Human breast adenocarcinoma (MCF-7) cells were cultivated in the RPMI 1640 medium supplemented with 10 mM L-glutamine and 50 μg/ml streptomycin (Sigma, USA) at 37°C in the presence of 5% fetal bovine serum (FBS; Biolot, Russia) in an atmosphere of 5% CO2 to a density of 0.7 × 107 cells per 4 wells of a 24-well plate.
Preparation of DNA and precursor
Human placental DNA (10 μg) fragmented to 500 bp was labeled by nick translation in the presence of Klenow fragment, three cold, and one hot dNTPs. The unincorporated precursors were removed by double precipitation with isopropanol according to the protocol described by Glover . The yield of labeled DNA upon purification amounted to 7–15 μg. DNA was dissolved in an appropriate volume of distilled water and added to wells of the plate (no more than 20 μl per well). Each well contained 250–350 μl of the medium. A 1-μl aliquot was taken from the working volume to determine radioactivity.
The aliquot of α-dNTP* was diluted in the appropriate volume of water, and 1 μl of the diluted triphosphate was sampled to count radioactivity. An approximately similar amount of α relative to DNA (with respect to radioactive count and volume) was added per each point.
The amounts of DNA (μg and cpm) and α-dNTP* (cpm) for the three experiments are listed in Table 1.
For cytological examination, DNA was labeled with the modified precursor containing FITC fluorochrome.
Restriction fragments were labeled at the sticky end formed by hydrolysis with SalGI restriction endonuclease in the presence of Klenow fragment, three cold triphosphates, and hot dATP*. Upon attachment of cold dATP to make the ends blunt, DNA was purified from the unincorporated precursors by double precipitation with isopropanol from 0.3 M NaAc.
DNA of the PCR fragments of 506 and 230 bp was labeled during PCR using specific primers .
Treating cells with fragmented DNA, preparing cell extracts, and assaying DNA in cell compartments
The labeled DNA treated as described above was added to the culture medium into the well where cells were cultivated. Cells were incubated with DNA at 37°C for the time required in an air thermostat. The following incubation times were chosen: 0 (labeled DNA was added to the medium in a titrator placed on ice; the medium was immediately sampled and supplemented with Triton X-100), 15, 30, 60, 120, and 180 min with variations in different experiments. The incubation time for α-dATP* was 180 min. The DNA amounts in the incubation medium per point are listed in Table 1.
After incubation, the plate was placed on ice. The medium was quantitatively sampled, and buffer A containing 2 mM CaCl2 and 0.5% Triton X-100 , was immediately added to cells. Each well was supplemented with 250 μl of lysing buffer, being 1 ml per point. Cells were kept with lysing buffer on ice for 10 min. The lysed cells were resuspended several times and loaded onto 1 ml of sucrose gradient in buffer A with 2 mM CaCl2 and centrifuged in conic tubes (25 ml) in a bucket rotor using a K23 centrifuge at 600 × g (2000 rpm) for 20 min. The supernatant, containing cytoplasmic fraction, was collected in a separate tube to precipitate with isopropanol from 0.3 M NaAc. The pellet of nuclei was washed twice with 250 μl of lysing buffer (buffer A containing 2 mM CaCl2 and 0.5% Triton X-100). After each washing, the preparation was centrifuged for 10 min in a bucket rotor in a K23 centrifuge at 600 × g (2000 rpm). The nuclei were resuspended in 500 μl of buffer A containing 2 mM CaCl2, examined cytologically, and transferred to the tubes of a JA-21 rotor of a J2–21 centrifuge. The suspension of nuclei was supplemented with 2 M NaCl and 1% SDS. Nuclear lysate was incubated at 65°C for 15 min until a complete clarification of the reaction mixture without any shaking. The nuclear lysate was then centrifuged at 21000 rpm (52000 × g) for 20 min at 35°C. The supernatant, being the nuclear sap or interchromosomal material, was collected preparatively with a yellow pipette tip and transferred to another tube. The supernatant was precipitated with 0.6 volume of isopropanol from 0.3 M NaAc. The lenticular precipitate, representing the chromosome fraction, was (1) supplemented with 100 μl of water and left to swell for 60 min before electrophoresis; (2) supplemented with 500 μl of water and left to swell for 10 min before precipitation with isopropanol; or (3) dissolved in the buffer containing 8 M urea and 8% glyoxal (denaturing conditions).
All the procedures were performed at 0°C. All the samples upon treatment were immediately precipitated with 0.6 volume of isopropanol from 0.3 M NaAc in 25-ml conic tubes to centrifuge in a bucket rotor in a K23 centrifuge at 4500 rpm for 20 min. The precipitate was dissolved in (1) 100 μl of water or (2) denaturing buffer at 65°C for 60 min. The amount of labeled material was determined routinely in Eppendorf tubes put into counting vials with a 1209 RackBeta counter (Finland). The samples were fractionated in 0.7% agarose gel. The DNA of chromosome fraction in the experiments under native conditions was not dissolved completely to reduce DNA degradation connected with dissolution but just left over 60 min for swelling. The "jellyfish" of undissolved, swollen precipitate was loaded into the well of agarose gel. During electrophoresis, chromosome DNA remained at the start. In the case of denaturing conditions, solution of nucleic acid material was loaded onto agarose gel without prior dialysis. Upon electrophoresis, the agarose gel was dried on a plate under a flow of hot air. X-ray film was exposed to the gel was overnight or for a period depending on the amount of labeled material.