A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein

Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin-dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for maintaining the orderly progression of the cell cycle. The B domain of Rb contains a site that recognizes and binds the LXCXE motif found in D-type cyclins. This interaction is important for phosphorylation of Rb by cdk4/D1, although in vitro the Rb C domain alone is efficiently phosphorylated by cdk4/D1. A mutation in the C domain of Rb, L901Q, has been identified that completely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contrast, the L901Q mutation has no effect on phosphorylation by either cyclin E/cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and cdk4/D1 is specific. Introduction of the L901Q mutation into Rb containing the A, B, and C domains results in phosphorylation becoming predominantly dependent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site within the C domain. The L901 binding site can supplant the LXCXE binding site for the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811. Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the isolated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in Rb and suggests a conservation of this C domain binding motif for cyclin D1/cdk4 kinase among the Rb family of proteins.     

Cell cycle progression of chronic lymphocytic leukemia cells is controlled by cyclin D2, cyclin D3, cyclin-dependent kinase (cdk) 4 and the cdk inhibitor p27.

B-CLL cells are arrested in G0/early G1 phase of the cell cycle and are characterized by a marked hyporesponsiveness towards a variety of polyclonal B cell activators. We have previously demonstrated that costimulation with CpG-ODN and IL-2 can overcome this proliferative defect. Cyclin D3 is the principal D-type cyclin which mediates G1 progression in normal B cells, but in B-CLL cells both cyclin D2 and cyclin D3, were strongly upregulated upon stimulation. Both cyclins were associated with cdk4 but not with cdk6, which is the catalytic partner of D-type cyclins in normal B cells. Moreover, immune complexes consisting of cyclin D2 and cdk4 or cyclin D3 and cdk4 were both functional and phosphorylated the RB protein in vitro. The cell cycle inhibitor p27 plays a pivotal role in cell cycle progression of B lymphocytes and has been shown to be overexpressed in B-CLL cells. P27 was rapidly downregulated in B-CLL cells even when stimulated with a non-CpG-ODN or IL-2 alone, while only moderate regulation could be observed in normal B cells. Taken together, our findings demonstrate that regulation of early cell cycle progression differs between B-CLL cells and normal B cells. These findings do not only contribute to the understanding of B-CLL pathophysiology, but might ultimately lead to the identification of new therapeutic targets.     

Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.     

A novel orally active small molecule potently induces G1 arrest in primary myeloma cells and prevents tumor growth by specific inhibition of cyclin-dependent kinase 4/6

Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of cyclin-dependent kinase 4 (Cdk4)-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of bone marrow myeloma cells in vivo. Here, we show that by specific inhibition of Cdk4/6, the orally active small-molecule PD 0332991 potently induces G(1) arrest in primary bone marrow myeloma cells ex vivo and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent, such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B-cell cancers.     

Overexpression of cdk4/cyclin D1, a possible mediator of apoptosis and an indicator of prognosis in human primary lung carcinoma

The relation between expression of cell cycle-regulator molecules and apoptosis was examined in surgical specimens and cultured human lung carcinoma cell lines. Immunohistochemical analysis for 133 cases revealed 2 types of staining pattern. The first group consisted of 95 cases (71.4%) characterized by apoptotic cells showing intensely positive staining for cdk4 and cyclin D1 but negative for other proteins (type A). In the second group (type B), comprising 38 cases (28.6%), apoptotic cells exhibited intense positive staining for any cyclins and cdks. Most of the latter cases had lost expression of Rb protein. When tumor cells retrieved from paraffin-embedded tissue were examined by flow cytometry, higher proportions of cells expressing only cdk4 or cyclin D1 in type A cases and of cells expressing any cyclin or cdk in type B cases showed a subdiploid DNA content. In survival analysis using the LI of apoptotic cells and cyclin/cdk-positive cells, the high-apoptosis/high-cyclin D1 group showed the poorest prognosis. Furthermore, forced overexpression of only cdk4 or cyclin D1 induced apoptosis in cultured cells with normal Rb protein, whereas overexpression of any cyclin or cdk induced apoptosis in cells defective for Rb protein. In conclusion, upregulation of cdk4/cyclin D1 may be a primary and critical factor in induction of apoptosis in human lung carcinomas in vivo. Moreover, inactivation of Rb protein renders cells more prone to apoptosis by abnormal expression of any cell-cycle protein.     

