Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor's pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.
We constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.
Our results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002.
Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.
Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.
Lewis (y) antigen; matrix metalloproteinases; tissue inhibitors of metalloproteinases; invasion
We have previously reported that Nodal, a member of the TGF-β superfamily, acts through activin receptor-like kinase 7 (ALK7) to inhibit ovarian cancer cell proliferation. To determine the mechanism underlying their effects, a cell cycle gene array was performed and cyclin G2 mRNA was found to be strongly up-regulated by Nodal and ALK7. To study the function and regulation of cyclin G2 in ovarian cancer cells, expression constructs were generated. We found that cyclin G2 protein level decreased rapidly after transfection, and this decrease was prevented by 26S proteasome inhibitors. Immunoprecipitation and pull-down studies showed that ubiquitin, Skp1, and Skp2 formed complexes with cyclin G2. Knockdown of Skp2 by siRNA increased, whereas overexpression of Skp2 decreased cyclin G2 levels. Nodal and ALK7 decreased the expression of Skp1 and Skp2 and increased cyclin G2 levels. Overexpression of cyclin G2 inhibited cell proliferation whereas cyclin G2-siRNA reduced the antiproliferative effect of Nodal and ALK7. Taken together, these findings provide strong evidence that cyclin G2 is degraded by the ubiquitin–proteasome pathway and that Skp2 plays a role in regulating cyclin G2 levels. Furthermore, our results also demonstrate that the antiproliferative effect of Nodal/ALK7 on ovarian cancer cells is in part mediated by cyclin G2.
This study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H.
The expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR.
Immunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (P < 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both P < 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by co-immunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (P < 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (P < 0.01). The mRNA level of CD44 in both cell lines was similar (P > 0.05).
Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.
Cyclins are regulatory subunits that bind to and activate catalytic Cdks. Cyclin E associates with Cdk2 to mediate the G1/S transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how cyclin E is normally downregulated during development in order to determine if disruption of similar mechanisms could either contribute to its overexpression in cancer, or be exploited to decrease its expression. In Xenopus laevis embryos, cyclin E protein level is high and constant until its abrupt destabilization by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT) and remodeling of the embryonic to the adult cell cycle. Since degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent, we examined the role of phosphorylation in Xenopus cyclin E turnover. We show that similarly to human cyclin E, phosphorylation of serine 398 and threonine 394 plays a role in cyclin E turnover at the MBT. Immunofluorescence analysis shows that cyclin E relocalizes from the cytoplasm to the nucleus preceding its degradation. When nuclear import is inhibited, cyclin E stability is markedly increased after the MBT. To investigate whether degradation of Xenopus cyclin E is mediated by the proteasomal pathway, we used proteasome inhibitors and observed a progressive accumulation of cyclin E in the cytoplasm after the MBT. Ubiquitination of cyclin E precedes its proteasomal degradation at the MBT. These results show that cyclin E destruction at the MBT requires both phosphorylation and nuclear import, as well as proteasomal activity.
Xenopus laevis; cyclin E; midblastula transition; phosphorylation; proteasomal degradation; ubiquitination
Lewis y (LeY) antigen is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Overexpression of LeY is frequently observed in epithelial-derived cancers and has been correlated to the pathological staging and prognosis. However, the effects of LeY on ovarian cancer are not yet clear. Previously, we transfected the ovarian cancer cell line RMG-I with the α1,2-fucosyltransferase gene to obtain stable transfectants, RMG-I-H, that highly express LeY. In the present study, we examined the proliferation, tumorigenesis, adhesion and invasion of the cell lines with treatment of LeY monoclonal antibody (mAb). Additionally, we examined the expression of TGF-β1, VEGF and b-FGF in xenograft tumors. The results showed that the proliferation and adhesion in vitro were significantly inhibited by treatment of RMG-I-H cells with LeY mAb. When subcutaneously inoculated in nude mice, RMG-I-H cells produced large tumors, while mock-transfected cells RMG-I-C and the parental cells RMG-I produced small tumors. Moreover, the tumor formation by RMG-I-H cells was inhibited by preincubating the cells with LeY mAb. Notably, the expression of TGF-β1, VEGF and b-FGF all increased in RMG-I-H cells. In conclusion, LeY plays an important role in promoting cell proliferation, tumorigenecity and adhesion, and these effects may be related to increased levels of growth factors. The LeY antibody shows potential application in the treatment of LeY-positive tumors.
