The overall purpose of the grant application is to define the genetic and biological parameters associated with the initiation and metastases of malignant prostate cancer. Tumor tissue will be obtained from Afro-American (75%) and caucasian (25%) patients (approximately 20 to 30/yr) at early and late Gleason stages of tumor progression. Cells will be grown up for culturing by subcutaneous (s.c.) implantation with Matrigel in severe combined immune deficient (SCID) mice. Both male and female SCIDs will be used in attempts to isolate androgen dependent and independent tumor variants. Primary tumor lines will be established from the s.c. tumor tissue utilizing procedures developed in our laboratory. The first goal is to classify the primary tumor lines based on their invasive ability in 'Boyden chemotactic assays' . Invasive and non-invasive sublines will be isolated from primary lines and maintained at low passage (less than 5). Further, SCID mice will be injected i.v. with the invasive cells to determine the metastatic potential of the sublines and to obtain cultures from any metastases that occur. The second goal is to karyotype the primary tumor lines, the non-invasive, the invasive and the metastatic sublines. By extensive karyotyping and comparison it should be possible to identify consistent chromosomal aberrations at early stages in tumorigenesis and perhaps in relation to the invasive and/or metastatic abilities of the culture. In extension of the karyotype work, a third goal is to use flow cytometry to measure DNA content and aneuploidy of the variants. In situ hybridization and fluorescence microscopy with cDNA probes to chromosomes (i. e. , chromosomes 2, 7, 10, 13, 15, 16, 17 and Y) will be used to assess whether specific chromosomes are gained or lost in the various sublines. Flow cytometry with univariate analysis of the chromosomal content of metaphase cells will help determine if deletions or gross translocations occur. A fourth goal, is to examine tumor lines for amplification and expression of oncogenes (i.e., c-myc, H-ras, Ki-ras, fos) by Southern and Northern blot analyses. In addition, in situ hybridization will show if oncogene amplification occurs in homogeneously staining regions or if oncogenes are associated with gross translocations, marker chromosomes or double minutes. Taken together, these experiments help identify specific chromosomal regions involved in prostate cancer. The consistency of any chromosomal changes observed will be determined by evaluation of cultures established from a variety of patients. The 'data manager' will provide information on the patients age, history, race, treatment and stage of progression of the cancer.
