tumour and corresponding normal tissues showed strong aberrant accumulation of cyclin D3 in a subset (about 10%) of breast carcinomas, whereas only weak-to-moderate expression was found in colorectal, head and neck and uterine carcinomas, melanomas and soft tissue sarcomas. The specificity of the immunohistochemical data was confirmed by immunoblotting analysis of tissue and tumour lysates. Our results indicate that over-abundance of cyclin D3 is considerably less frequent than that of cyclin D1, yet we identify subsets of breast tumours, and potentially lymphomas, as candidate tumour types with elevated cyclin D3 expression.
- MeSH
- buněčné jádro metabolismus patologie MeSH
- cyklin D3 MeSH
- cykliny * analýza MeSH
- imunoblotting MeSH
- imunohistochemie MeSH
- lidé MeSH
- nádorové biomarkery * analýza MeSH
- nádorové buňky kultivované MeSH
- nádory * metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
In an effort to elucidate the biological role played by cyclin D1, a candidate BCL-1 oncogene, in human B-cell tumours carrying the t(11;14) translocation, we have studied the properties of this cyclin protein in a series of human lymphoid lines with rearrangements in the BCL-1 locus. The BCL-1/cyclin D1 protein was easily detectable in both immunocytochemistry and immunoblotting, its abundance grossly correlating with the mRNA levels. The cyclin D1 protein was localised predominantly to nuclei and there was a striking variation of staining intensity among the exponentially growing cells, reflecting the maximum level reached in mid/late G1 and the lowest level in S-phase. This characteristic mode of cell cycle-dependent oscillation was confirmed by three independent approaches, demonstrating that even upon rearrangement, the expression of cyclin D1 is regulated in a cyclical manner. Antibody-mediated and anti-sense oligonucleotide 'knockout' experiments revealed that the aberrantly expressed BCL-1/cyclin D1 protein is required for G1 phase progression of all four B-cell tumours with the BCL-1 rearrangement. Consistent with the proposed oncogenic role of this cyclin, our data demonstrate that the BCL-1 deregulation caused by chromosomal rearrangement leads to expression of a functionally active cyclin D1 protein which subverts the G1 phase control in the human B-cell tumours carrying the t(11;14) translocation.
- MeSH
- B-buněčný lymfom * genetika patologie MeSH
- cyklin D1 MeSH
- cykliny * fyziologie MeSH
- G1 fáze MeSH
- leukemie B-buněčná * genetika patologie MeSH
- lidé MeSH
- lidské chromozomy, pár 11 * MeSH
- lidské chromozomy, pár 14 * MeSH
- molekulární sekvence - údaje MeSH
- nádorové buňky kultivované MeSH
- onkogenní proteiny * fyziologie MeSH
- protoonkogenní proteiny * fyziologie MeSH
- sekvence nukleotidů MeSH
- translokace genetická * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il. ; 32 cm
Cílem je objasnění genetických a z nich vyplývajících biochemických aberací antionkogenů a genů podílejících se na řízení buněčné proliferace.
The cyclin-dependent kinase 7 (CDK7) represents the 40-kDa catalytic subunit of the CDK-activating kinase, the enzyme responsible for activatory phosphorylation of multiple CDKs controlling G1, S and G2/M phases of the cell cycle. Here, we surveyed a wide range of normal and tumour cell types, in both cell culture and biopsy specimens, for abundance and subcellular localisation of the CDK7 protein. Immunoblotting and immunocytochemical analyses showed that CDK7 was (i) ubiquitously expressed in all cell types examined; (ii) exclusively nuclear; (iii) moderately elevated in tumour cells when compared with their normal cell counterparts; (iv) invariant throughout the cell cycle of normal lymphocytes, fibroblasts, breast epithelium and several cancer cell lines; and (v) clearly detectable even in quiescent cells, including highly differentiated cell types in situ. Our data are consistent with the emerging role for CDK7/CAK in multiple biological processes, possibly providing a link between cell-cycle control, transcriptional regulation and genomic integrity control.
