Transcript Detection of Chromosomal Translocation in Prostate Cancer

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
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
1
Prostate 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:
GENETIC
FACTORS
> genetic
susceptibility
> mutation
> gene
expression
FAMILIAL
HISTORY
> genes involved in
familial prostate cancer
(for example: HPC1 on
chromosome 1)
EPIGENETIC
FACTORS
> lifestyle, smoking
> ethnic origin
> dietary factors
(vitamin D, fats,)
> androgens
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
Aim 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 methods
Investigated groups
[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
Fig. 1. Standard cytogenetic procedure
antibiotics, PHA
X-rays
irradiation
colcemid
Incubation at
37ºCfor 72 hours
Donor’s peripheral 80% RPMI 1640,
blood samples
20% fetal calf serum
KC
L
Fixation methanol/acetic acid
(3:1)
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).
Materials and methods
Fig. 2. General FISH protocol
Labeled
probe
Fig. 3.
Hybridization 37ºC,
72 hours
detection
Fig. 4.
Place probe
on slide
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.
Materials and methods
2 types of parameters were
used to describe the extent of
chromosomal damage
t – frequency of chromosome 1
translocation
FG - the total genomic translation frequency
FG/100 – genomic frequency of
chromosome 1 translocation
FG=Fg/2.05fp(1-fp) [2].
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
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].
14,6
16
14
10,24
t
12
10
8
6
4
2
0
BPH
PC
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).
Results
Sig. 2. Genomic frequency of chromosome 1 translocation for patients with
prostate cancer [PCP] and benign prostatic hyperplasia [BPH].
0,55
0,6
0,5
0,38
FG/100
0,4
0,3
0,2
0,1
0
BPH
PC
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).
We want to study correlation between
occur cancer in family and frequency of
chromosome 1 translocation.
Results
Tab. 1. Correlations for patients with prostate cancer between age of donors,
existing cancer in the closely related members of family and t.
t
CiF – reported cancer in the immediate family
t – number of translocations/1000 cells
Age
R – correlation coefficient
CiF
R
0.50
p<
.001
R
0.11
p<
.001
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.
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 between predisposition to genetic instability
chromosome 1 and hereditary or familial conditioning
of prostate cancer.
However, more studies are necessary of other factors which
could affect genomic frequency of translocations such as: life
style, diet or genetic polymorphism.