Lower cyclin H and cyclin-dependent kinase-activating kinase activity in cell cycle arrest induced by lack of adhesion to substratum

Knowledge about adhesion checkpoints is important to counteract dissemination of cells from solid tumors. Lack of anchorage in adherent cells is associated with growth arrest and inhibition of cyclin-dependent kinases (cdks) required to drive cell cycle progression. Because cyclin-cdk complex activation requires CDK-activating kinase comprising cdk7 and cyclin H, we now investigated their relationship to decreased proliferation by lack of cell spreading. This report shows that either UV irradiation on an adhesive substrate or culture on a nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition of proliferation by UV primarily induced the cdk inhibitor p21WAF1 without a significant effect on cyclin H and cdk7. In contrast, lack of adhesion to substratum decreased cyclin H but not cdk7 with accumulation of a slower migrating, presumably unphosphorylated cdk4 isoform. These results were paralleled by decreased cdk7-mediated phosphorylation of GST-cdk2 and lower activation of a baculovirus-derived cdc2-cyclin B kinase complex. This is the first report showing that cyclin H-mediated down-regulation of cdk-activating kinase activity is involved in growth arrest induced by lack of anchorage.     

Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo.

Cyclin D1 regulates mitogen-dependent progression through G(1) phase in cultured cells, and its overexpression in malignant cells is thought to contribute to autonomous proliferation in vivo. However, previous studies in cell lines have not demonstrated that cyclin D1 is sufficient to trigger cell replication. In this study, we found that transient transfection of adult hepatocytes with cyclin D1 stimulated assembly of active cyclin D1/cdk4 complexes, robust hepatocyte proliferation, and liver growth in the intact animal. After several days, hepatocyte proliferation was inhibited despite the persistence of high levels of cyclin D1 and cyclin E, suggesting that endogenous antiproliferative mechanisms were induced. Our data suggest that this antiproliferative response includes the marked up-regulation of p21, which in turn inhibits cyclin D1/cdk4 and cyclin E/cdk2 complexes. This study offers further evidence that cyclin D1 plays a pivotal role in the regulation of hepatocyte proliferation in the liver. Furthermore, this model may offer a unique system to study the normal cellular response to cyclin D1 expression in vivo.     

Immunohistochemical analysis of cyclin D1 and p53 in multiple myeloma

Conflicting data are reported on the clinical significance of cyclin D1 deregulation in multiple myeloma. The aim of this study was to evaluate the incidence and prognostic significance of cyclin D1 expression and p53 mutations in multiple myeloma, as well as the relationship of their expression with selected clinical data, histological features, and proliferative activity of myeloma cells. We analyzed bone marrow biopsy specimens obtained from 59 patients with newly diagnosed multiple myeloma. Expression of cyclin D1 and p53 was analyzed using standard imunohistochemical method of B5-fixed and routinely processed paraffin-embedded bone marrow specimens. Cyclin D1 was overexpressed in 14/59 (27%) and p53 in 5/59 (8.5%) specimens. There was no significant correlation between cyclin D1 overexpression and age, gender, clinical stage (Durie-Salmon classification), extent of osteolytic lesions, type of monoclonal protein, hemoglobin concentration, platelet count, serum concentration of creatinine, calcium, C-reactive protein, and beta₂-microglobulin. No association was observed between the expression of cyclin D1 and the extent of bone marrow infiltration, histological grade, proliferative activity index (measured with Ki-67 immunoreactivity) and response to therapy. No significant difference was observed regarding overall survival between cyclin D1 positive and cyclin D1 negative patients (29 vs 36 mo, p=0.76). Results of this study did not revealed prognostic significance of cyclin D1 overexpression in multiple myeloma. Mutations of p53 gene are rare events in myeloma, suggesting their limited role in the pathogenesis of the disease.     

Immunohistochemical analysis of cyclin D1 and p53 in multiple myeloma: relationship to proliferative activity and prognostic significance

Conflicting data are reported on the clinical significance of cyclin D1 deregulation in multiple myeloma. The aim of this study was to evaluate the incidence and prognostic significance of cyclin D1 expression and p53 mutations in multiple myeloma, as well as the relationship of their expression with selected clinical data, histological features, and proliferative activity of myeloma cells. We analyzed bone marrow biopsy specimens obtained from 59 patients with newly diagnosed multiple myeloma. Expression of cyclin D1 and p53 was analyzed using standard immunohistochemical method of B5-fixed and routinely processed paraffin-embedded bone marrow specimens. Cyclin D1 was overexpressed in 14/59 (27%) and p53 in 5/59 (8.5%) specimens. There was no significant correlation between cyclin D1 overexpression and age, gender, clinical stage (Durie-Salmon classification), extent of osteolytic lesions, type of monoclonal protein, hemoglobin concentration, platelet count, serum concentration of creatinine, calcium, C-reactive protein, and beta2-microglobulin. No association was observed between the expression of cyclin D1 and the extent of bone marrow infiltration, histological grade, proliferative activity index (measured with Ki-67 immunoreactivity) and response to therapy. No significant difference was observed regarding overall survival between cyclin D1 positive and cyclin D1 negative patients (29 vs 36 mo, p = 0.76). Results of this study did not revealed prognostic significance of cyclin D1 overexpression in multiple myeloma. Mutations of p53 gene are rare events in myeloma, suggesting their limited role in the pathogenesis of the disease.     

