Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells

Mouse embryonic stem (ES) cells are known to express D-type cyclins at very low levels and these levels increase dramatically during in vitro and in vivo differentiation. Here, we investigate some of the signalling pathways regulating expression of cyclin D1 and progression to S phase, the Ras/Extracellular signal-regulated protein kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. We demonstrate that ERK phosphorylation is fully dispensable for the regulation of cyclin D1 level and for the progression from G1 to S phase in ES cells. By contrast, PI3-kinase activity is required for both. Differentiation induced by retinoic acid results in the gain of ERK-dependent control of cyclin D1 expression and of S phase progression. Differentiation is also paralleled by an increase in PI3-kinase activity. This leads (a) to an increase in the p70 S6 kinase-dependent regulation of the steady-state level of cyclin D1, and (b) to a concomitant decrease in the GSK3beta-dependent rate of cyclin D1 degradation. Altogether, these multiple pathways account for the dramatic increase in the level of cyclin D1 protein which parallels ES cell differentiation. Our studies suggest that PI3-kinase is an important regulator of the ES cell cycle and that its activity is not regulated by mitogen stimulation.     

Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells.

All-trans retinoic acid inhibits growth associated with downregulation of cyclin D1 and can cause low level apoptosis in estrogen receptor positive breast cancer cell lines. The cyclin D1 gene is amplified and/or the protein overexpressed in about one-third of breast cancers. Constitutive expression of cyclin D1 in estrogen receptor positive MCF-7 and ZR-75 breast cancer cells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S phase and reduced the G1 accumulation following retinoic acid treatment compared with control cells. However, culture of MCF-7(cycD1) with 1 microM all-trans retinoic acid resulted in about threefold greater growth inhibition compared with vector-transfected cells. Hoechst staining of DNA and in situ DNA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultures contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vector-transfected cells. Retinoic acid treatment of vector-transfected clones resulted in Bax protein activation as assessed by exposure of the NH(2)-terminus of Bax but the proportion of cells containing activated Bax was increased in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translocation of cytochrome c into the cytosol following RA-treatment. Retinoic acid markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingly, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to retinoic acid-induced apoptosis. We conclude that constitutive expression of cyclin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induced mitochondrial death pathway involving Bax activation, cytochrome c release and caspase-9 cleavage.     

Expression of D-type cyclins in colon cancer and in cell lines from colon carcinomas

Cyclins D1, D2 and D3 play important roles in cell proliferation and differentiation. Although their abnormal expression has been linked to cancer development and progression in a number of tissues, the expression of cyclin D2 and D3 proteins in colon cancer has not yet been characterised. In this study, we examined cyclin D1, D2 and D3 protein expression by Western blot analysis in tumour and adjacent normal colon tissues of 57 patients. In addition, we examined D-type cyclins protein expression in HT29 and LoVo39 cell lines from colon carcinomas, as a function of induced proliferation and differentiation. In both cell lines, the expression of the three D-type cyclins increased as a result of induced proliferation, whereas the expression of cyclin D3 increased as a result of induced differentiation. In colon tumours, cyclin D1 was overexpressed in 44%, cyclin D2 was overexpressed in 53% and cyclin D3 was overexpressed in 35% of the cases. We also found that in 16% of the cases, cyclin D3 protein expression was reduced in the tumour, as compared to the adjacent normal tissue. Examination of D-type cyclin protein overexpression in relation to the TNM stage of the tumours revealed that overexpression of cyclins D1 and/or D2, but not cyclin D3, is linked to colon carcinogenesis and that overexpression of cyclin D2 may be related to a higher TNM stage of the tumour.     

E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes

We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.     

Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer

D-type cyclins are important cell cycle regulators that promote cellular proliferation in response to growth factors by inactivation of the retinoblastoma protein (Rb). Cyclin D1 has been shown to be overexpressed in several cancer types and to act as an oncogene in breast cancers. As D-type cyclins are rate limiting for progression into S phase, the level at which they accumulate must be carefully regulated. Several mechanisms leading to overexpression of cyclin D1 have been reported including amplification, translocation and stabilization of the mRNA. Here, we present data showing elevated cyclin D1 protein in breast cancer samples in the absence of elevated mRNA level. Further, we found that in these cases, cyclin D3 protein also accumulates and that the coordinate increase in cyclin D1 and D3 occurs in 15% (7/47) of breast cancers. In addition we show that blocking the activity of the 26S proteosome results in the accumulation of cyclin D1 and D3, that both D-type cyclins are ubiquitinated and associate with Cul-1, a component of the SCF ubiquitin ligase complex. Finally, we show that the coordinated elevation of cyclin D1 and D3 is also observed in the breast cell line MCF-7 and demonstrate that the degradation of cyclin D1 and D3 is deficient in this cell line. These results indicate that cyclin D1 and cyclin D3 share a common mechanism of degradation and we propose that the coordinate increase of D-type cyclins observed in primary breast cancers reflects a defect in their proteolysis.     

Loss of cyclin E requirement in cell growth of an oral squamous cell carcinoma cell line implies deregulation of its downstream pathway

Cyclin E and Cdk2 have been shown to play an important role in G1/S transition of the cell cycle. Two E-type cyclins (E1 and E2) have been identified to date and share functionally similarities. Upregulation of these cyclins has been observed frequently in human cancers. We examined the expression profile of cyclin E1 and E2 in cell lines derived from human oral squamous cell carcinoma (SCC), and found that the expression of cyclin E1 protein was hardly detected in HSC-2 cells. Although cyclin E2 was abundantly expressed, histone H1 kinase activities of both E-type cyclins were virtually undetectable in this cell line. Inhibition of cyclin E1, but not that of E2, by using vectors expressing antisense-oriented their cDNAs induced drastic growth suppression on HOC313 cells that express both E-type cyclins. Inhibition of neither cyclin E1 nor E2 suppressed the growth of HSC-2 cells, and compensatory elevation of cyclin E1 was not evident in cyclin E2-inhibited HSC-2 cells. In contrast, HSC-2 cells expressed cyclin D1 and hyperphosphorylated forms of Rb family proteins, and were arrested in G1 by overexpression of p16(INK4), a specific inhibitor against D-type cyclin activity. These results indicate that HSC-2 cells lost proper growth control specifically mediated by cyclin E and suggest that deregulation of its downstream pathway may contribute to tumorigenesis of oral SCC.     

Activation of cyclin D1 and D2 promoters by human T-cell leukemia virus type I tax protein is associated with IL-2-independent growth of T cells

Our aim was to examine the involvement of G(1) cell-cycle regulators in cell growth dysregulation induced by HTLV-I. Compared to uninfected cells, higher expression levels of cyclin D1 and D2 mRNA were detected in HTLV-I-infected T-cell lines, which were at least in part mediated by the viral transforming protein Tax since Tax activated both cyclin D1 and D2 promoters in the human T-cell line Jurkat. A Tax mutant that did not activate NF-kappaB failed to activate cyclin D1 and D2 promoters. Inhibitors of NF-kappaB (dominant negative IkappaBs mutants) suppressed Tax-dependent activation of cyclin D1 and D2 promoters, indicating that Tax-induced activation was mediated by NF-kappaB. Wild-type and mutant Tax capable of activating NF-kappaB, but not Tax mutant incapable of activating NF-kappaB, converted cell growth of a T-cell line from being IL-2-dependent to being IL-2-independent; and this conversion was associated with IL-2-independent induction of cyclins D1 and D2. Our data suggest that induction of cyclins D1 and D2 by Tax is involved in IL-2-independent cell-cycle progression as well as IL-2-independent transformation of primary human T cells by HTLV-I. High expression levels of cyclin D1 and D2 mRNAs were also detected in some patients with ATL. Our findings link HTLV-I infection to changes in cellular D-type cyclin gene expression, transformation of T cells and subsequent development of T-cell leukemia.     

Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation, we used a low molecular weight compound, PD173074, that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415, MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression, inhibition of cyclin D/cdk4 activity and, as a consequence, reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells, suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells, both MAPK and PI3K signaling pathways were suppressed. In conclusion, results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells.     

Role of glycogen synthase kinase 3beta in rapamycin-mediated cell cycle regulation and chemosensitivity

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.     

