Monoterpenes inhibit proliferation of human colon cancer cells by modulating cell cycle-related protein expression

The monoterpene perillyl alcohol (POH) is a naturally occurring anti-cancer compound which is effective against a variety of rodent organ-specific tumor models. To establish the molecular mechanisms of POH and its major metabolite perillic acid (PA) as anti-proliferative agents, their effects on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in HCT 116 human colon cancer cells. POH, and to a lesser extent, PA, exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed that monoterpenes increased expression of cdk inhibitor p21(Waf1/Cip1) and cyclin E, and decreased expression of cyclin D1, cyclin-dependent kinase (cdk) 4 and cdk2. Our results suggest that monoterpenes induce growth arrest of colon cancer cells through the up-regulation of p21(Waf1/Cip1) and the down-expression of cyclin D1 and its partner cdk4.     

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.     

Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.     

Differential effects of transforming growth factor on cell cycle regulatory molecules in human myeloid leukemia cells.

In this report we have studied the mechanism by which Transforming Growth Factor beta (TGF beta) inhibits growth of human myeloid leukemia cell lines. TGF beta 1 arrested cells in G1 phase and significantly downregulated the expression of cyclin D2, cyclin D3, cdk4, cyclin A, and cdk2. The downregulation of the molecules resulted in approximately 50-90% decrease of the molecule-dependent kinase activity, varying with each molecule. Although treatment of cells with TGF beta 1 up-regulated accumulation of p27(kip1) in both nucleus and cytoplasm, the association of the p27(kip1) with cdk2, cyclin A, cyclin D2, cyclin D3, and cdk4 was markedly down-regulated, suggesting that p27(kip1) is not responsible for the downregulation of the kinase activity. In contrast, TGF beta 1 upregulated cyclin E-associated p27(kip1) with no effect on the expression of cyclin E. p27(kip1)-immunodepletion upregulated cyclin E-dependent kinase activity by more than 10-fold in TGF beta 1-treated cells but not in proliferating cells; whereas immunodepletion of p27(kip1) from cdk2-immunoprecipitates markedly downregulated cdk2 kinase activity in the lysates extracted from both proliferating and TGF beta-treated cells. Consistent with this observation, TGF beta 1 and p27(kip1) antisense cDNA had a synergistic or additive inhibitory effect on cdk2 but not cyclin E-dependent kinase activity. Our data suggest that (1) TGF beta 1-mediated growth inhibition is accomplished through multiple pathways and (2) p27(kip1) has opposing effects on cdk2 and cyclin E activity in response to TGF beta 1.     

The role of RhoA in vulvar squamous cell carcinoma: a carcinogenesis,progression, and target therapy marker

Ras homologue gene family member A (RhoA) is involved in tumor mobility, invasion, and metastasis. We detected RhoA expression in vulvar squamous cell carcinoma (VSCC) tissue, measured RhoA expression in the VSCC cell phenotype, and measured the expression of the relevant molecules after RhoA small interfering RNA (siRNA) transfection in SW962 cells. RhoA has a higher expression level in VSCC than normal vulva skin tissue and was positively associated with the International Federation of Gynecology and Obstetrics (FIGO) stage and differentiation; besides, VSCC patients with lymph node metastasis had higher positive RhoA expression. RhoA messenger RNA and protein expression was significantly reduced in the RhoA siRNA transfectants as compared with the negative control (NC) and mock-transfected cells (p?<?0.05). The RhoA siRNA transfectants lead to low growth, G1 arrest, high apoptosis, low migration and invasion (p?<?0.05), and suppressed lamellipodia formation as compared to NC and mock-transfected cells. Besides, matrix metalloproteinase-2 (MMP2), MMP9, and cyclinA1 protein expression was downregulated, while that of Bax was upregulated in the RhoA siRNA transfectants (p?<?0.05). SW962 cell proliferation rates were significantly lovastatin dose-dependent. Lovastatin caused G1 arrest, high apoptosis, low migration and invasion (p?<?0.05), and suppression of lamellipodia formation. Similar to the RhoA siRNA transfectants, lovastatin treatment downregulated RhoA, MMP2, MMP9, and cyclinA1 protein expression, while upregulating that of Bax as compared to that of the NC (p?<?0.05). Abnormal RhoA expression in vulvar carcinoma is involved in tumor proliferation and invasion and may be a treatment target. The RhoA inhibitor lovastatin alters VSCC cell migration and proliferation and may be effective for treating VSCC.     

