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Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population.

Kabzinski J, Mucha B, Cuchra M, Markiewicz L, Przybylowska K, Dziki A, Dziki L, Majsterek I - Oxid Med Cell Longev (2015)

Bottom Line: SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped.Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects.In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Plac Hallera 1, 90-647 Lodz, Poland.

ABSTRACT
DNA oxidative lesions are widely considered as a potential risk factor for colorectal cancer development. The aim of this work was to determine the role of the efficiency of base excision repair, both in lymphocytes and in epithelial tissue, in patients with CRC and healthy subjects. SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped. A radioisotopic BER assay was used for assessing repair efficiency and TaqMan for genotyping. Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects. In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.

No MeSH data available.


Related in: MedlinePlus

(I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-32P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗U: uracil; ∗∗AP: apurinic/apyrimidinic.
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Related In: Results  -  Collection


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fig1: (I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-32P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗U: uracil; ∗∗AP: apurinic/apyrimidinic.

Mentions: BER efficiency was evaluated according to Matsumoto et al. [5] with some minor modifications to improve the preparation of synthetic lesion site. A plasmid construct with radioactively labeled single-strand breaks was incubated with protein extract isolated from peripheral blood lymphocytes and slices of cancerous tissue removed during surgical procedures. Repair capability was assessed by densitometric analysis of DNA fragments which had been electrophoretically separated and depicted on X-ray film: these fragments vary with regard to length and can identify repaired or unrepaired fractions. The course of procedure is outlined in Figure 1.


Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population.

Kabzinski J, Mucha B, Cuchra M, Markiewicz L, Przybylowska K, Dziki A, Dziki L, Majsterek I - Oxid Med Cell Longev (2015)

(I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-32P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗U: uracil; ∗∗AP: apurinic/apyrimidinic.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4663340&req=5

fig1: (I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-32P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗U: uracil; ∗∗AP: apurinic/apyrimidinic.
Mentions: BER efficiency was evaluated according to Matsumoto et al. [5] with some minor modifications to improve the preparation of synthetic lesion site. A plasmid construct with radioactively labeled single-strand breaks was incubated with protein extract isolated from peripheral blood lymphocytes and slices of cancerous tissue removed during surgical procedures. Repair capability was assessed by densitometric analysis of DNA fragments which had been electrophoretically separated and depicted on X-ray film: these fragments vary with regard to length and can identify repaired or unrepaired fractions. The course of procedure is outlined in Figure 1.

Bottom Line: SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped.Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects.In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Plac Hallera 1, 90-647 Lodz, Poland.

ABSTRACT
DNA oxidative lesions are widely considered as a potential risk factor for colorectal cancer development. The aim of this work was to determine the role of the efficiency of base excision repair, both in lymphocytes and in epithelial tissue, in patients with CRC and healthy subjects. SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped. A radioisotopic BER assay was used for assessing repair efficiency and TaqMan for genotyping. Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects. In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.

No MeSH data available.


Related in: MedlinePlus