Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line.

Human small cell lung carcinoma (SCLC) tumours exhibit neuroendocrine differentiation, secreting hormones such as ACTH and related peptides. While glucocorticoids inhibit ACTH secretion from the pituitary, this does not occur in SCLC tumours and SCLC cell lines. Failure of glucocorticoids to suppress ACTH peptides is accompanied by a global lack of glucocorticoid action in a number of SCLC cell lines. In the human SCLC cell line, COR L103, activation of a human tyrosine aminotransferase (TAT3)-luciferase reporter gene is resistant to glucocorticoids despite similar glucocorticoid receptor (GR) expression to the glucocorticoid-sensitive A549 human lung cancer cell line; moreover, the GR is free of deleterious mutations. Over-expression of a wild-type GR restores glucocorticoid regulation of TAT3-luciferase, and this is enhanced when the activation function (AF)-2 domain is deleted but much reduced when the AF-1 domain is deleted. This suggests aberrant AF-2 activation domain function. We identified defective steroid receptor co-activator 1 (SRC1) recruitment to the GR AF-2 in COR L103 cells, although SRC1 was successfully recruited to the steroid X receptor with rifampicin, suggesting a defect in the GR. Analysis of other GR C-terminal co-factors identified increased expression of nuclear co-repressor (NCoR) in COR L103 cells. To determine the impact of this, NCoR was over-expressed in A549 cells, where it reduced GR transactivation by 55%. In summary, glucocorticoid resistance is associated with altered SRC protein recruitment and increased expression of NCoR in these SCLC cells, suggesting that glucocorticoid sensitivity may be modified by subtle changes in co-factor recruitment.

Difficulty in brainstem death testing in the presence of high spinal cord injury.

In the UK, when the standard brain death criteria are met, further investigations are not necessary. Confirmatory tests can be useful, however, when it is not possible to carry out all of the brainstem tests. We report the case of a patient with multiple trauma and a high spinal cord injury who was apnoeic. Confirmatory tests (EEG, brainstem, auditory evoked potential) were essential in supporting the diagnosis of brainstem death to allow withdrawal of artificial ventilation, as organ donation was being considered.

Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta.

Epidemiological studies of postmenopausal hormone replacement therapy show a reduction in the risk of developing colon cancer, and animal studies using 17beta-oestradiol (E(2)) demonstrate a decreased incidence of chemically-induced colon cancer. Using the colon cancer cell line, COLO205, we found that E(2) induced a dose-dependent increase in DNA fragmentation and nuclear condensation, significant effects being seen at 10(-12 )mol/l. BSA-conjugated E(2), which cannot enter cells, was ineffective at inducing apoptosis in COLO205 cells, indicating that E(2) was not acting through a cell-membrane receptor. E(2) did not induce the morphological changes characteristic of differentiation. Using RT-PCR we found that the oestrogen receptor alpha (ERalpha) isoform was absent in the COLO205 cell line in contrast to CACO-2, LoVo and SW620 cells, but mRNAs for ERbeta1, -beta2, -beta5 and -beta6 isoforms were detected. Western immunoblotting results showed full-length ERbeta protein but no detectable ERalpha in COLO205 cells. In normal human colon tissue samples immunoreactive ERbeta was found but ERalpha was barely detectable. Expression of ERbeta was lost in some colon cancer specimens and reduced in others. We conclude that E(2), through ERbeta, at concentrations found during replacement therapy, may inhibit the development of colon cancer by inducing apoptosis.