The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function

VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with > or =4N DNA content, followed by cell death. Here, we define the role of p53 and p21(Waf1/Cip1) in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21(Waf1/Cip1) and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21(Waf1/Cip1) within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21(Waf1/Cip1) is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21(Waf1/Cip1) expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21(Waf1/Cip1) in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21(Waf1/Cip1)-dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis.     

Expression of the cell cycle regulator p27(Kip1) in normal squamous epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma of the uterine cervix. Immunohistochemistry and functional aspects of p27(Kip1).

BACKGROUND: Abnormality of cell cycle regulators and tumor suppressors, such as cyclin dependent kinase inhibitors (cdkIs), has been reported in malignant tumors. The current study was undertaken to examine the involvement of a cdkI, p27(Kip1) (p27), in the neoplastic process of the uterine cervical epithelium. METHODS: Immunohistochemical staining of p27 was performed in samples of normal cervical tissue (30 samples), cervical intraepithelial neoplasias (CINs; 17 samples), and invasive squamous cell carcinoma (SCC; 25 samples). The results were compared with the expression levels of Ki-67, cdk2, and cyclin E. The functional aspects of the p27 protein, such as its ability to bind to cdk2 and the phosphorylation activity of p27-bound cdk2, also were evaluated with an immunoprecipitation and histone H1 kinase assay. RESULTS: In normal cervical epithelia, the expression of p27 was strong in the intermediate and superficial cells but very weak in the parabasal cells. In CIN samples, the expression of p27 was negligible. The expression of p27 in these tissues showed an inverse topologic correlation to that of Ki-67, cdk2, and cyclin E. However, it is noteworthy that the number of p27 positive cells increased in SCC samples that also showed increased expression of Ki-67, cdk2, and cyclin E. The p27 protein in SCC samples was bound to cdk2 and cyclin E. However, cdk2 that was bound to p27 still possessed histone H1 kinase activity. CONCLUSIONS: The expression of p27 may be involved in the growth regulation of the normal squamous epithelium in the uterine cervix. However, aberrant function of p27 expression may occur in invasive SCC of the cervix.     

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.     

UVB induced cell cycle checkpoints in an early stage human melanoma line, WM35

The activation of cell cycle checkpoints in response to genotoxic stressors is essential for the maintenance of genomic integrity. Although most prior studies of cell cycle effects of UV irradiation have used UVC, this UV range does not penetrate the earth's atmosphere. Thus, we have investigated the mechanisms of ultraviolet B (UVB) irradiation-induced cell cycle arrest in a biologically relevant target cell type, the early stage human melanoma cell line, WM35. Irradiation of WM35 cells with UVB resulted in arrests throughout the cell cycle: at the G1/S transition, in S phase and in G2. G1 arrest was accompanied by increased association of p21 with cyclin E/cdk2 and cyclin A/cdk2, increased binding of p27 to cyclin E/cdk2 and inhibition of these kinases. A loss of Cdc25A expression was associated with an increased inhibitory phosphotyrosine content of cyclin E- and cyclin A-associated cdk2 and may also contribute to G1 arrest following UVB irradiation. The association of Cdc25A with 14-3-3 was increased by UVB. Reduced cyclin D1 protein and increased binding of p21 and p27 to cyclin D1/cdk4 complexes were also observed. The loss of cyclin D1 could not be attributed to inhibition of either MAPK or PI3K/PKB pathways, since both were activated by UVB. Cdc25B levels fell and the remaining protein showed an increased association with 14-3-3 in response to UVB. Losses in cyclin B1 expression and an increased binding of p21 to cyclin B1/cdk1 complexes also contributed to inhibition of this kinase activity, and G2/M arrest. Oncogene (2000) 19, 4480 - 4490.     

Enhancement of hypoxia-induced apoptosis of human breast cancer cells via STAT5b by momilactone B

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.     

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.     

