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J Lukas

Publications and source records attributed to J Lukas.

At least 73 records · Page 4Linked to original sources
PubMed1996

Enhanced protein stability: a novel mechanism of D-type cyclin over-abundance identified in human sarcoma cells.

The mammalian D-type cyclins promote progression through a G1 checkpoint by phosphorylating the retinoblastoma protein (pRB), and can contribute to oncogenesis via their deregulated expression achieved through gene amplification, chromosomal rearrangement, or retroviral integration. We now report a novel mechanism of tumour-associated D-cyclin over-abundance, resulting from enhanced protein stability. In two human cell lines established from a single uterine sarcoma biopsy, pRB-positive SK-UT-1B and pRB-deficient SK-UT-1, aberrant accumulation of functional cyclins D1, and D2 and D3 occurred in the absence of gene amplification and/or elevated mRNA expression. The abundance of D-cyclin proteins remained elevated throughout the cell cycle, and pulse-chase experiments revealed six to 10-fold prolongation of their protein half-lives as compared with either diploid fibroblasts or control U-2-OS sarcoma cells. These results point to a critical regulatory role of D-type cyclin turnover, and contribute to refinement of current views of the role played by the cyclin D-CDK-p16-pRB pathway in cell cycle control and tumorigenesis.

Cyclin D↗
PubMed1996

The retinoblastoma protein modulates expression of genes coding for diverse classes of proteins including components of the extracellular matrix.

The product of the retinoblastoma susceptibility gene, pRb, is a negative regulator of cell growth. It functions by regulating the activity of transcription factors. Rb represses some genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some others by interacting with factors like Sp1 or ATF-2. However, there are only a few examples of genes which are positively regulated by pRb. In order to find out if there are common mechanisms for promoter regulation by pRb, we were interested to identify more genes which are either stimulated or repressed by pRb. Using the method of differential display (DDRT-PCR) in combination with nuclear run-on analyses we were able to detect a number of genes which are upregulated by ectopic expression of the Rb gene in Rb-deficient mammary carcinoma cells. We could demonstrate not only stimulation of the endogenous mutant Rb gene but also positive regulation of genes coding for diverse classes of proteins, including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40. As a second approach, we investigated gene expression in cell lines established from Rb deficient heterozygous and homozygous knockout mouse embryos and normal mice. We have identified several genes the expression of which correlates positively or negatively with the presence of Rb. These data provide further evidence for pRb being a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells.

Animals↗
PubMed1996

The retinoblastoma protein pathway and the restriction point.

The emerging role of the retinoblastoma protein (pRb) as a major controller of the restriction point has been supported by recent discoveries, including pRb's ability to repress gene transcription by all three RNA polymerases, which suggests a link between DNA replication and cell growth. Convergent genetic and biochemical data provide new insights into the molecular events that are upstream of, at, and downstream of pRb phosphorylation, which is regulated by G1-phase cyclins and cyclin-dependent kinases (Cdks) and their inhibitors (CKIs). Major advances have also been made in our understanding of a key role of the pathway involving cyclin D, Cdks, CKIs, pRb and E2F both in commitment to traversing the cell cycle and in restraining oncogenesis.

Cell Cycle↗
PubMed1996

Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint.

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.

Animals↗
PubMed1996

Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression.

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.

Animals↗
PubMed1995

Oncogenic aberrations of p16INK4/CDKN2 and cyclin D1 cooperate to deregulate G1 control.

The p16INK4/CDKN2, D-type cyclins, their partner cyclin-dependent kinases, and retinoblastoma protein constitute a G1 regulatory pathway commonly targeted in oncogenesis. We show that, unexpectedly, abnormalities of p16INK4/CDKN2 occur concomitantly in two-thirds of cancer cell lines harboring aberrations of cyclin D1. Gene and protein transfer experiments demonstrated that concurrent alterations of cyclin D1 and p16 levels cooperate to (de)regulate G1 control in diploid fibroblasts, and that both events influence growth of retinoblastoma (RB)-positive, but not RB-deficient cancer cells. These results show that biological consequences of deregulating individual components along the pathway are unequal, reflecting their hierarchical roles in the G1 checkpoint control. Whereas RB defects eliminate the checkpoint completely, aberrations of the upstream components, such as cyclin D1 and p16INK4/CDKN2, can cooperate in multistep tumorigenesis.

Base Sequence↗
PubMed1995

Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes.

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.

Animals↗
PubMed1995

Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16.

D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB). The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14). The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor. We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos. Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis.

Animals↗
PubMed1995

Cyclin D2 is a moderately oscillating nucleoprotein required for G1 phase progression in specific cell types.