- MeSH
- buněčná diferenciace genetika účinky léků MeSH
- buněčné jádro metabolismus MeSH
- buněčný cyklus * genetika MeSH
- cyklin-dependentní kinasy * MeSH
- fibroblasty metabolismus MeSH
- genetická transkripce MeSH
- lidé MeSH
- lymfocyty metabolismus MeSH
- nádorové kmenové buňky * metabolismus MeSH
- nádorové proteiny * biosyntéza genetika MeSH
- nádory genetika metabolismus patologie MeSH
- protein-serin-threoninkinasy * biosyntéza genetika MeSH
- prsy cytologie metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- regulace genové exprese * MeSH
- retina cytologie embryologie metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
D-type cyclins are proto-oncogenic cell cycle regulators implicated in the pathogenesis of several types of cancer. Amplification of the cyclin D1 gene has been described in 30-50% of human head and neck squamous cell carcinoma (HNSCC). Using immunohistochemistry on archival specimens of human HNSCC and a mAb DCS-6, which is specific for cyclin D1, strong positivity was found in nuclei of 9 (17%) of 52, a moderately elevated signal in 16 (31%) of 52, and weak staining comparable with normal tissues in 27 (52%) of 52 patients. Immunoblotting analysis of five HNSCC-derived cell lines showed three distinct spectra of D-type cyclin proteins: cyclin D1 only (in UMSCC-2 and UMSCC-22b cell lines with 11q13 amplification), cyclins D1 and D3 (in HN5 and HN6), or cyclins D1, D2, and D3 (in UMSCC-1). Electroporation of neutralizing antibodies demonstrated requirement for cyclin D1 in cell cycle progression of all five HNSCC cell lines. Cyclin D2 was essential and showed a cooperative effect with cyclin D1 in positive regulation of G1 in UMSCC-1 cells. These data are consistent with the proposed oncogenic role of cyclin D1 in HNSCC and open up the way for immunohistochemical assessment of cyclin D1 aberrations in archival clinical specimens. It is also suggested that excessive levels of cyclin D1 alone or cooperative effects of several D-type cyclin proteins may lead to deregulation of G1 control in distinct subsets of human HNSCC. These results are discussed in the context of possible functional redundancy of D-type cyclins and the role of the D-type cyclin/p16-CDKN2/pRB pathway in tumorigenesis.
- MeSH
- cyklin D1 MeSH
- cyklin D2 MeSH
- cykliny analýza fyziologie MeSH
- G1 fáze * fyziologie MeSH
- imunohistochemie MeSH
- lidé MeSH
- nádory hlavy a krku chemie patologie MeSH
- onkogenní proteiny analýza fyziologie MeSH
- spinocelulární karcinom chemie patologie MeSH
- zalévání tkání do parafínu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The PRAD-1/cyclin D1 proto-oncogene is localized on chromosome 11q13 and it is overexpressed in several tumour types as a consequence of gene amplification or chromosomal rearrangements. In this study, the abundance and patterns of cyclin D1 protein expression in normal/non-involved colon (n = 44), primary (n = 48) and metastatic (n = 9) colorectal carcinomas, and in a series of 4 colon cancer cell lines were investigated by immunochemical methods using the DCS-6 monoclonal antibody specific for cyclin D1. While examination of all normal colorectal tissue samples and 56% of the primary tumours revealed only weak to undetectable immunostaining signals, 23% of the primary carcinomas showed moderate and 21% showed strong aberrant accumulation of this cell-cycle regulatory oncoprotein. The immunohistochemical patterns in the secondary lesions were concordant with the matched primary tumours in all cases. The staining was nuclear both in the clinical specimens and in the colon cancer cell lines, in which the antibody-mediated knock-out experiments demonstrated a positive regulatory role of the cyclin D1 protein whose function was required for progression through the G1 phase of the cell cycle. These results indicate that the PRAD-1/cyclin D1 protooncogene may be deregulated in a significant subset of colorectal tumours, and warrant further analyses of such aberrations of the cyclin D1/retinoblastoma protein pathway to elucidate its potential involvement in the multistep pathogenesis of human colorectal cancer.
