Table 1: LOH analyses using microsatellite markers within C13, BRCA2 and RB1 at the chromosomal region 13q13. tumor. non tumorous. blood. Fig. 1: LOH at patient number 234 with the marker D13S765. LOH - loss of heterozygosity AI - allelic imbalance.
Table 1: LOH analyses using microsatellite markers within C13, BRCA2 and RB1 at the chromosomal region 13q13.
Fig. 1: LOH at patient number 234 with the marker D13S765.
LOH - loss of heterozygosity AI - allelic imbalance
LOH ANALYSES IN THE REGION OF THE PUTATIVE TUMOR SUPPRESSOR GENE C13 ON CHROMOSOME 13
U. Fiedler, W. Ehlers, Jana Herrmann, Jörg Stade and M. P. Wirth
Department of Urology, Technical University of Dresden, Germany
LOH analyses were carried out with DNA of 21 prostate carcinoma patients using 5 micro-satellite markers in the region of C13 (13q13), 3 at the BRCA2 and 3 at the RB1 locus. All together 15 of the 21 patients showed chro-mosomal alterations in the investigated regions (fig. 2). In 13/15 patients changes were detected in the region of C13, in 7/15 for BRCA2 and in 12/15 for the RB1 locus (table 1). The most frequently deleted region was within C13 at D13S1491. For BRCA2 most changes occurred at D13S171 and for RB1 at D13S168. LOHs or allelic imbalances did not focus on a single region but involved either the two regions C13 and RB1 (6/15) or all three locations (5/15).
By differential display analyses we isolated a new DNA fragment C13 which is downregulated in malignant prostate tissues. The corresponding gene was localized on chromosome 13q13 between the known tumor suppressor genes BRCA2 and RB1. Loss of heterozygosity (LOH) analyses were carried out in the region of C13, BRCA2 and BR1 in order to investigate the importance of the putative tumor suppressor gene for prostate cancer development.
Table 2: Chromosomal losses correlated to the tumor stage.
MATERIAL & METHODS
Isolation of genomic DNA
Genomic DNA was isolated from paraffin embedded prostate tissues (tumor and non tumorous) as well as from blood of 21 patients. Tumor tissues were only analyzed if they contained at least 60% tumor cells. Non tumorous tissue were completely free of tumor cells as shown by histopathology. DNA was extracted by a Proteinase K treatement carried out in DNA-buffer (50 mM Tris, pH 8,5; 1 mM EDTA; 0,5% Tween 20). From blood samples DNA was obtained by a salt extraction described by Lahiri and Nürnberger (1991).
LOH studies were carried out in the region of 13q13 using 5 microsatellite markers within the chromosomal region of C13 (D13S894, D13S1491, D13S1253, D13S765 und D13S1227) and 6 markers including the BRCA2 (D13S1699, D13S171, D13S1293) and the RB1 (D13S168, D13S1251, D13S1274) locus. For these analyses always one primer was labelled by Texas Red.
PCR reactions were carried out with approximately 200-300 ng tissue DNA or 50-100 ng blood DNA. PCR products were resolved by electrophoresis on a 6% sequencing gel and analyzed by the automated Vistra DNA sequencer (Amersham) applying the included software (fig. 1).
Allelic imbalance (AI) was defined as a 10-30% difference in peak hight between tumor and non tumorous tissue and an LOH if more than 30% difference occurred.
For prostate carcinoma chromosomal abberations are often described on 13q. In this region the already known tumor suppressor genes BRCA2 und RB1 are localized. Since deletions in these chromosomal regions often do not correlate with the gene expression of BRCA2 or RB1 further tumor suppressor genes are supposed in the region. The LOH studies showing DNA losses in the region of our newly identified C13 gene at more than 61% of the studied patients suggest that C13 may act as a tumor suppressor gene. Together with RB1 where 57% of the patients had chromosomal changes it could play a role during prostate cancer development. Therefore, C13 seems to be an interesting candidate for a new tumor suppressor gene which is supposed to be localized on 13q around BRCA2 and RB1. At the moment comparable analyses are carried out with microdissected tissues.
= AI (10-30% LOH)
= LOH (over 30%)
Fig. 2: Detailed overview of the LOH analyses obtained from the 21 PCa patients.