In order to study the role of the p16INK4A(MTS1/CDKN2a) tumor suppressor in breast cancer, we analyzed p16 protein expression in 60 breast cancer samples which were also analyzed for expression of Rb, Ki67, HER2/neu, and estrogen and progesterone receptors (ER, PR). P16 expression was investigated by two methods: western blotting (WB) followed by densitometry, and immunohistochemistry (IHC). The Rb status was studied by western blotting, and expression of Ki67, HER2/neu, ER, and PR was analyzed immunohistochemically. P16-negative results were found in 18% of the carcinomas by WB, but in only one case by IHC and were not associated with established prognostic parameters. In contrast, p16 overexpression which was detected by WB and IHC in 15% and 25% of the tumors, respectively, was significantly associated with unfavorable prognostic indicators. High p16 expression as detected by both methods correlated significantly with high grading and a negative estrogen receptor status. In addition, a significant association of p16 staining with inverse progesterone receptor status and high Ki67 expression was found with IHC. No correlation of p16 expression with clinical stage, HER2/neu immunostaining, Rb expression or Rb phosphorylation was found. Comparison of western blot results and immunohistochemistry suggests that both nuclear and cytoplasmic immunoreactivity in tumor cells is specific and due to p16 expression. We conclude that high p16 reactivity (both nuclear andcytoplasmic) is indicative of a more undifferentiated, malignant phenotype in mammary carcinomas.

Extracellular-regulated kinases (ERK1, ERK2) play important roles in the malignant behaviour of breast cancer cells in vitro. In our present study, 148 clinical breast cancer samples (120 cases with follow-up data) were studied for the expression of ERK1, ERK2 and their phosphorylated forms p-ERK1 and p-ERK2 by immunoblotting, and p-ERK1/2 expression in corresponding paraffin sections was analysed by immunohistochemistry. The results were correlated with established clinical and histological prognostic parameters, follow-up data and expression of seven cell-cycle regulatory proteins as well as MMP1, MMP9, PAI-1 and AP-1 transcription factors, which had been analysed before. High p-ERK1 expression as determined by immunoblots correlated significantly with a low frequency of recurrences and infrequent fatal outcome (P ¼ 0.007 and 0.008) and was an independent indicator of long relapse-free and overall survival in multivariate analysis. By immunohistochemistry, strong p-ERK staining in tumour cells was associated with early stages (P ¼ 0.020), negative nodal status (P ¼ 0.003) and long recurrence-free survival (P ¼ 0.017). In contrast, expression of the unphosphorylated kinases ERK1 and ERK2 was not associated with clinical and histological prognostic parameters, except a positive correlation with oestrogen receptor status. Comparison with the expression of formerly analysed cell-cycle-and invasion-associated proteins corroborates our conclusion that activation of ERK1 and ERK2 is not associated with enhanced proliferation and invasion of mammary carcinomas. Growth factors or cytokines exert positive and negative effects on cell proliferation and differentiation. After binding to their membrane receptors, the message is relayed to the nucleus by a complex system of intracellular signalling pathways. The majority of signalling molecules involved in these pathways are kinases, which are themselves activated by phosphorylation and repressed by their specific phosphatases. The mitogen-activated protein kinase (MAPK) signalling pathway includes a cascade of four groups of kinases (MAPKKKKs, MAPKKKs, MAPKKs and MAPKs). The MAPK kinase kinase kinases of the first level are phosphorylated in response to various extracellular stimuli through interaction with small GTP-binding proteins like Ras, Raf, etc. The activated enzymes then phosphorylate one of 14 kinases of the second level (the MAPKKKs, that is, Raf proteins, MEKK1-4, etc.), which themselves activate one of the MAPK kinases (MEK1 and 2, MKK3 -7) of the third level. Finally, these kinases (MAPKKs) activate MAP kinases, which are then able to phosphorylate transcription factors, which regulate the expression of genes involved in cell proliferation or differentiation. Three different, partly interacting signalling pathways have been identified in mammalian cells, leading to the activation of three types of MAP kinases: the MAP kinase JNK (Jun kinase) phosphorylates c-Jun, JunB, ATF2 and ELK1, etc., P38 activates ATF2, ELK-1 and MAX, whereas ERK1 and ERK2 phosphorylate...

We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.

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