- MeSH
- cyklin D1 MeSH
- cykliny * metabolismus MeSH
- imunohistochemie MeSH
- kolorektální nádory * metabolismus MeSH
- lidé MeSH
- lidské chromozomy, pár 11 MeSH
- nádorové buňky kultivované MeSH
- onkogenní proteiny * metabolismus MeSH
- protoonkogeny MeSH
- retinoblastomový protein metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The commitment of mammalian cells in late G1 to replicate the genome and divide in response to mitogenic growth factors operating via tyrosine kinase receptors depends on phosphorylation of the retinoblastoma protein (pRb), a process controlled by cyclin D-associated cyclin-dependent kinases (cdks) and their inhibitors. This study addressed the issue of whether also other mitogenic signalling cascades require activation of cyclin D-associated kinases or whether any mitogenic pathway can bypass the cyclin D-pRb checkpoint. We show that mitogenic signal transduction pathways from three classes of receptors, the membrane tyrosine kinase receptors activated by serum mitogens or epidermal growth factor, estrogen receptors triggered by estradiol, and the cyclic AMP-dependent signalling from G-protein-coupled thyrotropin receptors, all converge and strictly require the cyclin D-cdk activity to induce S phase in human MCF-7 cells and/or primary dog thyrocytes. Combined microinjection and biochemical approaches showed that whereas these three mitogenic cascades are sensitive to the p16 inhibitor of cdk4/6 and/or cyclin D1-neutralizing antibody and able to induce pRb kinase activity, their upstream biochemical routes are distinct as demonstrated by their differential sensitivity to lovastatin and requirements for mitogen-activated protein kinases whose sustained activation is seen only in the growth factor-dependent pathway. Taken together, these results support the candidacy of the cyclin D-cdk-pRb interplay for the convergence step of multiple signalling cascades and a mechanism contributing to the restriction point switch.
- MeSH
- buněčný cyklus * MeSH
- cyklin D MeSH
- cyklin-dependentní kinasy * metabolismus MeSH
- cykliny * metabolismus MeSH
- G1 fáze * MeSH
- lidé MeSH
- mitogeny * metabolismus MeSH
- nádorové buňky kultivované MeSH
- psi MeSH
- retinoblastomový protein * metabolismus MeSH
- signální transdukce * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.
- MeSH
- cyklin D1 MeSH
- cyklin-dependentní kinasa 4 MeSH
- cyklin-dependentní kinasy * MeSH
- cykliny antagonisté a inhibitory imunologie metabolismus MeSH
- DNA primery genetika MeSH
- fosforylace MeSH
- G1 fáze * fyziologie genetika MeSH
- geny retinoblastomu * MeSH
- kultivované buňky MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- neutralizační testy MeSH
- onkogenní proteiny antagonisté a inhibitory imunologie metabolismus MeSH
- protein-serin-threoninkinasy * metabolismus MeSH
- protoonkogenní proteiny * MeSH
- retinoblastomový protein genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The p16Ink4/MTS1/CDKN2 is a cell-cycle regulatory inhibitor of cyclin-dependent kinase 4 (cdk4), and a candidate tumour suppressor whose gene on chromosome band 9p21 is frequently deleted or mutated in diverse types of cancer. Cdk4 in association with its D-type cyclin partners, together with p16Ink4, and the product of the retinoblastoma tumour-suppressor gene (pRB), appear to constitute a G1-phase-controlling pathway which can become de-regulated through oncogenic aberrations of any of the components. In an attempt to elucidate the underlying molecular mechanisms, we have now surveyed expression of p16Ink4, at the protein and the mRNA levels, in 21 human cell types expressing normal pRB, as compared with another series of 21 cell lines whose pRB is mutant and/or inactivated through sequestration by DNA tumour virus onco-proteins. In contrast to aberrant lack of p16 expression in the majority of RB-positive cell types, expression of apparently normal (as shown by electrophoretic mobility and/or the ability to form protein-protein complexes with cdk4 in vivo) p16 was uniformly preserved in the cancer cell lines whose RB function was compromised. These data indicate that p16 operates upstream of pRB along the same pathway in G1. The results are discussed in view of the nature of a selective growth advantage potentially gained by cells through de-regulation of this key cell-cycle control mechanism.
- MeSH
- buněčný cyklus fyziologie MeSH
- chromozomální aberace * MeSH
- cyklin-dependentní kinasa 4 MeSH
- cyklin-dependentní kinasy * MeSH
- geny retinoblastomu * MeSH
- inhibitor p16 cyklin-dependentní kinasy MeSH
- inhibitory proteinkinas MeSH
- lidé MeSH
- messenger RNA analýza genetika MeSH
- mutace MeSH
- nádorové buňky kultivované MeSH
- nádory * genetika MeSH
- polymerázová řetězová reakce MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory MeSH
- protoonkogenní proteiny * MeSH
- transportní proteiny * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
