Cyclin-dependent kinases (Cdks) form a family of enzymes that coordinate the cell division cycle. The periodic activation of cdks requires their association with proteins called cyclins, and their dissociation from inhibitory subunits, called Ckis. Ubiquitin-proteasome-mediated proteolysis is a major mechanism by which the protein levels of cell cycle regulators are regulated in response to mitogenic and anti-mitogenic stimuli. They have previously shown that in normal diploid cells protein levels of the cki p27 are mainly regulated by degradation via the ubiquitin-proteasome system. Similarly, degradation of other G1 regulatory proteins (Cyclin E, Cyclin D1, E2F-1) is controlled by the ubiquitin-pathway. Over-activation of positive cell cycle regulators (e.g., cyclins, E2F-1, Cdc25) and inactivation of Ckis (e.g., p27, p16) play a significant role in oncogenesis. These changes inactivity can occur through mutation, or transcriptional silencing of the corresponding gene, or through changes in protein stability. Indeed, although p27 gene has never been found altered in human tumors, they have recently observed that aggressive human colorectal carcinomas have reduced expression of p27 and enhanced proteolytic activity specific for p27. To determine whether the abnormal expression of cell cycle regulators represents novel prognostic markers for human breast cancer, they will use immunohistochemistry and in situ hybridization to analyze the expression of cell cycle regulators (p27, Cyclin D1, Cyclin E, E2F-1, and Cdc25 B) and their mRNA in archival sections from approximately six hundred human breast cancers (Specific Aim 1). These tumor samples, obtained from three different tumor banks, have concordant normal tissues and a long term follow-up. Furthermore, each tumor bank has an extensive data base containing information on clinico-pathological features and standard molecular markers, allowing them to determine the prognostic value of cell cycle regulators. They will focus the study on breast carcinomas 1 cm in size, and tumors obtained from pre-menopausal women. They hypothesize that aggressive tumors have enhanced degradation of p27 and other cell cycle inhibitors and decreased degradation of cell cycle activators, thus acquiring a distinct growth advantage. To test whether deregulated proteolysis of cell cycle regulatory proteins is responsible for their abnormal expression in breast carcinomas, they will analyze tumor breast samples for the abundance of specific degradation activities and for the levels of various ubiquitin conjugating enzymes (Ubcs) involved in cell cycle regulation (Specific Aim 2).
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