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

Detection of Chromosomal Translocation in Detection of Chromosomal Translocation in Prostate Cancer and Benign Prostatic Hyperplasia Prostate Cancer and Benign Prostatic Hyperplasia

by Fluorescence in situ Hybridization (FISH)by Fluorescence in situ Hybridization (FISH)

Prostate cancer epidemiologyProstate cancer epidemiology

Benign Prostatic Hyperplasia [BPH] and Prostate Cancer [PC]

are the most common males diseases.

In Poland prostate cancer is the third

most common malignant cancer in

males.

Prostate cancer incidence varies widely between:

ethnic populations, countries and increases sharply with

older age.

Prostate cancer risk is strongly influenced by:Prostate cancer risk is strongly influenced by:

GENETIC FACTORS

> mutation

> genetic susceptibility

FAMILIAL HISTORY

EPIGENETIC FACTORS

> dietary factors (vitamin D, fats,)

> androgens

> ethnic origin

> lifestyle, smoking > genes involved in familial prostate cancer (for example: HPC1 on chromosome 1)

> gene expression

> 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

Becouse...

Aim of studyAim of study

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

Materials and methodsMaterials and methods

Investigated groups

[PCP] – 30 prostate cancer patients. (average age 62.4±5.3)

[BPH] – 27 persons from the control group with benign prostatic hyperplasia. (average age 68.9 ± 8.3)

Challenging doseIn the laboratory tubes with blood were irradited with X-rays doses of 2 Gy.

Fluorescent in situ hybridisation (FISH)

Biotin-labeled whole chromosome probes specific to chromosome 1 (Star Fish Cambio, UK).

Donor’s peripheral blood samples

Fixation methanol/acetic acid (3:1)

KCL

80% RPMI 1640, 20% fetal calf serum

antibiotics, PHA

Incubation at 37ºCfor 72 hours

colcemid

X-rays irradiation

Standard cytogenetic procedureFig. 1.

Culture were set up according to standard cytogetic procedure, then were harvested and followed by fixation procedure.

Materials and methodsMaterials and methods

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].

Labeled probe

Place probe on slide

Hybridization 37ºC, 72 hours

detection

General FISH protocolFig. 2.

[1] Tucker J.D. et al. A proposed system for scoring structural aberrations detected by chromosome painting. Cytog. Cell Genet. (1995), 211-221.

Fig. 3.

Fig. 4.

Fig. 5.

Materials and methodsMaterials 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

ResultsResults

t

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).

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 – number of translocations/1000 cells

10,24

14,6

0

2

4

6

8

10

12

14

16

BPH PC

ResultsResults

0,38

0,55

0

0,1

0,2

0,3

0,4

0,5

0,6

BPH PC

FG/

10

0

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).

FG/100 - genomic frequency of translocation

Sig. 2. Genomic frequency of chromosome 1 translocation for patients with prostate cancer [PCP] and benign prostatic hyperplasia [BPH].

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

ResultsResults

t

Age R 0.50

p< .001

CiF R 0.11

p< .001

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

R – correlation coefficient

t – number of translocations/1000 cells

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.

ConclusionsConclusions

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.

However, more studies are necessary of other factors which could affect genomic frequency of translocations such as: life style, diet or genetic polymorphism.

2. Our results might confirm hypothesis that exist an association between predisposition to genetic instability chromosome 1 and hereditary or familial conditioning of prostate cancer.