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DNA synthesis and methylation in normal and transformed cells
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...
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| Format: | Thesis |
| Language: | English |
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Division of Medical Biochemistry and Structural Biology
2018
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| _version_ | 1867613271858610176 |
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| access_status_str | Open Access |
| author | De Haan, Judy Bettina |
| author2 | Parker, M Iqbal |
| author_browse | De Haan, Judy Bettina Parker, M Iqbal |
| author_facet | Parker, M Iqbal De Haan, Judy Bettina |
| author_sort | De Haan, Judy Bettina |
| collection | Thesis |
| description | 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. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/27266 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:28.738Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Division of Medical Biochemistry and Structural Biology |
| publisherStr | Division of Medical Biochemistry and Structural Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/27266 DNA synthesis and methylation in normal and transformed cells De Haan, Judy Bettina Parker, M Iqbal DNA replication Mythylation Cell cycle 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. 2018-02-05T12:42:11Z 2018-02-05T12:42:11Z 1985 Master Thesis Masters MSc (Med) http://hdl.handle.net/11427/27266 eng application/pdf Division of Medical Biochemistry and Structural Biology Faculty of Health Sciences University of Cape Town |
| spellingShingle | DNA replication Mythylation Cell cycle De Haan, Judy Bettina DNA synthesis and methylation in normal and transformed cells |
| thesis_degree_str | Master's |
| title | DNA synthesis and methylation in normal and transformed cells |
| title_full | DNA synthesis and methylation in normal and transformed cells |
| title_fullStr | DNA synthesis and methylation in normal and transformed cells |
| title_full_unstemmed | DNA synthesis and methylation in normal and transformed cells |
| title_short | DNA synthesis and methylation in normal and transformed cells |
| title_sort | dna synthesis and methylation in normal and transformed cells |
| topic | DNA replication Mythylation Cell cycle |
| url | http://hdl.handle.net/11427/27266 |
| work_keys_str_mv | AT dehaanjudybettina dnasynthesisandmethylationinnormalandtransformedcells |