Aberrations in the G1/S transition of the cell cycle have been observed in many malignancies and seem to be critical in the transformation process. Few studies have delineated the presence of G1/S regulatory defects and their clinical relevance in renal cell carcinoma (RCC). Therefore, we have examined the protein contents of cyclin D1, D3, E, and p27 in 218 RCCs, using tissue microarray and immunohistochemistry. The results from a subset of tumours were confirmed by Western blotting and immunohistochemical staining of regular tissue sections. Interestingly, low protein contents of cyclin D1 and p27 were associated with high nuclear grade, large tumour size, and poor prognosis for patients with conventional tumours. We further observed substantial differences in the pattern of G1/S regulatory defects between the different RCC subtypes. The majority of both conventional and papillary cases expressed p27; however, chromophobe tumours generally lacked p27 staining. In addition, conventional RCCs often expressed high cyclin D1 protein levels, while papillary RCCs exhibited high cyclin E. In summary, we have shown that G1/S regulatory defects are present in RCC and are associated with clinico-pathological parameters. The pattern of cell cycle regulatory defects also differed between RCC subtypes.

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Aberrations in the G1-S transition have been observed in several malignancies, suggesting that cell cycle defects are linked to the activation of oncogenes and inactivation of suppressor genes involved in the transformation process. The frequency of G1/S aberrations in human renal cell carcinoma (RCC) has not been fully clarified. We have therefore analyzed the cyclin E content, using Western blotting, in 79 RCC and 12 corresponding kidney cortex tissues as well as the fraction of p27-positive cells in 73 RCCs, using immunohistochemistry. Most of the tumors (65%) exhibited higher cyclin E levels than corresponding normal kidney cortex tissues. However, only a small fraction of the tumors (3 of 80) had excessive levels of cyclin E when cyclin E levels were compared with proliferation. Cyclin E levels higher than the median value were associated with aneuploidy (p ‫؍‬ 0. Key words: renal cell carcinoma; G1/S transition; cell cycle; cyclin; p27; proteinHuman renal cell carcinoma (RCC) is a malignant disease with a variable clinical course. Tumor stage is the best predictor of prognosis and is correlated with tumor progression. 1 Other parameters that predict prognosis are tumor grade, tumor cell proliferation and DNA ploidy. 2 During the last decade a detailed genetic characterization has allowed a classification of RCC that correlates with recognizable histopathologic features. 3 Conventional RCC, the most common tumor type, is characterized by chromosome 3p deletions. Aberrations on chromosomes 8, 9, 13 and 14 and nonrandom alterations involving 5q, 6q and 10q have also been described in this tumor type. 4 Papillary RCCs generally have trisomies of chromosomes 7 and 17 and loss of the Y chromosome, often in combination with additional trisomies of chromosomes 12, 16 and 20. 3 Chromophobe RCC is characterized by a combination of monosomies of chromosomes 1, 2, 10, 13, 17 and 21. 5 Among these RCC types, patients with conventional RCC had the poorest outcome. 6 A complex machinery consisting of cyclin-dependent kinases (CDKs) and sets of activating and inhibitory molecules controls the G1-S transition in the cell cycle. Both cyclins D and E are involved in the sequential activation of various CDKs, which, if the specific CDK inhibitors are detached from the cyclin-CDK complexes, allows phosphorylation of key substrates such as the retinoblastoma protein and release of transcription factors like E2F. This control system governs the transfer of cells from G1 to S-phase and initiation of DNA-replication, which for most cells lead to completion of the cell cycle and division into two daughter cells. Defects in the G1-S transition have been proposed to be one of a rather limited number of key events in the process leading to transformation. 7,8 Cyclin E and p27 are two important G1/S regulatory molecules that are often deregulated in malignancies, indicating an association with tumor development, progression or proliferation. 9 -13 The possibility for tumor cells to respond properly to genetic damages, such as a p53-...

RESULTSOf the tumours, 75% expressed endoglin, and in conventional RCC the expression was inversely correlated to the Tumour-NodeMetastasis (TNM) stage ( P = 0.008) and nuclear grade ( P = 0.01). There was no correlation between endoglin expression and gender, age, tumour size or cell type. Patients with conventional RCC and high endoglin expression had a more favourable prognosis than those with tumours with lower expression ( P = 0.04). A multivariate analysis of prognostic factors showed that TNM stage and nuclear grade were independent predictors of prognosis. Endoglin expression did not add further prognostic information. CONCLUSIONThese results indicate that endoglin expression is inversely related to stage and grade in RCC, and that it is associated with prognosis.

