The D-type cyclins are positive regulators of the GI phase of the mammalian cell cycle. Cyclins D I 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 0 3 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 GI cyclin in a series of human cultured cell types and in I80 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 D I 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 overabundance of cyclin D3 is considerably less frequent than that of cyclin DI, yet we identify subsets of breast tumours, and potentially lymphomas, as candidate tumour types with elevated cyclin D3 expression. o 1996 Wiley-Liss, Inc. Cyclins D1, D2 and D3 comprise a distinct subfamily of cyclin genes whose products positively regulate progression through the GI phase of the mammalian cell division cycle (reviewed by Sherr, 1994). The D-type cyclin proteins can combinatorially bind with several of the cyclin-dependent kinases (CDKs), in particular with CDK4 and CDK6 (re-viewed by Sherr, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1995). According to current understanding, the cyclin D/CDK holo-enzymes control GI via phosphorylation of the retinoblastoma protein (pRB) (Kato et al., 1993; Lukas et al., 1995a; reviewed by Weinberg, 1995). This phosphorylation in mid-to-late GI cancels pRB's proliferation-constraining capacity through a conformational change resulting in release of a series of transcription factors from sequestration by the under-phosphorylated pRB. The liberated transcription factors , such as E2F, then regulate activities of a number of genes essential for initiation of DNA replication (reviewed by Weinberg, 1995). Expression of D-type cyclins is growth factor-inducible, thereby providing a link between various mitogenic signaling pathways and cell cycle machinery (Matsushime et al., 1991; Sherr, 1994). Consistent with the central role of the cyclin D family in growth control, and as a functional antagonist of the pRB tumour suppressor, a large body of evidence implicates deregulation of at least cyclin D1 as an oncogenic event involved in the pathogenesis of a wide range of neoplasms. Thus, the CCNDl gene on chromosome llq13, encoding cyclin D1, classifies as a proto-oncogene that can become...
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 GI, 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. lmmunoblotting and immunocytochemical analyses showed that CDK7 was (i) ubiquitously expressed in all cell types examined; (ii) exclusively nuclear: (iii) moderately elevated in turnour 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.o 1996 Wiley-Liss, Inc.Transitions through the ordered checkpoints that control the mammalian cell cycle are triggered by activation of a distinct class of serine/threonine kinases, the cyclin-dependent kinases (CDKs). To ensure proper timing and mutual coordination of the major cell-cycle events, the CDKs are carefully regulated by multiple mechanisms (Clarke, 1995;Doree and Galas, 1994;Morgan, 1995;Sherr and Roberts, 1995). One such mechanism is an activatory phosphorylation of a conserved threonine residue (Thr 160 in CDK2, Thr 161 in CDC2 and their equivalents in other CDKs) within the T loop of various CDKs (Clarke, 1995;Doree and Galas, 1994;Morgan, 1995). This phosphorylation appears to alter conformation of the T loop, to stabilize the CDK-cyclin interaction and probably to render the catalytic domain of CDKs accessible to their cellular protein substrates Pines, 1995). A kinase responsible for phosphorylating the threonine residue has been identified in various organisms and designated CAK, for CDK-activating kinase (Darbon et al., 1994; Fesquet etal., 1993 (Shuttleworth et al., 1990). The catalytic subunit of CAK itself, therefore, appears to be a member of the CDK family, and it requires binding to a regulatory cyclin subunit to become active (Clarke, 1995;Fisher and Morgan, 1994;Makela et al., 1994;Morgan, 1995). The CAK has therefore been recently renamed CDK7 and its novel cyclin partner termed cyclin H Makela et al., 1994).Accumulating evidence implicates deregulation of some of the components of the mammalian cell-cycle machinery in oncogenesis. In particular, over-expression of D-type cyclins or their CDK4 partner kinase appears to be involved in pathogenesis of several human cancer types, and these positive cell cycle regulators qualify as new proto-oncogenes (Bartkova et al., 19956;Hunter and Pines, 1994;Sherr and Roberts, 1995;Strauss et al., 1995). Since CDK7/CAK activates the key CDKs at various cell-cycle transitions, including...
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