Michael R. Norman

Prolactin and Growth Hormone: Molecular Heterogeneity and Measurement in Serum

The pituitary hormones prolactin and growth hormone are related single-chain polypeptides. Both hormones exist in the circulation in several molecular forms, and this heterogeneity may account for some of the complex and sometimes contradictory actions, in vivo and in vitro, of both hormones. It may also lead to problems with quantitation by immunoassays and discrepancies between the results given by assays using different antibodies. Modified forms of the hormones may have markedly different activity in bioassays from that of the parent hormone, but the clinical significance of this is unclear. In this review we summarize what is known about the molecular heterogeneity of the hormones and briefly discuss the implications for clinical biochemists.

A novel member of the SAF (scaffold attachment factor)-box protein family inhibits gene expression and induces apoptosis

The SLTM [SAF (scaffold attachment factor)-like transcription modulator] protein contains a SAF-box DNA-binding motif and an RNA-binding domain, and shares an overall identity of 34% with SAFB1 {scaffold attachment factor-B1; also known as SAF-B (scaffold attachment factor B), HET [heat-shock protein 27 ERE (oestrogen response element) and TATA-box-binding protein] or HAP (heterogeneous nuclear ribonucleoprotein A1-interacting protein)}. Here, we show that SLTM is localized to the cell nucleus, but excluded from nucleoli, and to a large extent it co-localizes with SAFB1. In the nucleus, SLTM has a punctate distribution and it does not co-localize with SR (serine/arginine) proteins. Overexpression of SAFB1 has been shown to exert a number of inhibitory effects, including suppression of oestrogen signalling. Although SLTM also suppressed the ability of oestrogen to activate a reporter gene in MCF-7 breast-cancer cells, inhibition of a constitutively active beta-galactosidase gene suggested that this was primarily the consequence of a generalized inhibitory effect on transcription. Measurement of RNA synthesis, which showed a particularly marked inhibition of [(3)H]uridine incorporation into mRNA, supported this conclusion. In addition, analysis of cell-cycle parameters, chromatin condensation and cytochrome c release showed that SLTM induced apoptosis in a range of cultured cell lines. Thus the inhibitory effects of SLTM on gene expression appear to result from generalized down-regulation of mRNA synthesis and initiation of apoptosis consequent upon overexpressing the protein. While indicating a crucial role for SLTM in cellular function, these results also emphasize the need for caution when interpreting phenotypic changes associated with manipulation of protein expression levels.

Prolactin-induced activation of STAT5 within the hypothalamic arcuate nucleus.

Prolactin signalling within hypothalamic areas associated with the control of fertility was examined in male and lactating female rats. Following exogenous prolactin treatment, phosphorylation of STAT5 (signal transducer and activator of transcription) within the arcuate nucleus was measured using a highly sensitive immunoblotting strategy. A significant increase in phosphorylated STAT5 was detected in the arcuate nucleus of female rats compared with same-sex controls. No such effect was apparent in the males. Phosphorylation of STAT5 was not observed in the liver of either males or females. These results show that prolactin-induced intracellular signalling within the hypothalamus involves activation of the Janus tyrosine kinase/signal transducer and activator of transcription pathway, and that this signalling mechanism can be readily triggered in lactating females where prolactin receptors are known to be upregulated and fertility impaired.

Multiple Actions of Glucocorticoids Studied in Cell Culture Systems

Glucocorticoids produce differing effects in various cells both in vivo and in vitro. The wide spectrum of effects of these steroids baffled endocrinologists for some time until the unifying concept of steroid- and tissue-specific receptors provided a means of recognizing those cells likely to respond (Thompson and Lippman 1974; King and Mainwaring 1974; Yamamoto and Alberts 1976). This clearing in the mystery, however, has proved to be more valuable for the sex steroids and for cells selected for resistance to growth inhibition by glucocorticoids than it has for glucocorticoid-sensitive cells and tissues in general. In fact, the majority of cells and tissues tested have been found to contain receptors for glucocorticoids. Nevertheless, specific cellular responses to these steroids remain varied, and the problem of explaining the differences in response in different cell and tissue types remains. These responses are as widely disparate as induction of a limited number of peptides or suppression of a limited number of functions (Ivarie and O’Farrell 1978) and cell lysis and death (Claman 1972; Baxter and Harris 1975). At present it is not clear whether there is a single unifying mechanism that can account for such widely differing effects. It does seem that in the vast majority of the systems studied cytoplasmic receptors seem to be required.

Serum Prolactin Bioactivity and Immunoactivity in Hyperprolactinaemic States

Serum prolactin concentrations were measured using a sensitive bioassay (Nb2 assay) and by radioimmunoassay in 11 patients with prolactin-secreting pituitary tumours (median serum immunoactive prolactin 5150 mU/L), and in 58 normal control subjects (median prolactin 190 mU/L). The mean ratio of serum prolactin bioactivity to immunoactivity was significantly lower in patients with prolactinomas than in normoprolactinaemic controls. Ten lactating women in the early post-partum period (median prolactin 3800 mU/L), studied as a model of physiological hyperprolactinaemia, also had reduced bioactivity to immunoactivity ratios. Overall, there was a significant negative correlation between bioactivity : immunoactivity ratio and serum immunoactive prolactin. The change in relative bioactivity of prolactin in serum samples from patients with prolactinomas and in women with physiological hyperprolactinaemia may reflect changes in the molecular heterogeneity of the hormone. Such changes may affect activity in both bioassays and radioimmunoassays.

Biological Activity of Serum Prolactin in Patients with Primary Hypothyroidism

Prolactin bioactivity was measured in sera from 22 patients with primary hypothyroidism and 13 euthyroid control subjects and compared with estimates of immunoactivity given by radioimmunoassay. The Nb2 rat lymphoma cell assay was modified to improve specificity for prolactin and used to measure bioactivity. Results of serum prolactin levels obtained using the bioassay were closely correlated with radioimmunoassay results in both hypothyroid patients and control subjects. Mean bioactivity/immunoactivity ratios in patients were not significantly different from those in control subjects. Mean prolactin concentration measured by both assays was significantly higher in patients than control subjects. Serum prolactin in patients with primary hypothyroidism appears to have essentially normal bioactivity as measured in the Nb2 assay, in contrast with a report of major differences between activity in radioreceptor assay and RIA in hypothyroid patients.

The production and use of monoclonal antibodies to measure apolipoprotein B by a competitive enzyme-linked immunoassay.

The production of monoclonal antibodies to human lipoproteins is described. One of these antibodies, which was shown to be specific to apolipoprotein B, was used to develop a competitive enzyme-linked immunoassay for apolipoprotein B in serum samples. The antibody selected recognises apolipoprotein B in both low density and very low density lipoprotein particles, but there is no cross-reactivity with high density lipoprotein. There is no requirement for labelling of antigen or antibody used in the assay, and results obtained correlate well (r=0·88) with measurements on serum samples using a radial immunodiffusion assay for apolipoprotein B.