Involvement of pRB-related p107 protein in the inhibition of S phase progression in response to genotoxic stress.

pRB family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G1 to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G1 restriction point, they are hyperphosphorylated by G1 cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G1 regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells.

Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression.

The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.

Ras and signal transducer and activator of transcription (STAT) are essential and sufficient downstream components of Janus kinases in cell proliferation.

Cytokines exert their activities in cell growth and differentiation by binding specific cell membrane receptors. Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and they play crucial roles in transducing signals triggered by the cytokine-receptor interaction. We have previously shown that conditional activation of JAK through membrane-proximal dimerization confers cytokine-independence on interleukin-3 (IL-3)-dependent Ba / F3 lymphoid cells and that the cytokine-independent proliferation is completely inhibited by dominant negative Ras. In this work, we demonstrate that ectopic expression of a dominant negative form of Stat5, a major signal transducer and activator of transcription (STAT) expressed in Ba / F3 cells, also inhibits JAK-triggered mitogenesis. In contrast, overexpression of constitutively active Ras or conditional activation of Stat5 by chemical dimerization fails to confer cytokine-independence. However, concomitant activation of ectopic Ras and Stat5 molecules in Ba / F3 cells suffices for cell proliferation in the absence of IL-3. Our results indicate that Ras and STAT are essential and sufficient components of JAK-triggered mitogenesis. Our findings further indicate that the cytokine signal bifurcates into Ras and STAT pathways following JAK activation.

Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex.

The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase. The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A.

beta3-endonexin as a novel inhibitor of cyclin A-associated kinase.

Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals. In this study, we demonstrate that beta3-endonexin, a molecule known to associate with the integrin beta3 cytoplasmic domain, specifically binds cyclin A. Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta3-endonexin to interact with cyclin A. In an in vitro kinase assay, beta3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected. Coexpression of beta3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase. Our results indicate that beta3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity.

Changes in levels of mucosal glycoprotein on cysteamine-induced duodenal ulcers in rats.

Duodenal ulcers were induced in rats by cysteamine administration. Time-course changes in duodenal hexosamine, blood flow and intragastric pH were measured, as well as changes in the susceptibility of Brunner's glands to concanavalin A (Con A) staining. Hexosamine contents in the duodenum decreased significantly at 5 and 24 h. In the control group, Brunner's glands were stained brown using Con A staining. One day after cysteamine administration, the extent of Con A staining markedly decreased. At 3 and 7 days, gland susceptibility to staining was essentially the same as that of the control group.

Follow-up endoscopic study of gastric mucosal changes secondary to gastric ulcer.

A long-term follow-up gastrocamera photographic study of 481 patients with gastric ulcer was conducted at intervals of 3 weeks to 6 months. It showed that gastric ulcers could remain healed for 2 months to 6 years of observation, yet, recurrence of ulceration occurred in 79% including 18% with multiple occurences. Gastric polyp developed in 8 patients whose initial gastrophotography showed no such lesion in the original study. Furthermore, 7 cancers of the stomach developed at sites away from the initial benign ulcer foci. Six of these cases proved to be mucosal cancer and one was an advanced cancer. In 5 of 7 cases of malignancy, the cancer occurred distal to the initial ulcer foci and anaplastic adenocarcinoma was seen in 3 of these. The others were well differentiated adenocarcinomas. No correlation was found among histopathological type of cancer, depth of malignant invasion, location of the new growth or gastric juice acidity in these 7 cases of malignancy. It is concluded that a thorough and routine gastroscopic examination is indicated for all sorts of gastric disorders whether they are initial or follow-up cases.