Sulindac sulfide alters the expression of cyclin proteins in HT-29 colon adenocarcinoma cells

Sulindac sulfide (SS), the active metabolite of the colon cancer chemopreventive compound sulindac, inhibits the proliferation of HT-29 colon cancer cells mainly by inducing cell quiescence. We determined by bivariate flow-cytometric analysis both the DNA and cyclin protein content of individual cells. Thus, we assessed in detail the expression of several cyclins during the cell-cycle phases and demonstrated that SS (i) decreases the expression of cyclins B1 and E and (ii) increases the expression of cyclins D1, D2 and D3, particularly in the G₁ phase of the cell cycle. SS-induced apoptotic cells expressed both E- and D-type cyclins but not cyclin B1. The changes in cyclin expression combined with reduced catalytic activity of cyclin-dependent kinases could explain in molecular terms the anti-proliferative effect of SS on HT-29 colon cancer cells. These changes may contribute to the chemopreventive effect of sulindac. Int. J. Cancer 76:99–104, 1998.© 1998 Wiley-Liss, Inc.     

Alternative mammary oncogenic pathways are induced by D-type cyclins; MMTV-cyclin D3 transgenic mice develop squamous cell carcinoma

The three human D-type cyclins, cyclin D1, D2 and D3 share the ability to bind to and activate cdk4 and 6. MMTV-cyclin D1 transgenic mice develop mainly adenocarcinoma, while MMTV-cyclin D2 mice show a lack of alveologenesis during pregnancy and only develop carcinoma at low frequency. The effect of cyclin D3 overexpression in mammary glands remains hitherto unknown. We generated MMTV-cyclin D3 transgenic mice and report here that they develop exclusively squamous cell carcinoma. We show that although cyclin D3 transgene expression was detected early in puberty, postnatal development and mammary gland proliferation were normal in virgin animals. In contrast, multiparous mice develop multiple foci of abnormal growth that correspond to various stages of squamous metaplasia. Therefore, our results support a role for cyclin D3 in squamous differentiation. In addition, we found that p16 expression during involution is abolished, while p27 expression increased in MMTV-cyclin D3 mice, two modifications that have been reported in the other MMTV-D-type cyclin transgenic models. Our observations indicate that despite biochemical redundancy in vitro and in vivo, D-type cyclins promote distinct oncogenic pathways.     

Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.     

Requirement for cyclin D3 in lymphocyte development and T cell leukemias

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3(-/-) animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.     

Expression of cyclin D2 and D3 in lymphoid lesions

The D-type cyclins, involved in the regulation of G1 progression of the cell cycle, are expressed in a lineage-specific manner. Normal hematopoietic cells express cyclin D2 and/or D3. In order to determine whether their expression pattern changes in lymphoid tumors, we examined cyclin D2 and D3 expression in non-neoplastic and neoplastic lymphoid lesions, using a sensitive immunohistochemical amplification method. Centroblasts in lymphoid follicles of reactive lymph nodes expressed exclusively cyclin D3 and no D2. Interfollicular areas contained scattered cyclin D3 and D2 positive cells. By double staining, cyclin D3 was detected in CD79a positive B cells, CD3 positive T cells and CD68 positive macrophages. Cyclin D2 was present only in CD3 positive T cells. Neoplastic lymphoid lesions included 33 B cell lymphomas, 9 T cell lymphomas and 12 Hodgkin's lymphomas. The B cell lymphomas comprised 9 follicular lymphomas (FL), 1 Burkitt lymphoma (BL), 22 diffuse large cell lymphomas (DL) and 1 chronic lymphocytic leukemia (CLL). All 9 FLs and the single BL expressed exclusively cyclin D3, similarly to germinal center B cells, that represent their cells of origin. Six DLs expressed both cyclin D2 and D3, while 6 expressed only D3. Among the 9 pleomorphic T cell lymphomas, medium and large cell type, 5 expressed cyclin D2. Cyclin D3 was also detected in scattered cells in 4 of 9 cases and was highly expressed in 2 of 9 T cell lymphomas. The majority of Hodgkin's lymphomas expressed both cyclin D2 and D3 in Hodgkin/Reed-Sternberg (HRS) cells. The high frequency of positive cells indicates that both cyclins were expressed in the same cells.