Lewis y; ovarian cancer; proliferation; tumorigenecity; adhesion; inhibition
The cell cycle is promoted by activation of cyclin dependent kinases (Cdks), which are regulated positively by cyclins and negatively by Cdk inhibitors. Proliferation of carcinoma is associated with altered regulation of the cell cycle. Little is known on the combined alterations of cyclins A, B1, D1 and E in breast cancer in relation to the tumour grade and other prognostic factors.
Immunohistochemical analysis of cyclins A, B1, D1 and E, estrogen receptor, progesterone receptor, Ki-67, Her-2/neu and CK5/6 was performed on 53 breast carcinomas. mRNA levels of the cyclins were analysed of 12 samples by RT-PCR. The expression of cyclins A, B1 and E correlated with each other, while cyclin D1 correlated with none of these cyclins. Cyclins A, B1 and E showed association with tumour grade, Her-2/neu and Ki-67. Cyclin E had a negative correlation with hormone receptors and a positive correlation with triple negative carcinomas. Cyclin D1 had a positive correlation with ER, PR and non-basal breast carcinomas.
Cyclin A, B1 and E overexpression correlates to grade, Ki-67 and Her2/neu expression. Overexpression of cyclin D1 has a positive correlation with receptor status and non-basal carcinomas suggesting that cyclin D1 expression might be a marker of good prognosis. Combined analysis of cyclins indicates that cyclin A, B and E expression is similarly regulated, while other factors regulate cyclin D1 expression. The results suggest that the combined immunoreactivity of cyclins A, B1, D and E might be a useful prognostic factor in breast cancer.
Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220NPAT/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21CIP1/WAF1, p27KIP1 and p57KIP2 attenuate CDK2 activity. Here we find that γ-irradiation induces p21CIP1/WAF1 but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220NPAT/HiNF-P complex. We also show that p21CIP1/WAF1 is less effective than p27KIP1 and p57KIP2 in inhibiting the CDK2 dependent phosphorylation of p220NPAT at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57KIP2 in blocking the p220NPAT/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220NPAT that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220NPAT/HiNF-P complex.
cell cycle; histone genes; transcription; chromatin; prliferation
cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.
A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27Kip1 and p21Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.
To investigate the expression of cyclin-dependent kinase 8 (CDK8) and β-catenin in colon cancer and evaluate the role of CDK8 in the proliferation, apoptosis and cell cycle progression of colon cancer cells, especially in HCT116 cell line.
Colon cancer cell line HCT116 was transfected with small interfering RNA (siRNA) targeting on CDK8. After CDK8-siRNA transfection, mRNA and protein expression levels of CDK8 and β-catenin were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay in HCT116 cells. Cell proliferation was measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide Methylthiazolyl tetrazolium (MTT) assay, and cell cycle distribution and apoptosis were analyzed by flow cytometry analysis (FACS). CDK8 and β-catenin protein levels were also examined by real-time PCR and immunohistochemistry (IHC) in colon cancer tissues and adjacent normal tissues.
After CDK8 specific siRNA transfection, mRNA and protein expression levels of CDK8 and β-catenin in HCT116 cells were noticeably decreased (P < 0.05). CDK8 specific siRNA transfection inhibited HCT116 cells' proliferation and facilitated their apoptosis significantly (P < 0.05). In addition, the proportion of HCT116 cells in the G0/G1 phase was remarkably increased after CDK8-siRNA transfection (P < 0.05). The expression levels of CDK8 and β-catenin in adjacent normal tissues were lower than in tumor tissues (P < 0.05). Moreover, the expression of CDK8 was correlated with the expression of β-catenin in both tumor and adjacent normal tissues (P < 0.05).
CDK8 and β-catenin were expressed in colon cancer at a high frequency. CDK8 specific siRNA transfection down-regulated the expression of CDK8 in colon cancer cells, which was also associated with a decrease in the expression of β-catenin Moreover, CDK8 specific siRNA inhibited the proliferation of colon cancer cells, promoted their apoptosis and arrested these cells in the G0/G1 phase. Interference of CDK8 might be an effective strategy through β-catenin regulation of colon cancer.
LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TβRI/TβRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TβRI and TβRII contain the LeY structure, and the level of LeY in TβRI and TβRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF-β receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.
Lewis Y; p42/44 mitogen-activated protein kinase; phosphoinositide 3-kinase; Smad; transforming growth factor β type I (II) receptor
Pancreatic cancer is the 4th leading cause of death from cancer in the U.S. The opioid growth factor (OGF; [Met5]-enkephalin) and the OGF receptor form an inhibitory growth regulatory system involved in the pathogenesis and treatment of pancreatic cancer. The OGF-OGFr axis influences the G0/G1 phase of the cell cycle. In this investigation, we elucidate the pathway of OGF in the cell cycle.
Using BxPC-3 cells, OGF decreased phosphorylation of retinoblastoma (Rb) protein without changing total Rb. This change was correlated with reduced cyclin-dependent kinase protein (Cdk) 2 kinase activity, but not total Cdk2. OGF treatment increased cyclin-dependent kinase inhibitor (CKI) p21 protein expression in comparison to controls, as well levels of p21 complexed with Cdk2. Naloxone abolished the increased expression of p21 protein by OGF, suggesting a receptor-mediated activity. p21 specific siRNAs blocked OGF's repressive action on proliferation in BxPC-3, PANC-1, and Capan-2 cells; cells transfected with negative control siRNA had no alteration in p21 expression, and therefore were inhibited by OGF.
These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human pancreatic cancer is a p21 CKI pathway, expanding strategies for diagnosis and treatment of these neoplasias.
Partial hepatectomy (PH) endorses quiescent hepatocytes to reenter the cell cycle. The regenerating liver returns to its preresection weight after 7 days, following one or two cell division and maintains nearly its original volume after then. We focused on the inhibition of further hepatocyte proliferation, hypothesizing possible involvement of cell cycle upregulators and inhibitors. We studied protein levels in expression of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs), and their in situ hepatic lobular distributions in partial hepatectomized rat liver. Cyclin E was expressed in the same levels in normal liver and after PH. Expression of cyclin A, not detected in normal liver, increased in following times after PH and reached a maximum at 7 day. CDK2 and 4 showed increased expression toward terminal period. Contradictory findings of cyclin A and these CDKs might play an important role in the inhibition of further cell division, although still unclear. Constitutively expressed CDK6 decreased after 1 day. p18 showed peak expression within 1 day, and p16, p21, p27 and p57 were stronger at terminal periods. During the expected period of their activity, intranuclear translocations were observed in cyclin E, p18 and p16. There was no evidence of regional distribution in hepatic lobular architecture, instead, diffuse in situ expression, corroborating synchronous event, was found.
Epithelial carcinomas of the ovary exhibit the highest mortality rate among gynecologic malignancies. Studies found that the metabolism of glycolipids or carbohydrates is associated with acquirement of anticancer drug-resistance by cancer cells. This study was to characterize possible involvement of Lewis Y (LeY) antigen in the drug-resistance of cancer cells. We transfected the α1,2-fucosyltransferase gene into human ovarian carcinoma-derived RMG-1 cells and established RMG-1-hFUT cells with enhanced expression of LeY. We determined the effects of docetaxel on the survival of cells by MTT assaying and observed the apoptosis of cells in the presence of docetaxel by flow cytometric analysis and by transmission electron microscopy. Plasma membranes and intracellular granules in RMG-1-hFUT cells were stained with anti-LeY antibody, the intensity of the staining was higher than that in control cells. The RMG-1-hFUT cells exhibited higher resistance to docetaxel than the control cells with regard to the docetaxel concentration and time course. After treatment with 10 μg/mL docetaxel for 72 h, the control cells, but not RMG-1-hFUT, contained abundant positively stained cell debris due to disintegration of the cytoskeleton. On transmission electron microscopy, although the control cells treated with docetaxel as above showed the following morphology, i.e., absence of villi, cells shrunken in size, pyknosis, agglutinated chromatin and cell buds containing nuclei in the process of apoptosis, the RMG-1-hFUT cells showed only agglutinated chromatin and vacuoles in the cytoplasm. In summary, cells with enhanced expression of LeY were shown to acquire docetaxel-resistance, indicating the possible involvement of glycoconjugates in the drug-resistance.
ovarian cancer; Lewis Y antigen; docetaxel; drug resistance
During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration and/or downregulation of the CDK inhibitor p21Waf1/Cip1, without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression, which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins E1 and E2 are not always coordinately regulated.