Inhibition of cdk2 kinase activity by methylselenocysteine in synchronized mouse mammary epithelial tumor cells

Methylselenocysteine (MSC), an organic selenium compound has significant anticarcinogenic activity against mammary tumorigenesis. Previous experiments have demonstrated that MSC and inorganic selenite inhibit mammary cell (TM6 cell line) growth through different pathways. The present investigation demonstrated that MSC arrested cells in S phase during the TM6 cell cycle, which was followed by cells entering apoptosis at 48 h. Methylselenocysteine specifically affected the cdk2 kinase activity of the TM6 cells (54% reduction) at 16 h after release from growth arrest. The cdk4 kinase activity did not change during the cell cycle, confirming that cells had passed the G1 checkpoint and had entered S phase. The amount of cyclin E associated with cdk2 was increased by MSC by the 12 h time point, thereby facilitating entry of cells into S phase. Afterwards, cyclin E and cyclin A associated with cdk2 did not change for the remainder of the cell cycle. The data demonstrate that inhibition of mammary cell growth by MSC is mediated by alterations in progression of cells through S phase. The decrease in cdk2 kinase activity is coincident with prolonged arrest in S phase. One consequence of prolonged arrest may be apoptosis.      

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines.

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1 h, 42 degree C). In IGROV-1 cells, a low concentration (15 microg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27(Kip1) and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

HMG-CoA reductase inhibitor mevastatin enhances the growth inhibitory effect of butyrate in the colorectal carcinoma cell line Caco-2.

Mevastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis. Butyrate, a short-chain fatty acid, reduces proliferation and induces differentiation of human colon cancer cells. The aim of our study was to determine the effect of mevastatin, alone or in combination with butyrate, on proliferation, the cell cycle and apoptosis in the human colorectal carcinoma cell line Caco-2. In this report we show that mevastatin combined with butyrate synergistically suppressed growth of Caco-2 cells in a dose- and time-dependent manner. In addition, incubation with mevastatin arrested cells in the G1 phase of the cell cycle after 24 h with a switch to the G2/M phase after 72 h. This was accompanied by a down-regulation of cyclin-dependent kinases (cdk) 4 and cdk 6 as well as cyclin D1, while cdk 2 and cyclin E protein levels remained unchanged during mevastatin treatment. Cell cycle inhibitors p21 and p27 were significantly upregulated by mevastatin. The proapoptotic properties of mevastatin were further enhanced by co-incubation with butyrate. Lastly, the effects of mevastatin could be reversed by addition of mevalonate, but not farnesyl- or geranylgeranylpyrophosphate, intermediate products of cholesterol synthesis, to the medium. These results suggest that HMG-CoA reductase inhibitors like mevastatin may enhance the antiproliferative effect of butyrate in colon cancer cells via induction of apoptosis together with a G0/G1 cell cycle arrest.     

Resveratrol causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells.

Resveratrol (trans-3,4',5,-trihydroxystilbene), a phytoalexin found in grapes, nuts, fruits, and red wine, is a potent antioxidant with cancer-preventive properties. The mechanism by which resveratrol imparts cancer chemopreventive effects is not clearly defined. Here, we demonstrate that resveratrol, via modulations in cyclin-dependent kinase (cdk) inhibitor-cyclin-cdk machinery, results in a G(1)-phase arrest of the cell cycle followed by apoptosis of human epidermoid carcinoma (A431) cells. Resveratrol treatment (1-50 microM for 24 h) of A431 cells resulted in a dose-dependent (a) inhibition of cell growth as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, (b) G(1)-phase arrest of the cell cycle as shown by DNA cell cycle analysis, and (c) induction of apoptosis as assessed by ELISA. The immunoblot analysis revealed that resveratrol treatment causes a dose- and time-dependent (a) induction of WAF1/p21; (b) decrease in the protein expressions of cyclin D1, cyclin D2, and cyclin E; and (c) decrease in the protein expressions of cdk2, cdk4, and cdk6. Resveratrol treatment was also found to result in a dose- and time-dependent decrease in kinase activities associated with all of the cdks examined. Taken together, our study suggests that resveratrol treatment of the cells causes an induction of WAF1/p21 that inhibits cyclin D1/D2-cdk6, cyclin D1/D2-cdk4, and cyclin E-cdk2 complexes, thereby imposing an artificial checkpoint at the G(1)-->S transition of the cell cycle. This series of events results in a G(1)-phase arrest of the cell cycle, which is an irreversible process that ultimately results in the apoptotic death of cancer cells. To our knowledge, this is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of cancer cells by resveratrol.     

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.