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The effects of cell cycle inhibition on the expression of the multidrug resistance transporter P-glycoprotein (P-gp) as well as of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p21WAF-1 were investigated in DU-145 prostate tumor spheroids. With increasing spheroid size the number of cells in the G0/G1 phase augmented, whereas the number of cells in the G2/M phase and the S phase of the cell cycle declined. The number of G0/G1 cells was elevated after incubation with either mimosine, staurosporine or serum-free medium. Mitomycin C and roscovitine increased the number of S phase cells. Roscovitine additionally increased cells in the G2/M phase. Incubation in serum-free medium upregulated p21WAF-1, p27Kip1 and P-gp. Mimosine treatment resulted in upregulation of p27Kip1 and P-gp, whereas p21WAF-1 remained unchanged. Upon roscovitine treatment p27Kip1 and p21WAF-1 were downregulated, whereas P-gp was unaltered. Mitomycin C treatment resulted in downregulation of p27Kip1 and p21WAF-1; no significant change in P-gp levels was observed. Staurosporine induced upregulation of p21WAF-1 whereas p27Kip1 remained unaltered. P-gp was downregulated upon staurosporine treatment, which was owing to an elevation of intracellular reactive oxygen species by this compound. It is concluded that upregulation of P-gp in G0/G1 phase cells requires coexpression of the CDK inhibitor p27Kip1 but not the CDK inhibitor p21WAF-1. [Copyright &y& Elsevier]

It has been demonstrated that exquisite regulation of the cell cycle between the activation and inhibition is crucial to maintain the transparency of the ocular lens. While it is generally recognized that the sugar cataract is accompanied by the enhanced proliferation of lens epithelial cells (LECs), it is unclear whether or not an inhibitory mechanism against the lens proliferation is involved, except for TGF-β. In this study, the authors demonstrated the enhanced expression of p21WAF-1/CIP-1, a potent inhibitor against cell cycle progression, and its specific temporal and regional expression profiles in the LECs during the development of sugar cataract. Sugar cataract was induced in 6-week-old male Sprague-Dawley rats by feeding them on a 50% galactose-rich diet, and then the expression patterns of p21WAF-1/CIP-1 mRNA and protein with the advance of the sugar cataract were studied. Western blot analyses showed that p21WAF-1/CIP-1 expression increased throughout the period of galactose exposure, up to 21 days. Also, a gradual increase in the number of p21WAF-1/CIP-1 positive cells was observed immunohistochemically in the course of the galactose exposure. Interestingly, p21WAF-1/CIP-1 was significantly expressed in the multi-layered epithelium, which was observed typically in the advanced cataract. Proliferating cell nuclear antigen (PCNA), an indicator of cell proliferation, was also positive in the most multi-layered epithelial cells. In addition, transient expression of PCNA mRNA and its protein was noticed throughout the lens epithelium in the course of the sugar cataract development. Prior to the elevation of p21WAF-1/CIP-1 mRNA expression, PCNA mRNA expression increased greatly and reached a peak according to the semiquantitative analyses using either the real time reverse transcription–polymerase chain reaction (RT–PCR) or the Southern blot analyses. Based on these observations, it is possible that p21WAF-1/CIP-1 is elevated and exerts its inhibitory action against the proliferating epithelial cells during the development of the sugar cataract. [Copyright &y& Elsevier]

Parathyroid adenomas (PTAs) are the main cause of primary hyperparathyroidism. Cell cycle regulation in normal parathyroid tissue (NPT) and PTA remains largely unknown. We have systematically explored several components involved in the p53/MDM2/p19ARF pathway in PTA and compared the results were with NPT. Forty-six PTA and 12 NPT were immunostained with anti-p21WAF-1, MDM2, p53, and p27KIP1 antibodies. The slides were processed by cytometry and the results were statistically analyzed using nonparametric methods (Mann-Whitney test). p21WAF-1 and MDM2 expression were significantly higher in PTA compared with NPT ( p<0.05). The opposite results were found for p27KIP1 ( p<0.05). Occasional p53 staining was found in some PTA, albeit no significant difference was found in comparison with NPT. In conclusion, MDM2 and p21WAF-1 are the proteins more overexpressed in PTA. These findings are surprising taking into account the benign nature of PTA, making them suitable candidates for further molecular analysis. [ABSTRACT FROM AUTHOR]

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitinconjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and coimmunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by cotransfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21waf-1 expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53- dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1. [ABSTRACT FROM AUTHOR]

Multiple genetic alterations, several of which may be important prognostic markers, characterize the development of cancer in pancreas. We review our findings from previously published studies with regard to molecular alterations associated with survival differences in patients treated with conventional radiation and chemotherapies used as adjuvant or palliative therapy. K- ras-negative patients with pancreas cancer show improved survival with radiation therapy compared to K- ras-positive patients with pancreas cancer. p53 expression is associated with shorter survival when compared to no p53 expression in pancreas cancer patients treated with radiation therapy or chemotherapy. Pancreas cancer patients whose tumors express p21 show significant survival advantages when treated with chemotherapy or radiation therapy. An inverse relationship is observed with respect to p21 and p53 expression and clinical stage. Although stage and surgical resectability remain the most important variables with respect to pancreas cancer survival, these findings suggest promising opportunities for gene therapies designed to enhance p21 expression or restore wild-type K- ras or p53 function in pancreatic tumors. [ABSTRACT FROM AUTHOR]