RESUMO
Size and termini of cell-free DNA molecules circulating in blood plasma and being bound with blood cell surface of healthy females and untreated breast cancer patients were investigated. The size and concentration of circulating blood DNA were analyzed by Agilent 2100 Bioanalyser TM and TaqMan PCR. The termini of circulating DNA were examined by ligation using biotinylated double-stranded oligonucleotide adapters with random 1-3 b overhangs of both chains and subsequent quantification by PCR. Short (180 bp) and longer (>8 kbp) DNA fragments were found in cell free DNA from both groups, but short were less represented in primary breast cancer patient plasma. Predominantly high molecular weight DNA was found in cell surface bound DNA both in healthy females and breast cancer patients with a minor fraction of short fragments. Heterogeneous DNA molecules with diverse 5'- and 3'- protruding as well as blunt ends were found both in plasma DNA and cell bound DNA from healthy individuals. Cell surface bound DNA from breast cancer patients mainly contains 5'-protruding ends, whereas 5'- and 3'-protruding ends are equally presented in cell free DNA from these patients. The data obtained obviously reflect over-representation of specific nucleases in breast cancer.
Assuntos
Neoplasias da Mama/genética , Fragmentação do DNA , DNA de Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , DNA de Neoplasias/sangue , DNA de Neoplasias/metabolismo , Feminino , Humanos , Estadiamento de NeoplasiasRESUMO
In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.