Inactivated tumor suppressor Rb by nitric oxide promotes mitosis in human breast cancer cells

Inactivation of tumor suppressor protein retinoblastoma (Rb) is important mechanism for the G1/S transition during cell cycle progression. Human breast cancer cells T47D release great amount of nitric oxide (NO), but its relation to tumor suppressor Rb is unknown. In this study, it is shown that NO induces phosphorylation and inactivation of Rb tumor suppressor protein, increasing G2/M phase and cell proliferation of breast cancer cells T47D. NO did not induce changes in p53 ser-15 phosphorylation, the most phosphorylated site of p53 during its activation. These data indicate that NO induces cell proliferation through the Rb pathway. NO phosphorylates and inactivates tumor suppressor protein Rb inducing mitosis by the p53 independent pathway in breast cancer cell.     

The phosphorylation of retinoblastoma gene product in human myeloid leukemia cells during the cell cycle.

Counterflow centrifugal elutriation and immunoblotting techniques were used to study the expression of the retinoblastoma (RB) gene during the cell cycle of BV173 chronic myeloid leukemia (CML) cells. Our data showed that Rb protein started to be phosphorylated at early G1 phase, became hyperphosphorylated when cells progressed to late G1 and S phases during cell cycle, and remained hyperphosphorylated throughout S and G2/M phases. Our data suggest that Rb phosphorylation starts at a more distal point to the G1/S phase boundary in human myeloid leukemia BV173 cells rather than at a point more proximal to the G1/S boundary, as seen in HeLa cells.     

Speedy/Ringo C regulates S and G2 phase progression in human cells

Cyclin-dependent kinases (CDKs) control cell cycle transitions and progression. In addition to their activation via binding to cyclins, CDKs can be activated via binding to an unrelated class of cell cycle regulators termed Speedy/Ringo (S/R) proteins. Although mammals contain at least five distinct Speedy/Ringo homologues, the specific functions of members of this growing family of CDK activators remain largely unknown. We investigated the cell cycle roles of human Speedy/Ringo C in HEK293 cells. Down-regulation of Speedy/Ringo C by RNA interference delayed S and G2 progression whereas ectopic expression had the opposite effect, reducing S and G2/M populations. Double thymidine arrest and release experiments showed that overexpression of Speedy/Ringo C promoted late S phase progression. Using a novel three-color FACS protocol to determine the length of G2 phase, we found that the suppression of Speedy/Ringo C by RNAi prolonged G2 phase by ~30 min whereas ectopic expression of Speedy/Ringo C shortened G2 phase by ~25 min. In addition, overexpression of Speedy/Ringo C disrupted the G2 DNA damage checkpoint, increased cell death and caused a cell cycle delay at the G1-to-S transition. These observations indicate that CDK-Speedy/Ringo C complexes positively regulate cell cycle progression during the late S and G2 phases of the cell cycle.     

Disruption of the Rb--Raf-1 interaction inhibits tumor growth and angiogenesis

The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G(1) phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.     

Human cytomegalovirus infection inhibits G1/S transition.

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.     

蛋白激酶C与细胞周期

近年的研究表明,PKC涉及到细胞的周期调节。在酵母细胞和哺乳动物细胞均发现PKC参与细胞周期调控,从而提示PKC可能在进化上是一种保守的细胞周期调节子。一般认为PKC在两个点上对细胞周期起作用,即G1期和G2期到M期的过渡期（G2／M）。在G1期,PKC分别在早G1期和晚G1期作用有所不同,主要作用表现在使细胞停留在G1期的中末阶段,这一过程,主要涉及到抑制肿瘤抑制因子－成视网膜细胞瘤（Rb)蛋白的磷酸化。PKC的主要作用是降低周期素依赖激酶CDK2的活性、降低周期素E和A的表达和增加周期素依赖的周期抑制蛋白p21^WAF1和p27^KIP1的表达;在G2／M期,PKC对细胞周期的调节主要与Cdc2(CDK1)的活性抑制有关。     

Functional expression of the Na/K pump is controlled via a cyclosporin A-sensitive signalling pathway in activated human lymphocytes.

An immunosuppressant cyclosporin A (CsA) inhibits T-cell proliferation by blocking the nuclear factor of activated T-cells (NFAT) required for expression of the interleukin-2 (IL-2) gene. This work has demonstrated for the first time that in human blood lymphocytes (HBLs) activated by phytohemagglutinin (PHA), CsA at anti-proliferative doses inhibits the late sustained increase in ouabain-sensitive Rb(K) influxes, which accompanies the growth phase of G0/G1/S transition. CsA affects neither the initial, transient activation of the pump in response to PHA nor the ouabain-resistant ion fluxes during cell cycle progression. When the HBLs were rendered competent to proliferate by phorbol 12,13-dibutyrate ester and ionomycin in the presence of CsA, the exogenous IL-2 did not bypass the initial inhibitory effect of CsA on the long-term pump enhancement. When applied after the competence induction, CsA produced no effect on the sustained increase in ouabain-sensitive Rb influxes during the IL-2-induced progression phase. These results indicate that in activated HBLs, (1) IL-2 is involved in functional expression of the Na/K pump during cell transition from quiescence to proliferation, (2) the cell cycle-associated upregulation of the pump is related to a CsA-sensitive signalling pathway.     

Regulation of synthesis of p34cdc2 and its homologues and their relationship to p110Rb phosphorylation during cell cycle progression of normal human T cells.

In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions. The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells. Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro. Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis. Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives. In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin. The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated. It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable. Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition. Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34. Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date. A species of protein with a mobility slightly less than true p34 was found, even in resting T cells. Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen. Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.     

Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1.

We have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to reenter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G1. These results demonstrate that expression of cyclin A is rate limiting for the G1-to-S transition and suggest that cyclin A can function as a G1 cyclin. Although the level of exogenous cyclin A was constant throughout the cell cycle, its associated kinase activity increased as cells approached S phase. Low kinase activity in early G1 was found to correlate with the presence of p27Kip1 in cyclin A-associated complexes, while high kinase activity in late G1 was correlated with its absence. These results suggest that a function of p27Kip1 in G1 is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G1 led to phosphorylation of the product of the retinoblastoma susceptibility gene (pRb). Thus, cyclin A expression can be rate limiting for pRb phosphorylation, implicating pRb as a physiological substrate of the cyclin A-dependent kinase. Taken together, these results demonstrate that deregulated expression of cyclin A can perturb the normal regulation of the G1-to-S transition.     

Role of p53 tumor suppressor in ageing: regulation of transient cell cycle arrest and terminal senescence.

In this study we investigated the function of p53 as a regulator of cell cycle progression in cycling and senescent cells. Using the conditional temperature-sensitive (ts) mutant we could prevent the detrimental effect of constitutive expression of high levels of wt p53 protein. High levels of wt p53 inhibited cell proliferation by blocking the cells to progress from G1 to S phase of the cell cycle. Flow cytometric analysis revelaed a maintenance of G1 cell population for a longer time depending on the prolonged expression of wt p53 protein. The p53 mediated inhibition of cell proliferation and of the cycle was reversible. However, a spontaneous increase of wt p53 occurring in ageing normal human MRC-5 fibroblasts was associated with irreversible reduction of proliferative potential. The accumulation of G1 cells was detected by flow cytometry. By the measurement of DNA content it is not possible to discriminate between cells arrested in G1 and G0 phase, therefore, the expression of G1 markers was determined. Analysis of the expression of distinct cell cycle regulators revealed that quiescent MRC-5 cells were in G0 phase. Our results indicate that cell cycle arrest occurring in senescent cells is associated with the G0 transition.