Cebulska-Wasilewska A. 1,2 , Miszczyk J. 1 , Dobrowolska B. 3 , Dobrowolski Z. 3

1. Detection of Chromosomal Translocation in Prostate Cancer and Benign Prostatic Hyperplasia by Fluorescence in situ Hybridization (FISH) Cebulska-Wasilewska A.1,2, Miszczyk J.1, Dobrowolska B.3, Dobrowolski Z.3 1 Environmental and Radiation Biology Department, The H. Niewodniczański Institute of Nuclear Physics PAN, Cracow, Poland, 2 Epidemiology and Preventive Medicine Departmen CM UJ, Poland, 3Urology Department and Clinic CM UJ, Poland

2. Prostate cancer epidemiology Benign Prostatic Hyperplasia [BPH] and Prostate Cancer [PC] are the most common males diseases. Prostate cancer incidence varies widely between: ethnic populations, countries and increases sharply with older age. In Poland prostate cancer is the third most common malignant cancer in males.

3. Prostate cancer risk is strongly influenced by: GENETIC FACTORS FAMILIAL HISTORY EPIGENETIC FACTORS > genetic susceptibility > lifestyle, smoking > genes involved in familial prostate cancer (for example: HPC1 on chromosome 1) > ethnic origin > mutation > dietary factors (vitamin D, fats,) > gene expression > androgens

4. Becouse... > genetic alteration on multiple chromosomes including especially chromosome 1, > many susceptibility loci have been reported at this chromosome, > many types of cancers are associated with specific types of chromosomal aberrations

5. Aim of study Compare the vulnerability to the induction in chromosome 1 translocation in lymphocytes from prostate cancer with that from benign prostatic hyperplasia.

6. antibiotics, PHA colcemid Incubation at 37ºCfor 72 hours X-rays irradiation Donor’s peripheral blood samples 80% RPMI 1640, 20% fetal calf serum KCL Fixation methanol/acetic acid (3:1) Materials and methods Investigated groups Fig. 1. Standard cytogenetic procedure [BPH] – 27 persons from the control group with benign prostatic hyperplasia. (average age 68.9 ± 8.3) [PCP] – 30 prostate cancer patients. (average age 62.4±5.3) Challenging dose In the laboratory tubes with blood were irradited with X-rays doses of 2 Gy. Culture were set up according to standard cytogetic procedure, then were harvested and followed by fixation procedure. Fluorescent in situ hybridisation (FISH) Biotin-labeled whole chromosome probes specific to chromosome 1 (Star Fish Cambio, UK).

7. Labeled probe Hybridization 37ºC, 72 hours detection Place probe on slide Materials and methods Fig. 2. General FISH protocol Fig. 3. Fig. 4. Fig. 5. The slides were examined at 1000x magnification of the epifluoescence microscope (Nicon Eclipse E400). Donors were examined for presence in their in peripheral blood lymphocytes of chromosome translocations according to the criteria of Protocol for Aberration Identification and Nomenclature-PAINT [1]. [1] Tucker J.D. et al. A proposed system for scoring structural aberrations detected by chromosome painting. Cytog. Cell Genet. (1995), 211-221.

8. Materials and methods 2 types of parameters were used to describe the extent of chromosomal damage t – frequency of chromosome 1 translocation FG/100 – genomic frequency of chromosome 1 translocation FG=Fg/2.05fp(1-fp) [2]. FG - the total genomic translation frequency Fg – the translocation frequency measured by FISH after painting Fp – the fraction of th genome represented the painted chromosome, for chromosome 1 = 0.084 fraction of the genome 8.4%) [2] Lucas J.N., Sachs R.K. Using three-color chromosomepainting to test chromome aberration models. Proc. Natl. Sci90, 1484-14

9. Results Sig. 1. X-rays effect on frequency of chromosome 1 translocation determined by FISH in peripheral lymphocytes in patients with prostate cancer [PCP] and benign prostate hyperplasia [BPH]. t T – number of translocations/1000 cells Number of translocations/1000 cells (t) was significantly higher in patients with prostate cancer (14.60±0.91) than in the control group (10.24 ±1.10; p<0.01).

10. Results Sig. 2.Genomic frequency of chromosome 1 translocation for patients with prostate cancer [PCP] and benign prostatic hyperplasia [BPH]. FG/100 FG/100 - genomic frequency of translocation Percentage of FG/100 was significantly higher in patients with prostate cancer (0.55±0.03) that obtained for the reference group (0.38 ±0.04, p<0.01).

11. We want to study correlation between occur cancer in family and frequency of chromosome 1 translocation.

12. Results Tab. 1. Correlations for patients with prostate cancer between age of donors, existing cancer in the closely related members of family and t. CiF – reported cancer in the immediate family t – number of translocations/1000 cells R – correlation coefficient High and significant correlation between age of donors and frequency of chromosome 1 translocation was observed (0.50; p<0.001). Furthermore, there was also correlation between frequency of chromosome 1 translocation observed in patients who had reported other cancers in family.

13. Conclusions 1. These studies, although preliminary, are suggesting that frequency of translocation detected in the response to challenging treatment might be used as predictor of susceptibility for prostate cancer patients. 2. Our results might confirm hypothesis that exist an association betweenpredisposition to genetic instability chromosome 1 and hereditary or familial conditioning of prostate cancer. However, more studies arenecessary of other factors which could affect genomic frequency of translocations such as: life style, diet or genetic polymorphism.