Vairapandi Mariappan
The Fels Institute for Cancer Research and Molecular Biology, School of Medicine, Temple University, Philadelphia, Pennsylvania 19140, USA.
J Cell Biochem. 2004 Feb 15;91(3):572-83. doi: 10.1002/jcb.10749.
DNA methylation/demethylation constitutes a major consequence in all biological processes involving transcription, differentiation, development, DNA repair, recombination, and chromosome organization. Our earlier studies established that demethylation of CpG rich sequence by human DNA demethylase activity (5-methylcytosine-DNA glycosylase (5MeC-DNA glycosylase)) resembles "base excision DNA repair activity" and creates single-strand breaks on DNA that is associated with proliferating cell nuclear antigen (PCNA). Here in this report, we have identified differential DNA demethylation targets (hemi-methylated vs. fully-methylated) in normal cell lines and cancerous cell lines, and a shortened G(0)/G(1) resting time in cancerous cell lines than the normal cell lines. We have identified that in normal HFL1 fibroblast cell line, DNA demethylase activity targets hemi-methylated CpG specific sites on DNA. This normal cell line DNA demethylase activity associates with PCNA immune complex that is inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. While in cancerous LnCap and BT20 cell lines DNA demethylase activity targets fully-methylated CpG specific sites on DNA. This cancer cell line DNA demethylase activity is not associated with PCNA immune complex and is not inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. We have also identified that the fully-methylated CpG specific DNA demethylase activity from cancerous cell lines to associate with p300/CBP protein. These significant observations of variable targets of DNA demethylation and alternate partner proteins for DNA demethylase activity in cancerous cell lines are discussed in terms of double-strand DNA breaks versus single-strand DNA breaks and their role in the exit of G(1)/G(2) cell cycle stages. Also, the inability of cell cycle regulatory proteins like PCNA, p21(waf1), and Gadd45 to control DNA demethylase activity in cancerous cell lines is discussed in terms of accelerated G(1)/G(2) cell cycle stage exit to facilitate unregulated cellular proliferation, loss of control of chromosomal organization, and the development of oncogenesis in cancerous cell lines.
DNA甲基化/去甲基化是所有涉及转录、分化、发育、DNA修复、重组和染色体组织的生物学过程中的一个主要结果。我们早期的研究表明，人类DNA去甲基化酶活性（5-甲基胞嘧啶-DNA糖基化酶（5MeC-DNA糖基化酶））对富含CpG序列的去甲基化类似于“碱基切除DNA修复活性”，并在与增殖细胞核抗原（PCNA）相关的DNA上产生单链断裂。在本报告中，我们确定了正常细胞系和癌细胞系中不同的DNA去甲基化靶点（半甲基化与全甲基化），以及癌细胞系中比正常细胞系缩短的G(0)/G(1)静止时间。我们发现，在正常的HFL1成纤维细胞系中，DNA去甲基化酶活性靶向DNA上的半甲基化CpG特定位点。这种正常细胞系的DNA去甲基化酶活性与PCNA免疫复合物相关，该复合物被CDKI蛋白p21(waf1)/Gadd45α和Gadd45β抑制。而在癌细胞系LnCap和BT20中，DNA去甲基化酶活性靶向DNA上的全甲基化CpG特定位点。这种癌细胞系的DNA去甲基化酶活性不与PCNA免疫复合物相关，也不被CDKI蛋白p21(waf1)/Gadd45α和Gadd45β抑制。我们还发现，癌细胞系中全甲基化CpG特异性DNA去甲基化酶活性与p300/CBP蛋白相关。本文根据双链DNA断裂与单链DNA断裂及其在G(1)/G(2)细胞周期阶段退出中的作用，讨论了癌细胞系中DNA去甲基化可变靶点和DNA去甲基化酶活性替代伴侣蛋白的这些重要观察结果。此外，并根据加速的G(1)/G(2)细胞周期阶段退出以促进不受调控的细胞增殖、染色体组织控制丧失以及癌细胞系中肿瘤发生的发展，讨论了细胞周期调节蛋白如PCNA、p21(waf1)和Gadd45无法控制癌细胞系中DNA去甲基化酶活性的问题。