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Intratumoural microvessel density (MVD) has prognostic significance in selected neoplasms. To evaluate the prognostic information of MVD in renal cell carcinoma (RCC) we assessed the immunohistochemical expression of CD31 in 208 tumours using the tissue microarray technique. The expression was related to tumour cell type, TNM stage, nuclear grade and survival. CD31 expression was significantly (p < 0.001) higher in 167 conventional RCCs (clear cell) compared to 28 papillary RCCs. In conventional RCC, CD31 expression was inversely correlated to TNM stage (p = 0.024) and nuclear grade (p = 0.010). To evaluate the impact of CD31 expression on tumour-specific survival, the conventional RCC tumours were subdivided into quartiles according to the CD31 expression. In univariate analysis, patients with tumours in the quartile with the highest expression had a more favourable prognosis (p = 0.01) compared to those with lower CD31 expression. A multivariate analysis of prognostic factors in conventional RCC showed that TNM stage and nuclear grade were independent predictors of prognosis, but CD31 expression did not add further prognostic information.

Background: Strong oestrous symptoms in the mare can cause problems with racing, training and handling. Since long-acting progesterone treatment is not permitted in mares at competition (e.g. according to FEI rules), there is a need for methods to suppress unwanted cyclicity. Spontaneous dioestrous ovulations in the late luteal phase may cause a prolongation of the luteal phase in mares.

The retinoblastoma gene product (pRb) is the main substrate for cyclin-dependent kinases (CDKs) during the G1/S transition. Aberrations in cell cycle regulatory proteins, which have been observed in many malignancies, can theoretically cause increased phosphorylation of pRb due to unbalanced CDK activities. The expression and phosphorylation of pRb and potential associations to cell cycle aberrations in renal cell carcinomas (RCC) has only partly been clarified. We therefore evaluated the presence of pRb and the level of pRb-phosphorylation in 216 RCCs arranged in tissue microarrays by using different pRb-antibodies, including pRb-phosphospecific antibodies. Most RCCs (95%) expressed pRb, while cases with the low pRb levels, potentially indicative for pRb-inactivation, were few. In order to detect secondary alterations to a potential pRb-inactivation, the p16 expression was also monitored. None of the tumors exhibited increased p16 levels, confirming that pRb-inactivation is rare in RCC. Phosphorylated pRb was detected in approximately 50% of the RCCs, using Western blotting or immunohistochemistry. The immunohistochemical ppRb ser807/811 levels were associated with high proliferation, cyclin D1, cyclin E and p27 protein content. Surprisingly, there was no association between pRb-phosphorylation and clinicopathological data. In summary, pRb seemed to be functional and aberrations in G1/S-regulatory proteins were associated with increased phosphorylation of pRb and proliferation. The data supports that pRb might be one of the main cell cycle regulators in RCC. © 2003 Wiley-Liss, Inc. Key words: retinoblastoma protein; phosphorylation; cell cycle; G1/ S-cyclins; renal cell carcinomaCell cycle progression and proliferation is controlled by sets of checkpoints with the G1/S-checkpoint as the main regulator. Essential cell cycle regulatory gene products are the cyclin-dependent kinases (CDKs) that are sequentially activated by various cyclins and inactivated by CDK inhibitors. Cyclin D1 is activated during early G1 and forms a complex with CDK4 or CDK6. 1 This complex initiates cell cycle progression by phosphorylation of the retinoblastoma gene product (pRb), which is followed by E2F release and activation of the cyclin E/CDK2 complex, which further phosphorylate pRb. 2 Many of the interplayers of this cascade, also called the "Rb-pathway," are deregulated in malignancies. High cyclin D1 levels could, theoretically, cause an increased CDK-activity and unbalanced phosphorylation of pRb. Cyclin D1 is highly expressed in several malignancies, 3-6 either due to amplification of the CCND1 gene or as a result of impaired degradation. [7][8][9] In RCC, the majority of the tumors expressed higher cyclin D1 levels compared to normal kidney cortex, suggesting that cyclin D1 was overexpressed in a fraction of the tumors. We showed in a previous study that high cyclin D1 in RCC was associated with good prognosis, small and diploid tumors, suggesting that activation of cyclin D1 is a characteristic for low malignant features in ...