Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon.

We report that CpG island methylation, an epigenetic modification of DNA known to correlate closely with silencing of gene transcription, appears in the oestrogen receptor (ER) gene in a subpopulation of cells which increases as a direct function of age in human colonic mucosa. This same methylation change characterizes virtually all cells in all 45 colorectal tumours examined, including the earliest stages of tumour formation. ER gene expression is diminished or absent in colorectal tumours, and introduction of an exogenous ER gene in cultured colon carcinoma cells resulted in marked growth suppression. Our data suggest that methylation associated inactivation of the ER gene in ageing colorectal mucosa could be one of the earliest events that predispose to sporadic colorectal tumorigenesis.

Programmed cell death in an estrogen-independent human breast cancer cell line, MDA-MB-468.

Previous studies have demonstrated that estrogen-responsive human breast cancer cells can be induced to undergo an energy-dependent, genetically programmed series of biochemical changes that result in the active suicide of the cells following estrogen ablation. In contrast, estrogen-independent human breast cancer cells do not activate this programmed cell death pathway following estrogen ablation. This could be due either to the absence of the cellular machinery required for programmed cell death or simply to the inability of estrogen ablation to activate this machinery. To discriminate between these two possibilities, the MDA-MB-468 estrogen-independent human mammary adenocarcinoma cell line was used as a model system to study the mechanism of cell death following cytotoxic drug treatment. Exposure of these cells to the fluorinated pyrimidines, 5-fluoro-2'-deoxyuridine or trifluorothymidine, resulted in growth inhibition and loss of proliferative capacity within 24 h. These changes occurred while cell membrane integrity was intact as measured by either cellular morphology or trypan blue exclusion. After 48 h of drug treatment, loss of cell membrane integrity was followed by cell lysis and a rapid decline in cell number. The addition of 16 microM thymidine prior to drug treatment prevented cell death, but thymidine did not rescue these cells once drug treatment was initiated. Analysis of DNA revealed the characteristic fragmentation into nucleosomal oligomers that is a hallmark of programmed cell death. Associated with this death pathway was a 15-fold induction of transforming growth factor beta 1 gene expression that has been previously observed in a variety of cellular systems undergoing programmed cell death. These results indicate that MDA-MB-468 estrogen-independent human mammary carcinoma cells retain the ability to undergo programmed cell death after treatment with cytotoxic drugs that induce a "thymineless" state.

Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells.

Breast cancer is the most common malignancy in women and hormone resistance is a challenging problem in its treatment. Loss of estrogen receptor expression is an important means of hormone resistance, but the mechanisms involved are poorly understood. We now demonstrate a potential role for abnormal DNA methylation in transcriptional inactivation of the estrogen receptor gene. Estrogen receptor-negative human breast cancer cells growing in culture lack estrogen receptor mRNA, have a higher capacity to methylate DNA, and display extensive methylation of the CpG island in the 5' promoter region of the estrogen receptor gene, which would correlate with silencing of expression. These results suggest that abnormal methylation could account for transcriptional inactivation of the estrogen receptor gene and subsequent hormone resistance in some human breast cancers.

Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells.

MDA-MB-468 human breast cancer cells lack estrogen receptors, overexpress epidermal growth factor (EGF) receptors, and are growth inhibited by EGF. We show that treatment of MDA-MB-468 cells with EGF leads to inhibition of cell proliferation, fragmentation of DNA into nucleosomal oligomers, and the development of apoptotic morphology. This treatment is associated with increased expression of c-myc, c-fos, jun family members, and transforming growth factor beta 1 mRNA and with partial proteolytic cleavage of poly(ADP-ribose) polymerase and lamin B. The observation that EGF can mediate apoptosis in EGF receptor-overexpressing cells has important implications for clinical efforts directed at the EGF receptor.

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors.

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.

Acute renal failure due to mannitol intoxication.

A case of mannitol-induced renal failure is described and previously reported cases are reviewed. Possible mechanisms of the etiology are discussed.