To explore regulation and function of cyclin D2, a candidate cell cycle-regulatory proto-oncogene, we examined subcellular localisation, cell type- and cell cycle-dependent expression, and requirement of cyclin D2 protein for G1 progression, in a panel of 40 human normal and cancer cell types. Except for lymphoid cells and sarcoma cell lines, expression of cyclin D2 was considerably more restricted than that of cyclin D1, whereas both D-type cyclin proteins were low or undetectable in cells lacking functional retinoblastoma gene product. In G1 cells, the cyclin D2 protein was more resistant to extraction and localised predominantly to nuclei, whereas it became more soluble and distributed in both nuclei and cytoplasm from G1/S transition onwards. Centrifugal elutriation and multiparameter flow cytometry analyses of several cell types showed moderate cell cycle oscillation with maximum levels of the cyclin D2 protein reached in late G1. Microinjection and/or electroporation of antibodies to cyclin D2 during G1 arrested the cyclin D2-expressing lymphocytes, breast myoepithelium, and U-2-OS sarcoma cells in G1 phase, whereas cyclin D2-negative cell types were unaffected by such treatment. Consistent with the putative proto-oncogenic role of cyclin D2 in specific cell types, our data show that this G1 cyclin has properties closely resembling those of cyclin D1, including the essential positive role in regulation of G1.

Animals↗
PubMed1995

Aberrations of p16Ink4 and retinoblastoma tumour-suppressor genes occur in distinct sub-sets of human cancer cell lines.

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.

Carrier Proteins↗
PubMed1995

Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis.

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.

Blotting, Western↗
PubMed1995

Abnormal patterns of D-type cyclin expression and G1 regulation in human head and neck cancer.

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.

Carcinoma, Squamous Cell↗
PubMed1995

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

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.

Animals↗
PubMed1995

Regulated expression of the retinoblastoma susceptibility gene in mammary carcinoma cells restores cyclin D1 expression and G1-phase control.

The product of the retinoblastoma susceptibility tumour suppressor gene, pRb, is a negative regulator of cell proliferation. In order to investigate the interaction between pRb and the cell cycle machinery in more detail, a functional Rb gene was reintroduced into the Rb-deficient human mammary carcinoma cell line Bt549. Since constitutive high level expression of Rb turned out to be difficult to maintain, the tetracycline-dependent gene expression system was used. A number of clones was generated which all showed low level expression in the noninduced state. Considerable induction rates were obtained. The low level of noninduced Rb expression was sufficient to induce the expression of cyclin D1 the level of which was not further increased by upregulation of Rb expression. Concomittantly, an increase in cell doubling time was observed due to retardation of the cell cycle in the G1-phase. The data suggest that limiting amounts of cyclin D1 determine, at least partly, the extent of growth-repressing properties of pRb. The inducible system allows for maintenance of Rb-reconstituted cells at a low level of expression and for their use in the investigation of downstream functions of pRb.

Breast Neoplasms↗
PubMed1994

The PRAD-1/cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinomas.

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.

Chromosomes, Human, Pair 11↗
PubMed1994

Cyclin D1 protein expression and function in human breast cancer.

Cyclin D1 is a cell-cycle regulator essential for G1 phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumour types, including breast carcinomas. In spite of the accumulating genetic evidence, however, there are no data regarding abundance and properties of the cyclin D1 protein in breast cancer. We now report aberrant nuclear overexpression/accumulation of the cyclin D1 protein in about half of the 170 primary breast carcinoma specimens analyzed by monoclonal antibody immunohistochemistry, indicating that the frequency of cyclin D1 abnormalities may be considerably higher than previously deduced from DNA amplification studies. A comparison of the expression patterns in matched lesions at different stages of tumour progression revealed that the cyclin D1 protein aberration appears to reflect a relatively early event and that, when acquired by a tumour, it is maintained throughout breast cancer progression including metastatic spread. In both tumour tissues and breast cancer cell lines, the abundance of this protein shows characteristic variations consistent with a cell-cycle oscillation and the peak levels expressed in G1. In all 7 cell lines whose retinoblastoma (Rb) protein is mutant or complexed to SV40 T antigen, exceptionally low levels of cyclin D1 protein and mRNA were found. Antibody-mediated and anti-sense oligonucleotide knockout experiments demonstrate the requirement for the cell-cycle regulatory function of cyclin D1 in breast cancer lines with single or multiple copies of the gene and reveal the absence of such a requirement in the cell lines with Rb defects. Our data are consistent with the notion that the emerging "Rb-cyclin D1 pathway" represents a frequent target of oncogenic abnormalities in breast cancer.

Base Sequence↗
PubMed1994

Efficient transfer of antibodies into mammalian cells by electroporation.

An optimized protocol for electroporation is described which is suitable for introducing antibodies into mammalian cells. The method results in the uptake of detectable amounts of antibodies in 80% of the cells and in 40% large amounts are introduced. As an example, cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells. This specific antibody mediated inhibition of cellular function did not affect the viability of the cells since they recovered from the inhibition after some time. While approaching the efficiency of microinjection, the new protocol for electroporation of antibodies additionally permits treatment of the larger number of cells which are required for biochemical analyses.

Animals↗