Cells are maintained in a quiescent state by members of the retinoblastoma protein family, pRb and p130. Both are phosphoproteins and hypophosphorylated forms of pRb and p130 bind and repress the activity of E2F transcription factors, thereby preventing entry into the cell cycle. Mitogenic stimulation causes activation of cyclin dependent kinases (cdk) that phosphorylate both pRb and p130, thereby releasing E2F factors which stimulate the transcription of a number of genes that are required for DNA synthesis and for regulating the cell cycle. In non-dividing cells, cdks are maintained in an inactive state by cdk inhibitor proteins such as p27(Kip1). The aim of our study was to determine how E2F complexes are regulated during the differentiation of human primary granulosa lutein cells (GLC) of the corpus luteum (CL). The CL is formed in the ovary after ovulation at the terminal stage of folliculogenesis after completion of maturation and differentiation of Graafian follicles. As shown by flow cytometry GLC are not dividing, being predominantly in the G(0)/G(1) phase of the cell cycle and, consistent with this, they contain the cdk inhibitor protein, p27(Kip1), but not E2F-1 which is normally expressed only in proliferating cells. The GLC do express E2F-4, hypophosphorylated pRb, p130 forms 1 and 2 and, surprisingly, hypophosphorylated p107. p107 is normally present only in dividing cells where it regulates E2F activity during the cell cycle. These forms of pRb, p130 as well as p107, together with E2F-4 are all active in that they can bind an E2F DNA-binding site in a pull-down assay. Immunocytochemistry shows that these proteins are expressed in almost all GLC but have different sub-cellular distribution: p107 is concentrated in nucleoli, while p130 and E2F-4 show relatively even nuclear and cytoplasmic distributions. Both pRb and p130 have been implicated previously in repressing E2F activity in many different cell types during cell cycle arrest in G(0)/G(1). We conclude that p107 is active in human primary GLC but its nucleolar localisation would suggest that it represses ribosomal RNA synthesis rather than E2F activity.
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- Author: C. R. Thomas x
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C Green, R Chatterjee, HH McGarrigle, F Ahmed, and NS Thomas
D L Russell-Jones, R M Leach, J P T Ward, and C R Thomas
Rats were maintained in chambers and breathed air (control, n=8) or an atmosphere containing 10% oxygen (hypoxic, n=10) for 35 days. On completion of the experiment the hypoxic animals weighed less than the controls (hypoxic, 290 ± 11.7g; control, 339 ± 19.2g; means ± S.E.M., p<0.05). No differences in the left ventricular weights were found between groups but the right ventricular weights were greater in the hypoxic rats (hypoxic, 0.39 ± 0.02g; control, 0.27 ± 0.08g; p<0.01). The amount of mRNA for IGF-I in the ventricles was quantified by Northern blot analysis. There was no difference between groups in IGF-I mRNA levels in the left ventricles (hypoxic, 1.07 ± 0.41 absorbance units (AU); control, 0.73 ± 0.33 AU). In the right ventricles, IGF-I mRNA was greater in hypoxic than in control rats (hypoxic, 2.37 ± 0.75 AU; control, 0.64 ± 0.11 AU; p<0.05). This study demonstrates that expression of IGF-I mRNA is increased in the hypertrophied right ventricle of hypoxic rats; IGF-I may play a central role in the initiation and maintenance of this process.
D. L. Russell-Jones, M. Rattray, V. J. Wilson, R. H. Jones, P. H. Sönksen, and C. R. Thomas
There is evidence that the hormonal control of hepatic IGF-I production is mediated by GH and insulin. To elucidate the role of these hormones further we administered s.c. or i.p. insulin (at 2·5 and 5·0 IU/day) and/or GH (0·8 IU/day) to rats made diabetic with streptozotocin 16 days previously. Hepatic IGF-I production was then assessed by quantifying hepatic IGF-I mRNA levels by autoradiography of Northern blots. Diabetes resulted in a fivefold reduction in hepatic IGF-I mRNA levels (optical density (OD) of the 0·7–1·1 kb band: controls, 1·3±0·09; diabetics, 0·28±0·08; P<0·01), which was not significantly changed by treatment with s.c. insulin (OD: low dose, 0·55±0·05; high dose, 0·58±0·05) or low dose i.p. insulin (OD: 0·40±0·03). High dose i.p. insulin enhanced hepatic IGF-I mRNA levels (OD: 0·93±0·23) compared with diabetic rats (P<0·01) and those given high dose s.c. insulin (P<0·04), despite the blood glucose values being similar in the treated groups (i.p., 4·72±0·29 mmol/l; s.c., 3·32±0·03 mmol/l). Administration of GH alone partially restored the hepatic IGF-I mRNA level (OD: GH-treated, 1·00±0·05; diabetic, 0·28±0·08; P<0·01), whilst having no effect on blood glucose values (diabetic, 36·35±0·45 mmol/l; GH-treated, 38·65±2·39 mmol/l). Additional administration of s.c. insulin completely restored IGF-I mRNA levels to those of controls (OD: low dose, 1·35±0·14; high dose, 1·27 ± 0·18). These observations indicate that insulin and GH are required for full expression of hepatic IGF-I mRNA and that insulin given i.p. is more potent than that given s.c. at stimulating hepatic synthesis of IGF-I.