Alterations of the cyclin D1/pRb/p16(INK4A) pathway in multiple myeloma.

The retinoblastoma protein (pRb), p16(INK4A), D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G(1) regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5' CpG island hypermethylation of the p16(INK4A)gene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16(INK4A) was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fisher's test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16(INK4A) hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16(INK4A) hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16(INK4A) hypermethylation is frequent, inactivation of p16(INK4A) seems not to be involved in the pathogenesis of plasma cell disorders.     

Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma

The oncogene c-maf is translocated in approximately 5%-10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin beta7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin beta7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.     

Differential regulation of cdk2 and cyclin D1 in irradiated human glioma cells

We have utilized a series of glioma cell lines to study the effects of ionizing radiation on the regulation of proteins that contribute to cell cycle progression. While no alterations of cyclin E or cdk4 were detected, a high percentage of glioma cell lines exhibited constitutive overexpression of cdk2 protein and aberrant patterns of cyclin D1 protein. The fraction of glioma cells expressing cdk2 was similar to that observed in normal astrocytes, but individual glioma cells overexpressed cdk2. In response to ionizing radiation, both cyclin D1 and cdk2 accumulated in control cells but not in gliomas with overexpressed cdk2 or aberrant cyclin D1. These novel findings provide the first evidence of altered cyclin-cdk regulation in gliomas in response to ionizing radiation.     

Expression of G1 phase regulators in MG-63 osteosarcoma cell line.

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.     

Neoplastic hepatocyte growth associated with cyclin D1 redistribution from the cytoplasm to the nucleus in mouse hepatocarcinogenesis

Cyclin D1 overexpression is a frequent change in hepatocellular carcinomas (HCCs). Our present study demonstrated that cyclin D1 overexpression with abundant cyclin E, cdk4, cdk2, and p27Kip1 (p27) occurred in neoplastic hepatocytes from the early stage of mouse hepatocarcinogenesis. While cyclin D1 expression was mainly found in the cytoplasm of the tumor cells, it shifted to the nucleus in association with cell proliferation after the animals were subjected to a partial hepatectomy (PH), and then returned once more to the cytoplasm when the cells became quiescent. Inhibition of PI3 kinase (PI3K) by Ly294002 in mouse HCC cells in vitro suppressed the nuclear shift of cyclin D1 as well as cell proliferation, while PI3K activation by PTEN suppression failed to induce nuclear shift of cyclin D1, suggesting that PI3K activation is essential but not sufficient for the cyclin D1 nuclear shift. While MEK-ERK1/2 inhibition by PD98059 and mTOR inhibition by rapamycin affected the cyclin D1 nuclear shift and cell proliferation to a lesser extent, both these inhibitors reduced cyclin D1 levels. Finally, although p27, cdk4 and calmodulin (CaM) were detected in the cyclin D1 immunoprecipitates from both quiescent and proliferating HCC cells, Hsc70 and SSeCKS were detected only in the immunoprecipitate from quiescent cells, and p21Waf1/Cip1 (p21) was detected only in that from proliferating cells, suggesting that the cyclin D1 complex is different in quiescent and proliferating cells. These observations indicate that the nuclear/cytoplasmic localization of cyclin D1 plays an important role in the proliferation/quiescence of neoplastic hepatocytes.     

Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene

Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.     

Expression of p16Ink4a compensates for p18Ink4c loss in cyclin-dependent kinase 4/6-dependent tumors and tissues

Cell cycle progression from G(1) to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16(Ink4a) and p18(Ink4c) showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16(Ink4a) and p18(Ink4c) resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16(Ink4a-/-);p18(Ink4c-/-) mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18(Ink4c-/-) mice and in MEFs from p16(Ink4a-/-), p18(Ink4c-/-), or p16(Ink4a-/-);p18(Ink4c-/-) mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16(Ink4a) and p18(Ink4c) coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice.