The Effect of DNase I on Free DNAs and Its Relationship with Metastasis: A Preliminary Results

Year 2020, Volume: 13 Issue: 2, 153 - 159, 15.08.2020

Aylin Dal Didem Turgut Coşan

Abstract

In breast cancer, p53 is generally mutant and plays an important role in the development of metastasis, including bone metastasis. However, mutations are not always responsible for regulation disorders in cancer-related genes. Epigenetic changes such as hyper-methylation-related gene silencing errors and chromatin remodeling in CpG islands contribute to the development of cancer and metastasis. Free DNAs play an important role in cancer and metastasis formation. Studies show that DNA fragment levels are low in normal conditions and an increase in malignancy. In our study, invasive breast cancer cells MDA-MB-231 and hFOB 1.19 cells representing bone tissue, which is one of the most metastasized tissues of breast cancer, were cultured in the same environment (co-culture). In both cells, the presence of hyper-methylation in the mutant p53 exon 8 region and APC1A, APC1B, and RASSF1 genes in MDA-MB-231 cells was observed. Appropriate primer sequences were selected for p53 exon8 amplifications and APC1A, APC1B, and RASSF1A methylation analysis. After PCR treatment applied for mutant p53 exon8 determinations, the products were subjected to electrophoresis in a 30% homogeneous polyacrylamide gel. Methylation Specific PCR (MSP) was performed by the APC1A, APC1B, and RASSF1A gene regions. PCR products were analyzed by conducting 2% agarose gel electrophoresis. The results were also evaluated in environments where DNase I was added. The results of our study provide researchers with a model that shows that some gene and structural features of tumor cells can be transported to normal cells via free DNA. Also, it is aimed to reveal the effect of DNase I in providing or preventing this transport.

