| Popis: |
The work described in this thesis involves a series of studies on the hormonal and intracellular regulation of the expression of the prolactin (PRL) and growth hormone (GH) genes. The studies are based on both rat pituitary tissue and human pituitary adenoma tissue, and the regulation of both rat and human genes is discussed. In initial studies cDNA/mRNA hybridization assays were used to assess the regulation of messenger RNA production, first in primary cell cultures of rat pituitary tissue, and then in a series of GH or PRL-secreting human pituitary adenomas. The effects of dopamine and dopaminergic drugs, somatostatin, hypothalamic releasing hormones and intracellular signalling systems were variable among different human tumours, but in general hPRL and hGH mRNA levels in tumour cells were less responsive to inhibition than rPRL or rGH mRNA levels in normal rat pituitary tissue. Secondly, the potential mechanisms for PRL gene regulation by intracellular signals were studied using the clonal rat pituitary GH3 cell line. Transfection analysis was used to assess the role of the promoter region of the rat PRL gene in calmodulin-dependent regulation of transcription: a defined region of proximal 5'-flanking DNA conferred marked calcium/calmodulin responsiveness onto a bacterial reporter gene. The role of nuclear protein binding to this region of the gene was studied using gel mobility shift assays, and this technique was used to study both the possible mechanisms for the calcium/calmodulin regulation of nuclear protein binding to DNA in PRL gene expression. The results showed that nuclear protein binding to DNA is affected by calmodulin antagonists, and suggest that the mechanisms for intracellular regulation of the PRL gene overlap with those of its tissue-specific expression. Finally, preliminary attempts were made to apply the transfection and gel shift techniques to human pituitary tumour tissue, and showed that such techniques may indeed by applicable even to small quantities of pituitary tumour tissue, and may throw some light on possible pathogenetic mechanisms for pituitary adenoma formation. |
