instability. Chromosomal abnormality is one of early genetic disorders resulting in the induction of the cell
genome instability and, as a consequence, its malignant transformation. The pattern of neoplastic cells
methylation changes significantly in comparison with normal cells; total demethylation is accompanied by an
increase in the activity of DNA methyltransferase DNA MTase and local hypermethylation of CpG islands. The
mechanism of local hypermethylation is not completely clear. Apparently, the important role in this process is
played by an increase in methyltransferase activity [6, 7]. The interrelation of genes, chromosomal and
epigenetic disorders in induction of genomic instability in the development of BC is of great importance .
MATERIAL AND METHODS
During the research, DNA samples obtained from peripheral blood leukocytes of BC patients (15 females) were
used. The blood of BC patients was received at the Department of Mammalogy of the National Center for
Cancer Research of the Ministry of Health (MoH) of Uzbekistan. As a control, DNA from peripheral blood
leukocytes taken from healthy donors was used (10 donors). Generally accepted clinical and morphological
prognostic criteria were evaluated: the tumor histological type, tumor receptor status, HER2/neu expression.
All of them were studied using the biopsy material.
The review board and ethics committee of National Center for Cancer Research under the MoH Tashkent,
Uzbekistan AND Bioorganic Chemistry Institute named after A.S. Sadyko. Tashkent, Uzbekistan. Academy of
Sciences, Tashkent, Uzbekistan approved the study protocol and gave permission.
Extraction of eDNA from serum / plasma
One ml of peripheral blood taken from the ulnar vein was transferred to plastic tubes with Na2-EDTA
sprayed. The blood was centrifuged at 40 °C sequentially at 1500 rpm for 10 minutes, at 3000 rpm for 15
minutes, at 5000 rpm for 15 minutes. After centrifugation, 400 μl of blood serum were taken from the tubes and
transferred to new sterile tubes. The serum was pretreated with the RNA (100 μg/ml), incubated at 37 °C for 1 h,
then resuspended with proteinase K (50 μg/ml), incubated for 1 hour at 37 °C. After enzymatic treatment, the
blood serum was added to 200 μl of lysis buffer (100 mM Tris- HCl, pH 8.0; 25 mM EDTA, pH 8.0; 0.15 M NaCl;
0.7 M β-Mercaptoethanol; SDS) to a final concentration of 2%. The lysis was carried out in the cold for 3 minutes
(on ice). The aliquots were deproteinized for 15 minutes in 1.5 ml phenol / chloroform mixture (1: 2) followed by
15 min. centrifugation at 5000 rpm at 40 °C. The supernatant was transferred to new test-tubes, 1/10 volume of
3M sodium acetate pH 5.2 was added, as well as 2.5-volume of cooled 96% ethanol, and the tubes were left
overnight at -200 °C. The denatured eDNA preparations were centrifuged at 5000 rpm for 30 min at 40⁰C. The
eDNA precipitate was washed in 1 ml with cooled 70% ethanol for 15 minutes, and then centrifuged at 13.000
rpm for 15 min at 40 °C, then it was dried in vacuum desiccator for 15 minutes and dissolved in 300 μl of TE
buffer, pH 8.0 and stored at -200 °C. The eDNA aliquots were analyzed in 2% agarose gel containing 0.5 μg/ml
ethidium bromide. Electrophoresis was conducted for 1 hour at 100V; the gel was photographed in UV rays.
Cultivation of lymphocytes
The culture medium consisting of (per vial): 6 ml of RPMI 1640 medium with glutamine (PANECO, Russia),
1 ml of fetal bovine serum (produced in France-Germany), 40 μg/ml gentamicin and 20 μg/ml mitogen -
phytohemagglutinin (PHA DifcоP) was added to 0.8 ml of whole blood. The prepared cell culture was incubated
in thermostat at 370°C and was periodically shaken gently (1-2 times per day). This procedure prevents
excessive agglutination of erythrocytes. The cultivation time under the experimental conditions was 72 hours.
The cells were fixed for 72 hours after the initiation of cultivation. Two hours prior to fixation, colchicine (0.4
μg/ml) was injected into the culture medium that destroyed the spindle microtubules and prevented
chromosome divergence. In consequence, the cellular mitosis stopped at the metaphase stage. The cultured
cells suspension was poured into centrifuge tubes, centrifuged at 1000 rpm for 7 minutes. Then, the
supernatant was removed, the precipitate was shaken, 7 ml of hypotonic solution 0.075 1M KCl pre-heated to 37
°C were added. After that the tubes were again placed in thermostat for 20 minutes. Hypotonic treatment is
carried out for the best spread of chromosomes of lymphocytes. After the end of hypotonic treatment of the
cells, they were fixed in three stages. At the first stage, the cell suspension, after treatment in KCl hypotonic
To cite this paper: Zakirova LT, Alimkhodjaeva LT and Kadyrova DA. 2018. Chromosomal Disorders and Aberrant DNA Methylation as Early Biomarkers of Breast