Cell Cycle Regulatory Molecules Research Articles

NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells.

The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.

Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system; potential targets for therapy?

In the preceding sections we have described the potential for using cell cycle regulatory molecules as targets for drug development within the cardiovascular system. Opportunities for affecting the expression and activities of selected cell cycle regulatory molecules exist in interventional cardiological procedures such as PTCA to limit specifically the intimal hyperplasia of vascular smooth muscle cells that occurs following angioplasty. In addition, the potential for targeting the cardiac myocyte cell cycle to re-initiate cell division in a controlled manner would provide a suitable approach for repairing damaged areas of myocardial tissue following an infarct. Although this approach has not been demonstrated to date in vivo, data from transgenic mouse models and in vitro studies have implicated the cell cycle as a suitable target for manipulation. The next few years will enable the feasibility of this approach to be demonstrated.

Activation of telomerase and its association with G1-phase of the cell cycle during UVB-induced skin tumorigenesis in SKH-1 hairless mouse.

Telomerase is a ribonucleoprotein enzyme that adds hexanucleotide repeats TTAGGG to the ends of chromosomes. Telomerase activation is known to play a crucial role in cell-immortalization and carcinogenesis. Telomerase is shown to have a correlation with cell cycle progression, which is controlled by the regulation of cyclins, cyclin dependent kinases (cdks) and cyclin dependent kinase inhibitors (cdkis). Abnormal expression of these regulatory molecules may cause alterations in cell cycle with uncontrolled cell growth, a universal feature of neoplasia. Skin cancer is the most prevalent form of cancer in humans and the solar UV radiation is its major cause. Here, we investigated modulation in telomerase activity and protein expression of cell cycle regulatory molecules during the development of UVB-induced tumors in SKH-1 hairless mice. The mice were exposed to 180 mjoules/cm2 UVB radiation, thrice weekly for 24 weeks. The animals were sacrificed at 4 week intervals and the studies were performed in epidermis. Telomerase activity was barely detectable in the epidermis of non-irradiated mouse. UVB exposure resulted in a progressive increase in telomerase activity starting from the 4th week of exposure. The increased telomerase activity either persisted or further increased with the increased exposure. In papillomas and carcinomas the enzyme activity was comparable and was 45-fold higher than in the epidermis of control mice. Western blot analysis showed an upregulation in the protein expression of cyclin D1 and cyclin E and their regulatory subunits cdk4 and cdk2 during the course of UVB exposure and in papillomas and carcinomas. The protein expression of cdk6 and ckis viz. p16/Ink4A, p21/Waf1 and p27/Kip1 did not show any significant change in UVB exposed skin, but significant upregulation was observed both in papillomas and carcinomas. The results suggest that telomerase activation may be involved in UVB-induced tumorigenesis in mouse skin and that increased telomerase activity may be associated with G1 phase of the cell cycle.

Clear cell carcinoma has an expression pattern of cell cycle regulatory molecules that is unique among ovarian adenocarcinomas.

The purpose of this study was to identify biologic differences between ovarian clear cell carcinoma and other ovarian adenocarcinomas by comparing the expression of cell cycle regulatory molecules and by analyzing the survival of the patients. In 51 cases of epithelial ovarian carcinoma, the expression of the cell proliferation marker Ki-67 and that of the cell cycle regulatory molecules p53, p16, p21, p27, cyclin E, and cyclin A was studied using immunohistochemical techniques. The correlations among clinical stage, histologic subtype, labeling index for Ki-67, and expression of these cell cycle regulators were examined statistically. Multivariate survival analysis was performed using these factors in the Cox proportional hazards model. Clear cell carcinoma revealed such trends as low expression of both p53 and cyclin A and significantly increased expression of both p21 and cyclin E (compared with the other histologic subtypes). In all ovarian carcinomas, a very strong positive correlation (correlation coefficient 0.79; P < 0.0001) between p53 positive staining and cyclin A positive staining and a weak positive correlation (correlation coefficient 0.47; P < 0.01) between p21 positive staining and cyclin E positive staining were recognized at the level of expression of cell cycle regulatory molecules. Clinical stage was the only independent predictive factor for the survival of the patients. Among ovarian adenocarcinomas, clear cell carcinoma exhibits a unique pattern of expression of cell cycle regulatory molecules, though in this study the survival of the patients did not correlate with histologic subtype, only with clinical stage.

Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation.

Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation.

The cell cycle and apoptosis.

Which decisions can be more fundamental to the cell of a living organism than to divide or to die? Hence the tremendous efforts by biologists to understand both these processes. All somatic cells proliferate using the common mechanism of mitosis following progression through the cell cycle. Apoptosis, or programmed cell death, also appears to be a ubiquitous process in multi-cellular organisms where it serves to eliminate unwanted or damaged cells. In this chapter, we shall address the question of whether these two most fundamental of all biological pathways are interlinked. We shall discuss the differences between cell death in cycling cells from resting or quiescent cells. We shall examine the evidence that cell cycle regulatory molecules influence apoptosis and, that similarly, some of the molecules known to regulate apoptosis can impinge on cell cycle control. Finally we shall discuss models that attempt to interconnect the two processes.

Implication of transcription factor E2F in regulation of DNA replication.

The transcription factor E2F plays crucial roles in induction of S phase in mammalian cells by regulating the expression of genes that encode molecules involved in cell cycle progression. E2F exerts a repressive effect on E2F-responsive genes in G0/G1 phase by associating with the retinoblastoma tumor suppressor gene product pRb and the related protein p130. This repression is relieved by phosphorylation of the pRb family proteins by G1 cyclin (cyclin D and cyclin E) -dependent kinases, resulting in expression of E2F-responsive genes in late G1 with a peak at the G1/S boundary. One group of genes influenced by E2F encode cell cycle regulatory molecules, including members of the E2F family and cyclin E, demonstrating a loop-type regulation of activities of E2F and cyclin E-dependent kinase at this stage in the cell cycle. Another group is involved in DNA replication, including genes for molecules regulating initiation of DNA replication. Overexpression of E2F is sufficient to induce DNA synthesis in serum starved fibroblasts. In addition, overexpression of cyclin E, which is essential for entry into S phase, overcomes G1 arrest caused by inhibition of E2F activity without resuming E2F mediated transcription, suggesting the mergence of the two pathways. Thus, E2F target-gene products and cyclin E-dependent kinase activity apparently co-operate to initiate replication of DNA.

Raf-1 physically interacts with Rb and regulates its function: a link between mitogenic signaling and cell cycle regulation.

Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell surface receptors to the cell cycle regulatory molecules are not yet clear. In this study, we show that the signaling molecule Raf-1 can physically interact with Rb and p130 proteins in vitro and in vivo and that this interaction can be detected in mammalian cells without overexpressing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in proliferating cells. Raf-1 can inactivate Rb function and can reverse Rb-mediated repression of E2F1 transcription and cell proliferation efficiently. The region of Raf-1 involved in Rb binding spanned residues 1 to 28 at the N terminus, and functional inactivation of Rb required a direct interaction. Serum stimulation of quiescent human fibroblast HSF8 cells led to a partial translocation of Raf-1 into the nucleus, where it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction of Raf-1 with Rb is a vital step in the growth factor-mediated induction of cell proliferation and that Raf-1 acts as a direct link between cell surface signaling cascades and the cell cycle machinery.

Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy.

The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and in vitro kinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1 phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1 phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21(CIP1)- and p27(KIP1)- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1 phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.

Role of G1 phase cyclins and cyclin-dependent kinases during cardiomyocyte hypertrophic growth in rats.

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1-3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly from day 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased approximately 70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P < 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1 to G2/M phase progression in AC myocyte nuclei (22.0 +/- 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 +/- 0.8% in G2/M; P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

Control of vascular smooth muscle cell growth and its implication in atherosclerosis and restenosis (review).