Cell cycle regulators such as cyclins, cyclin dependant kinases (CDKs) or Rb play important roles in the differentiation of adipocytes. In the present paper we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here, that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to PPARγ, the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.
3T3-L1 Cells; Adipocytes; cytology; metabolism; Adipogenesis; genetics; Animals; Cells, Cultured; Cyclin G2; genetics; metabolism; Fluorescent Antibody Technique; Immunoprecipitation; Male; Mice; Mice, Inbred C57BL; PPAR gamma; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Up-Regulation; adipogenesis; cyclin G2; PPARγ
AIM: To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation.
METHODS: IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3β and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments.
RESULTS: Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3β, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3β induced by serum stimulation and decreased Cyclin D1 stability in the nucleus.
CONCLUSION: Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.
c-Fos; Cyclin D1; GSK-3; Cell growth; Cell cycle; Hepatoma; Epidermal growth factor
Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.
The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb) complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-dependent kinase 9) as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4+ T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1.
We used shRNA lentiviral vectors to stably deplete either Cyclin T2 or Cyclin T1 in HeLa cells. Total RNA extracted from these cells was subjected to cDNA microarray analysis. We found that 292 genes were down- regulated by depletion of Cyclin T2 and 631 genes were down-regulated by depletion of Cyclin T1 compared to cells transduced with a control lentivirus. Expression of 100 genes was commonly reduced in either knockdown. Additionally, 111 and 287 genes were up-regulated when either Cyclin T2 or Cyclin T1 was depleted, respectively, with 45 genes in common.
These results suggest that there is limited redundancy in genes regulated by Cyclin T1 or Cyclin T2.
Cyclin E, a G1 cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Δ48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.
Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.
Cyclin E is essential for progression through the G1 phase of the cell cycle and initiation of DNA replication by interacting with, and activating its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). We found a substantial increase in cyclin E mRNA, accompanied by increased production of cyclin E protein and cyclin E/Cdk2 kinase activity in multiple myeloma and lymphoma cells following irradiation. Cyclin E expression increased early in a time and dose-dependent manner, with a threefold induction reached 8 h following γ-irradiation. Run-on analyses indicated a predominantly transcriptional regulation of cyclin E. Stable overexpression of cyclin E, but not cyclin D1, sensitized IM-9 cells to γ-irradiation-induced apoptosis; in contrast, a dominant-negative Cdk2, prevented apoptosis. Irradiation of cyclin E overexpressing cells led to an enhanced caspase activation and exposure of the phosphatidylserine on the plasma membrane, two key markers of apoptosis, events which were completely abolished in cells expressing a dominant-negative Cdk2. This study identifies cyclin E as a target for activation by ionizing radiation and which plays a functional role in apoptosis of hematopoietic cells.
Cyclin E; cyclin dependent kinase 2; apoptosis; ionizing radiation; caspase-3
Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.
To observe whether cyclin D1 siRNA-mediated inhibition of cyclin D1 represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non–small cell lung cancers. To stably transfect the A549 cell line with a cyclin D1–targeted siRNA to downregulate cyclin D1 expression and observe the effects on protein expression, and tumor growth in vitro and in vivo. Expression of cyclin D1–targeted siRNA resulted in a decrease in cyclin D1, MMP-2, RhoA, and Rac1 protein levels, as detected by Western blot and immunofluorescence studies. Transfected cells also exhibited a marked decrease in the rate of cell growth, and decreased invasive capacity, compared to cells transduced with a scrambled siRNA plasmid and untransduced A549 cells. siRNA-mediated inhibition of cyclin D1 expression represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non–small cell lung cancers. It is the reason for inhibiting tumor growth so that cyclin D1 siRNA can inhibit the cell cycle progression. In addition, the mechanism of inhibiting tumor metastasis was related to the decrease in the expression of MMP-2, RhoA, and Rac1 after cyclin D1 was decreased by cyclin D1 siRNA.