DNA synthesis and methylation in normal and transformed cells

 

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dc.contributor.advisor Parker, M Iqbal en_ZA
dc.contributor.author De Haan, Judy Bettina en_ZA
dc.date.accessioned 2018-02-05T12:42:11Z
dc.date.available 2018-02-05T12:42:11Z
dc.date.issued 1985 en_ZA
dc.identifier.citation De Haan, J. 1985. DNA synthesis and methylation in normal and transformed cells. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/27266
dc.description.abstract In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication. en_ZA
dc.language.iso eng en_ZA
dc.subject.other DNA replication en_ZA
dc.subject.other Mythylation en_ZA
dc.subject.other Cell cycle en_ZA
dc.title DNA synthesis and methylation in normal and transformed cells en_ZA
dc.type Master Thesis
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Health Sciences en_ZA
dc.publisher.department Division of Medical Biochemistry and Structural Biology
dc.type.qualificationlevel Masters
dc.type.qualificationname MSc (Med) en_ZA
uct.type.filetype Text
uct.type.filetype Image
dc.identifier.apacitation De Haan, J. B. (1985). <i>DNA synthesis and methylation in normal and transformed cells</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology. Retrieved from http://hdl.handle.net/11427/27266 en_ZA
dc.identifier.chicagocitation De Haan, Judy Bettina. <i>"DNA synthesis and methylation in normal and transformed cells."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 1985. http://hdl.handle.net/11427/27266 en_ZA
dc.identifier.vancouvercitation De Haan JB. DNA synthesis and methylation in normal and transformed cells. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 1985 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/27266 en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - De Haan, Judy Bettina AB - In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication. DA - 1985 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1985 T1 - DNA synthesis and methylation in normal and transformed cells TI - DNA synthesis and methylation in normal and transformed cells UR - http://hdl.handle.net/11427/27266 ER - en_ZA


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