Nauka innov. 2012, 8(6):60–66

І.Ya. Skrypkina1, О.G. Kondratov1, L.O. Tsyba1, G.V. Panasenko1, О.V. Nikolaienko1, А.М. Romanenko2, O.O. Kolesnyk3, D.Ye. Morderer1, K.A. Nekrasov1, V.І. Каshuba1, S.O. Vozianov2, I.B. Shchepotin3, А.V. Rynditch1
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv
2State Institution «Institute of Urology, Academy of Medical  Ukraine», Kyiv
3National Cancer Institute, Kyiv


Detection of Cell-Free DNA and Gene-Specific Methylation in Blood Plasma of Patients with Renal and Colon Cancer

Section: Scientific and Technical Innovative Projects of National Academy of Sciences of Ukraine
Language: Ukrainian
Abstract: Gene-specific methylation and concentration of cell-free DNA in blood can be important tools for the early diagnosis of cancer. Here, we have shown that total cell-free DNA levels were higher in patients with renal and colon cancer compared to healthy individuals. The results indicated that cellfree DNA can be used for detection of hypermethylation of cancer marker genes. CpG islands of LRRC3B, APC and FHIT genes were hypermethylated in 33,3 %; 27,8 % and 33,3 % of patients with renal cancer and in 14,3 %; 28,5 % and 19 % of patients with colon cancer, respectively. Hypermethylation of LRRC3B, APC and FHIT was not detected in cell-free DNA of healthy individuals. We have proposed the methods for quantitative analysis of cell-free DNA in blood plasma and detection of the methylation of marker genes, which could be useful for diagnostics of cancer.
Key words: cell-free DNA, renal cancer, colon cancer, methyl-specific PCR, tumor marker genes.

1. Garcia J.A., Rini B.I. Recent progress in the management of advanced renal cell carcinoma. C.A. Cancer. J. Clin. 2007. V. 57, No. 2. P. 112-125.
2. Rynditch A., Pekarsky Y., Schnittger S. et al. Leukemia breakpoint region in 3q21 is gene rich . Gene. 1997. V. 193, No. 1. P. 49-57.
3. Pekarsky Y., Rynditch A., Wieser R. et al. Activation of a novel gene in 3q21 and identification of intergenic transcripts with EVI1 in leukaemia . Cancer. Research. 1997. V. 57, No. 18. P. 3914-3919.
4. Kashuba V.I., Skripkina I.Ja., Saraev D.V. i dr. Ispol'zovanie NotI-mikrochipov dlja identifikacii izmenenij v lokusah genov, potencial'no uchastvujushhih v razvitii raka shejki matki . Ukr. Biokhim. Zh. 2006. T. 78, No. 2. S. 49-56 [in Russian].
5. Skrypkina I.Ja., Kashuba V.I., Gordyjuk V.V. ta in. Identyfikacija zmin v lokusah geniv, jaki potencijno zadijani v rozvytku raku nyrok, za dopomogoju novoi' tehnologii' NotI-mikrochypiv . Dopovidi akademii' nauk Ukrai'ny. 2006. No. 11. S. 188-192 [in Ukrainian].
6. Gerashchenko G.V., Gordiyuk V.V., Skrypkina I.Y. et al. Screening of epigenetic and genetic disturbances of human chromosome 3 genes in colorectal cancer. Ukr.Biokhim. Zh. 2009. V. 81, No. 4. P. 81-87.
7. Leon S.A., Shapiro B., Sklaroff D.M. and Yaros M.J. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977. V. 37, No. 3. P. 646-650.
8. Chang H.W., Goodman S.N., Singer G. et al. Assessment of plasma DNA levels, allelic imbalance, and CA 125 as dia g nostic tests for cancer . J Natl. Cancer Inst. 2002. V. 94. P. 1697-1703.
9. Ellinger J., Bastian P.J., Haan K.I. et al. Noncancerous PTGS2 DNA fragments of apoptotic origin in sera of prostate cancer patients qualify as diagnostic and prognostic indicators. Int J Cancer. 2008. V. 122, No. 1. P. 138-143.
10. Jung K., Fleischhacker M., Rabien A. Cell-free DNA in the blood as a solid tumor biomarker A critical appraisal of the literature. Clinica. Chimica. Acta. 2010. V. 411. P. 1611-1624.
11. Fleischhacker M., Schmidt B. Circulating nucleic acids (CNAs) and cancer A survey. Biochimica et Biophy sica Acta. 2007. V. 1775. P. 181-232.
12. Vlassov V.V., Laktionov P.P., Rykova E.Y. Circulating nucleic acids as a potential source for cancer biomarkers. Curr. Mol. Med. 2010. V. 10. P. 142-165.
13. Schwarzenbach H., Hoon D.S., Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer. 2011. V. 11, No. 6. P. 426-437.
14. Czeiger D., Shaked G., Eini H. et al. Measurement of circulating cell-free DNA levels by a new simple fluorescent test in patients with primary colorectal cancer. Am. J. Clin. Pathol. 2011. V. 135, No. 22. P. 64-70.
15. Martino M., de Klatte T., Haitel A. and Marberger M. Serum cell-free DNA in renal cell carcinoma: a diagnostic and prognostic marker. Cancer. 2012. V. 18, No. 1. P. 82-90.
16. Hauser S., Zahalka T., Ellinger J. et al. Cell-free Circulating DNA: Diagnostic Value in Patients with Renal Cell Cancer. Anticancer Research. 2010. V. 30. P. 2785-2790.
17. Tian X.Q., Zhang Y., Sun D. et al. Epigenetic silencing of LRRC3B in colorectal cancer. Scand J Gastroenterol. 2009. V. 44, No. 1. P. 79-84.
18. Kim M., Kim J.H., Jang H.R. et al. LRRC3B, encoding a leucine-rich repeat-containing protein, is a putative tumor suppressor gene in gastric cancer. Cancer Res. 2008. V. 68, No. 17. P. 7147-7155.
19. Kondratov A.G., Stoliar L.A., Kvasha S.M. et al. Methylation pattern of the putative tumor-suppressor gene LRRC3B promoter in clear cell renal cell carcinomas. Mol. Med. Report. 2012. V. 5, No. 2. P. 509-512.
20. Pekarsky Y., Garrison P.N., Palamarchuk A. et al. Fhit is a physiological target of the protein kinase. Src. Proc Natl Acad. Sci. USA. 2004. V. 101, No. 11. P. 3775-3779.
21. Kvasha S., Gordiyuk V., Kondratov A. et al. Hyper methyla tion of the 5'CpG island of the FHIT gene in clear cell renal carcinomas. Cancer Lett. 2008. V. 265, No. 2. P. 250-257.
22. Brock M.V., Hooker C.M., Ota-Machida E. et al. DNA methylation markers and early recurrence in stage I lung cancer. N. Engl. J. Med. 2008. V. 358, No. 11. P. 1118-1128.
23. Rowan A.J., Lamlum H., Ilyas M. et al. APC mutations in sporadic colorectal tumors: A mutational «hotspot» and interdependence of the «two hits». Proc. Natl. Acad. Sci. USA. 2000. V. 97, No. 7. P. 3352-3357.