Keywords

cell-free DNA, breast cancer cell, metastasis, bone cell, DNase I

Project Number

2014-575

References

• [1] Weinberg R A (2013) The Biology of Cancer, Second International Student Ed., WW Norton & Company. [2] Garcia-Olmo D, Garcia-Olmo D, Ontanon J, Martinez E, Vallejo M J H (1999) Tumor DNA circulating in the plasma might play a role in metastasis, The hypothesis of the genometastasis. Histology and Histopathology 14(4):1159-1164 [3] Garcı́a-Olmo D, Garcı́a-Olmo D C, Ontañón J, Martinez E J B (2000) Horizontal transfer of DNA and the" genometastasis hypothesis", Blood 95(2):724-725 [4] Pulciani S, Santos E, Lauver A V, Long L K, Aaronson S A, Barbacid M J N (1982) Oncogenes in solid human tumours. Nature 300(5892):539 [5] Diehl F, Li M, Dressman D, He Y, Shen D, Szabo S, et al (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumors. PNAS 102(45):16368-16373 [6] García-Olmo D C, Gutiérrez-González L, Samos J, Picazo M G, Atiénzar M, García-Olmo D (2006) Surgery and hematogenous dissemination: comparison between the detection of circulating tumor cells and of tumor DNA in plasma before and after tumor resection in rats. Annals of Surgical Oncology 13(8):1136-1144 [7] Silva J M, Dominguez G, Garcia J M, Gonzalez R, Villanueva M J, Navarro F, et al (1999) Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Annals of Surgical Oncology 59(13):3251-3256 [8] Silva J M, Garcia J M, Dominguez G, Silva J, Miralles C, Cantos B, et al (2002) Persistence of tumor DNA in plasma of breast cancer patients after mastectomy. Annals of Surgical Oncology 9(1):71-76 [9] Lujambio A, Esteller M J C c (2009) How epigenetics can explain human metastasis: a new role for microRNAs. Journal Cell Cycle 8(3):377-382 [10] Bukholm I, Nesland J, Karesen R, Jacobsen U, Børresen A L (1997) Relationship between abnormal p53 protein and failure to express p21 protein in human breast carcinomas. The Journal of Pathology 181(2):140-145 [11] Hartmann A, Blaszyk H, McGovern R, Schroeder J, Cunningham J, De E V et al (1995) p53 gene mutations inside and outside of exons 5-8: the patterns differ in breast and other cancers. Oncogene 10(4):681-688 [12] Lacroix M, Toillon R-A, Leclercq G J E (2006) p53 and breast cancer, an update. Endocr Relat Cancer 13(2):293-325 [13] Runnebaum I B, Nagarajan M, Bowman M, Soto D, Sukumar S (1991) Mutations in p53 as potential molecular markers for human breast cancer. Proc Natl Acad Sci U S A 88(23):10657-10661. [14] van Slooten H J, van de Vijver M J, Børresen A L, Eyfjörd J E, Valgardsdóttir R, Scherneck S, et al (1999)Mutations in exons 5–8 of the p53 gene, independent of their type and location, are associated with increased apoptosis and mitosis in invasive breast carcinoma. The Journal of Pathology 189(4):504-513 [15] Walerych D, Napoli M, Collavin L, Del Sal G J C (2012) The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carsinogenesis 33(11):2007-2017 [16] Gasco M, Shami S, Crook T J (2002) The p53 pathway in breast cancer. Breast Cancer Research 4(2):70 [17] Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian S V, Hainaut P, et al (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Human Mutation 28(6):622-629 [18] E. Powell, D. Piwnica-Worms, H. Piwnica-Worms (2014) Contribution of p53 to metastasis. Cancer Discov 4(4):405-414 [19] Yang P, Du C, Kwan M, Liang S, Zhang G J (2013) The impact of p53 in predicting clinical outcome of breast cancer patients with visceral metastasis. Scientific Reports 3:2246 [20] Esteller M (2008) Epigenetics in cancer. N Engl J Med 358(11):1148-1159 [21] Rishi V, Bhattacharya P, Chatterjee R, Rozenberg J, Zhao J, Glass K, et al (2010) CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes. PNAS 107(47):20311-20316 [22] Rozenberg J M, Shlyakhtenko A, Glass K, Rishi V, Myakishev M V, FitzGerald P C, et al (2008) All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues. BMC Genomics 9(1):67 [23] Kulis M, Esteller M (2010) Methylation and cancer. Adv. Genet. 70:27-56 [24] Müller H M, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, et al (2003) DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Research 63(22):7641-7645 [25] Lewis C M, Cler L R, Bu D-W, Zöchbauer-Müller S, Milchgrub S, Naftalis E Z, et al (2005) Promoter hypermethylation in benign breast epithelium in relation to predicted breast cancer risk. Clinical Cancer Research 11(1):166-172 [26] de Lamirande G (1961) Action of deoxyribonuclease and ribonuclease on the growth of Ehrlich ascites carcinoma in mice. Nature 192(4797):52 [27] Lahm A, Suck D (1991) DNase I-induced DNA conformation: 2 Å structure of a DNase I-octamer complex. Journal of Molecular Biology 222(3):645-667 [28] Leon S, Shapiro B, Sklaroff D, Yaros M. J (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Research 37(3):646-650 [29] Leon S A, Shapiro B, Servi P, Parsons R G (1981) A comparison of DNA and DNA-binding protein levels in malignant disease. European Journal of Cancer 17(5):533-538 [30] Gahan P, Stroun M (2010) The biology of circulating nucleic acids in plasma and serum (CNAPS) Extracellular Nucleic Acids, Springer [31] Lazarovici A, Zhou T, Shafer A, Machado A C D, Riley T R, Sandstrom R, et al (2013)Probing DNA shape and methylation state on a genomic scale with DNase I. PNAS 110(16):6376-6381 [32] Armenante F, Merola M, Furia A, Tovey M, Palmieri M (1999) Interleukin-6 repression is associated with a distinctive chromatin structure of the gene. Nucleic Acids Research 27(22):4483-4490 [33] Lengner C J, Steinman H A, Gagnon J, Smith T W, Henderson J E, Kream B E, et al (2006) Osteoblast differentiation and skeletal development are regulated by Mdm2–p53 signaling. The Journal of Cell Biology 172(6):909-921 [34] Papadopoulou E, Davilas E, Sotiriou V, Georgakopoulos E, Georgakopoulou S, Koliopanos A, et al (2006) Cell‐free DNA and RNA in plasma as a new molecular marker for prostate and breast cancer. Ann N Y Acad Sci 1075(1):235-243. [35] Zhu J, Zhang F, Du M, Zhang P, Fu S, Wang L (2017) Molecular characterization of cell-free eccDNAs in human plasma. Scientific Reports 7(1):10968. [36] Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genetics 33(3s):245-254 [37] Jeronimo C, Monteiro P, Henrique R, Dinis-Ribeiro M, Costa I, Costa V L, et al (2008) Quantitative hypermethylation of a small panel of genes augments the diagnostic accuracy in fine-needle aspirate washings of breast lesions. Breast Cancer Research and Treatment 109(1):27-34 [38] Swellam M, Abdelmaksoud M D, Sayed Mahmoud M, Ramadan A, Abdel‐Moneem W, Hefny M M (2015) Aberrant methylation of APC and RARβ2 genes in breast cancer patients. IUBMB Life 67(1):61-68 [39] Dulaimi E, Hillinck J, de Caceres I I, Al-Saleem T, Cairns P (2004) Tumor suppressor gene promoter hypermethylation in serum of breast cancer patients. Clin Cancer Res 10(18):6189-6193 [40] Hoque M O, Feng Q, Toure P, Dem A, Critchlow C W, Hawes S E, et al (2006) Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 24(26):4262-4269 [41] Hu X-C, Wong I H, Chow L W C (2003) Tumor-derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications. Oncology Reports 10(6):1811-5181 [42] Urrutia G, Laurito S, Marzese D M et al (2015) Epigenetic variations in breast cancer progression to lymph node metastasis. Clin Exp Metastasis 32:99–110 [43] Skvortsova T, Rykova E, Tamkovich S, Bryzgunova O, Starikov A, Kuznetsova N, et al (2006) Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation. Br J Cancer 94(10):1492 [44] Yazici H, Terry M B, Cho Y H, Senie R T, Liao Y, Andrulis I, et al (2009) Aberrant methylation of RASSF1A in plasma DNA before breast cancer diagnosis in the Breast Cancer Family Registry. Cancer Epidemiol Biomarkers Prev 18(10):2723-2725 [45] Fackler M J, Bujanda Z L, Umbricht C, Teo WW, Cho S, Zhang Z, et al (2014) Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer. Cancer Res 74(8):2160-2170 [46] Matuschek C, Bölke E, Lammering G, Gerber P, Peiper M, Budach W, et al (2010) Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease. Eur J Med Res 15(7):277 [47] Tan S-H, Ida H, Lau Q-C, Goh B-C, Chieng W-S, Loh M, et al (2007) Detection of promoter hypermethylation in serum samples of cancer patients by methylation-specific polymerase chain reaction for tumour suppressor genes including RUNX3. Oncol Rep 18(5):1225-1230 [48] Van der Auwera I, Elst H, Van Laere S, Maes H, Huget P, Van Dam P, et al (2009) The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients. Br J Cancer 100(8):1277-1286 [49] Kristiansen S, Nielsen D, Sölétormos G (2016) Detection and monitoring of hypermethylated RASSF1A in serum from patients with metastatic breast cancer. Cancer markers 8(1):35 [50] Golonka R M, Yeoh B S, Petrick J L, Weinstein S J, Albanes D, Gewirtz A T, et al (2019) Deoxyribonuclease I Activity, Cell-Free DNA, and Risk of Liver Cancer in a Prospective Cohort. JNCI Cancer Spectrum 2(4):pky083 [51] Lu R, Wang Y, Xu X, Xie S, Wang Y, Zhong A, et al (2018) Establishment of a detection assay for DNA endonuclease activity and its application in the screening and prognosis of malignant lymphoma. BMC Biochem 19(1):6 [52] Kochanek S, Renz D, Doerfler W (1993) Differences in the accessibility of methylated and unmethylated DNA to DNase I. Nucleic Acids Res 21(25):5843-5845 [53] Van Der Vaart M, Pretorius P J (2008) Circulating DNA: its origin and fluctuation. Annals of the New York Academy of Sciences 1137(1):18-26 [54] Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, et al (2000) The origin and mechanism of circulating DNA. Ann N Y Acad Sci 906(1):161-168 [55] Barták B, Nagy Z, Spisák S, Tulassay Z, Dank M, Igaz P, et al (2018) In vivo analysis of circulating cell-free DNA release and degradation. Orv Hetil 159(6):223-233 [56] Trejo-Becerril C, Pérez-Cardenas E, Gutiérrez-Díaz B, De La Cruz-Sigüenza D, Taja-Chayeb L, González-Ballesteros M, et al (2016) Antitumor effects of systemic DNAse I and proteases in an in vivo model, Integrative Cancer Therapies. 15(4) NP35-NP43