Abnormal proliferation of vascular smooth muscle cells (SMCs) is thought to contribute to neointimal thickening during spontaneous atherosclerosis and vessel renarrowing (restenosis) after angioplasty. This review will focus on the mechanisms underlying cell-cycle control in SMCs and its implication in atherosclerosis and restenosis. Therapeutic approaches that targeted specific cell-cycle control genes or growth regulatory molecules which effectively inhibited neointimal lesion formation will also be discussed.

Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor.

Intermediate trophoblast (IT) rarely gives rise to a placental site trophoblastic tumor (PSTT) To examine the different growth mechanisms present in normal and neoplastic IT, the expression of cell cycle regulatory molecules was compared at normal implantation sites and in PSTTs. Normal implantation sites in early gestation (19 patients) and PSTTs (6 patients) were immunohistochemically studied using antibodies against cytokeratin, human chorionic gonadotropin, and human placental lactogen to identify IT, and antibodies against Ki-67, cyclins (A, B, D1, and E), cyclin-dependent kinases (cdks), and p53 to investigate the proliferative activity of the trophoblast. Marked proliferative activity was observed in the trophoblast of the cell columns. Normal IT exhibited a very low labeling index for Ki-67, with negative expression for cdks and cyclins, except for cyclins B and E. The tumor cells of PSTT exhibited a high labeling index for Ki-67 with positive expression for all the cyclins and cdks examined. Expression of p53 was identified in tumor cells of PSTTs and the distribution of p53-positive cells correlated topographically with that of the cyclin A-positive cells. The transformed IT of PSTT has high proliferative activity with an abnormal expression of cell cycle regulatory molecules, which is not observed in normal IT.

Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells.

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.

Cyclin Dependent Kinase Inhibitors and Dominant Negative Cyclin Dependent Kinase 4 and 6 Promote Survival of NGF-Deprived Sympathetic Neurons

Neuronal apoptosis plays a critical role in both normal development and disease. However, the precise molecular events controlling neuronal apoptosis are not well understood. Previously, we hypothesized that cell cycle regulatory molecules function in controlling the apoptotic pathways of trophic factor-deprived neurons. To test this hypothesis, we used the RNA alphavirus Sindbis to express three known cyclin dependent kinase inhibitors (CKIs), p16(ink4), p21(waf/cip), and p27(kip1), and dominant negative mutant forms of four known G1 cyclin dependent kinases (CDKs), Cdk2, Cdk3, Cdk4, and Cdk6, in primary cultured rat superior cervical ganglion sympathetic neurons. We demonstrate that expression of each of the CKIs protects the postmitotic cultured neurons from apoptotic death evoked by withdrawal of NGF. In addition, we show that expression of dominant negative forms of Cdk4 or Cdk6, but not Cdk2 or Cdk3, protects NGF-deprived sympathetic neurons from death. Such findings suggest the participation of several CDKs and their cognate cyclins in a neuronal apoptotic pathway.

Control of cell cycle regulatory proteins and modulation of STAT1 proteins by IFN-gamma in human prostatic JCA-1 cells

The addition of human recombinant IFN-gamma (10(2) and 10(3) IU/ml) inhibited human prostatic JCA-1 cell growth by 27% and 64%, respectively. Since the high dose of IFN-gamma elicited an increase in G(1) concomitant with a decrease in G(2)/M phases of the cell cycle, changes in the expression of cell cycle regulatory protein molecules were analyzed by Western blots. Results of these experiments show that IFN-gamma down regulated the G(1)/S transition molecules, e.g., cyclin D1, the cyclin-dependent protein kinase Cdk4, and the retinoblastoma gene product (pRB), but increased the G(2)/M transition molecules, e.g., cyclin B1 and p34(cdc2) (Cdk1). Possible modulation of Cdk-inhibitors (CDKIs), e.g., p53 and p21(WAF1), which have checkpoint functions in the cell cycle, by IFN-gamma, was also studied. The p53 was induced by both 10(2) and 10(3) IU/ml IFN-gamma. At 10(3) IU/ml, IFN-gamma inhibited p21(WAF1), increased the expression of STAT1 alpha, and sustained the elevated STAT1 alpha for up to 96 h. Thus several mechanisms may be involved in the antiproliferative effects of IFN-gamma.

