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A concerted genomic and proteomic approach to understand the early stages of prostate carcinogenesis Ma, Stephanie

Abstract

Cancer of the prostate (CaP) is the most common cancer in men and the second leading cause of death from cancer. To improve survival rates, an understanding of the molecular events that govern this disease is critical. We approach this goal through a concerted effort to identify the critical, early genomic and proteomic changes that cause the transformation of normal cells into their pre- and cancerous stages. In the past however, these molecular studies were greatly hindered by the heterogeneity of the tissue and the small size of the early stage lesions. To overcome these problems, a protocol for microdissection of progressionspecific cells from sections of both paraffin-embedded and fresh frozen radical prostatectomy samples that would allow the extraction of high quantity and quality DNA was developed. Further, a standardized protocol for DNA quantitation tailored for these minute microdissected samples was established. Using these protocols, 75 radical prostatectomy specimens were studied. 28 of these specimens (totaling 28 normal, 15 PIN and 30 early invasive carcinoma - Gleason grades 2 to 4) yielded sufficient DNA of favorable quantity and quality for SMAL (Scanning of Microdissected Archival Lesions)-PCR fingerprinting. Polymorphic DNA fingerprint fragments correlated with early prostate cancer progression were identified and mapped to 1p32, 2p23, 3p26, 4q35, 5q23, 8q22, 8q24, 9q22, 11p13, 12p12-13, 16q12, 18q21. Regions of allelic imbalance were reviewed by literature analysis and candidate genes were proposed. As a complementary approach, a working protocol for the use of Laser Capture Microdissection (LCM) coupled with the SELDI ProteinChip™ was also developed and standardized. Using this protocol, a pilot experiment was then conducted by comparing SELDI generated protein profiles from lysates of microdissected normal and early tumor cells from 5 fresh frozen human prostatic specimens. A recurrent gain of a 24kDa protein peak in cancer was observed in 2 out of 5 patients. Groundwork has been laid for others to fine-map and defines the boundaries of the potential prostate cancer genes. Furthermore, using the same method in the proteomic approach, others can now screen more patients for similar protein alterations that may signal the initiation of a developing cancer.

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