Different points of action of retinoids and anti-estrogens in G1 phase identified in synchronized T-47D breast cancer cells.

Both retinoids and anti-estrogens inhibit breast cancer cell proliferation with accumulation of cells in the G1 phase of the cell cycle, but the effect of retinoids is delayed compared to that of anti-estrogens. To determine whether this temporal difference is due to a simple delay in the action of retinoids on a common site or to different sites of action within the G1 phase, we studied the cell cycle effects of retinoic acid (RA) and the anti-estrogen ICI 164384 (ICI) in T-47D cells partially synchronized by mevalonic acid rescue of lovastatin-induced cell cycle arrest. We found that cells entering the cell cycle semi-synchronously after mevalonic acid rescue of lovastatin treatment were immediately susceptible to ICI but not RA. This suggests that RA may act at a point up-stream and ICI at a point down-stream of lovastatin action. Consistent with this, cells recommencing cell cycle progression after RA treatment were susceptible to the effects of lovastatin, while cells pre-treated with ICI then rescued with estradiol were not. In addition, cells rescued from cell cycle arrest induced by either RA, ICI or lovastatin entered S phase with the same kinetics. Our findings suggest, first, that within G1, RA acts before and ICI acts after the point of lovastatin action and, second, that despite these differences in the initiation of cell cycle arrest, the final nature of the cell cycle arrest is similar. Hence, retinoids and anti-estrogens may be expected to target different cell cycle-regulatory molecules to initiate cell cycle arrest, while overcoming this arrest may be accomplished by the activation of a common molecular pathway.

Cell-free ubiquitination of cell cycle regulators in budding yeast extracts

Publisher Summary This chapter discusses the cell-free ubiquitination of cell cycle regulators in budding yeast extracts. Proteolysis of key cell cycle-regulatory molecules is one method of ensuring the irreversibility of phase transitions within the eukaryotic cell cycle. Degradation of such molecules is typically mediated by the ubiquitin pathway, and requires the concerted action of a complex array of proteins including ubiquitin-activating (El), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. The action of these enzymes results in the assembly of multiubiquitin chains on the substrate, which is then targeted to the 26S proteosome where it is cleaved into small peptides. To define the mechanism of action of the ubiquitin-dependent proteolyric pathways that regulate the eukaryotic cell cycle, one needs to have cell-free systems that faithfully reproduce these reactions.

Relationship between the expression of cyclins/cyclin-dependent kinases and sex-steroid receptors/Ki67 in normal human endometrial glands and stroma during the menstrual cycle.

Cell cycle regulatory molecules were analysed in normal human endometrial tissue using antibodies against cyclins D1, E, A, and B1 and cyclin-dependent kinases (CDKs) cdk4, cdk2, and cdc2. The expression of these regulatory molecules in gland cells and stromal cells was compared with the expression of oestrogen receptors (ER), progesterone receptors (PR), and Ki67 (a growth-related molecule). In general, a substantially higher percentage of the gland cells stained positive for cyclins and CDKs during the proliferative phase of the menstrual cycle. Cyclin E, cdk2 and/or cdk4 were especially apparent in the cytoplasm of most of the gland cells as well as in the stromal cells. In contrast, most of the regulatory molecules were undetectable in the gland cells by the end of the secretory phase of the cycle, but they did not decline in the stromal cells. The data also revealed that ER, PR, and Ki67 in both gland cells and stromal cells follow the same basic pattern of expression as the cyclins and CDKs. These results suggest that cyclins and CDKs are functionally involved in the rhythmic proliferation of normal human endometrial tissue, and the action of these agents may be related to the endometrial levels of sex steroids and Ki67.