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F. F. Bolander and M. E. Blackstone

ABSTRACT

The envelope protein, gp52, of the mouse mammary tumour virus (MMTV) binds to a cell-surface receptor as a first step in infection. A protein with characteristics of this receptor was measured on freshly isolated cells using, as ligand, 125I-labelled gp52 purified from C3H/HeN mice. The gp52-binding protein was found in all mouse tissues examined, but was present at highest concentrations in the mammary gland and spleen where it reached 4.2±0.3 (s.e.m.) pmol/mg protein; the dissociation constant was 30±7 pm. Binding to mammary epithelium could be displaced by either the RIII or 34I-R strains of MMTV, and binding was blocked by antibodies to gp52. Levels in the liver and adrenal glands were only 25% of those in the mammary gland, while the concentrations in the ovary and salivary gland were intermediate. Scatchard analyses of the binding data suggested that there was only a single set of high-affinity binding sites. During late pregnancy and lactation, receptor levels in mammary epithelium rose threefold, while those in the liver and salivary gland were unchanged. This induction would result in the mammary gland having 12 times the gp52-binding protein than other tissues and may result in the preferential reinfection of this tissue during lactation, with subsequent tumour formation.

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F. F. Bolander and M. E. Blackstone

ABSTRACT

A radioimmunoassay was developed and validated for the major glycoprotein (gp58) of the mouse mammary tumour virus (MMTV). Using this assay, the expression of gp58 during pregnancy and lactation was found to parallel that for MMTV RNA. In particular, there was a very rapid induction in late pregnancy and a decline in late lactation, although some residual expression persisted well into involution. In cultures of normal mouse mammary tissue, induction of gp58 occurred after a 24-h lag period and began to reach a plateau after 3 days. Both the insulin and prolactin dose—response curves for gp58 resembled those for MMTV RNA; in contrast, the effects of steroid hormones on gp58 and MMTV RNA were disparate. Although progesterone stimulated the RNA, it only slightly increased gp58 levels; however, the presence of cortisol greatly augmented this stimulation, despite the inability of cortisol to induce RNA at physiological concentrations. These results suggest that insulin, prolactin and progesterone act primarily at the level of RNA accumulation in normal mammary epithelium, while cortisol affects some more distal event.

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F. F. Bolander, M. E. Blackstone, and B. M. Bradham

ABSTRACT

The role of cyclic AMP (cAMP), calcium, calmodulin and protein kinase C (PKC) in the expression of both mouse mammary tumour virus (MMTV) RNA and an MMTV glycoprotein, gp58, was investigated in normal mammary epithelium in culture. None of these second messengers had any effect on MMTV RNA. Dibutyryl cAMP alone had no effect on gp58 levels but, at low concentrations (0·05–0·1 mm), it nearly doubled the induction seen with insulin, cortisol and prolactin; higher concentrations were inhibitory. Although a calcium ionophore (A23187), either alone or with hormones, was ineffective, a calcium channel blocker (verapamil) reduced hormonal induction of gp58 by 80%, and a calmodulin inhibitor (W-13) reduced it by 90%. Two PKC activators, a phorbol ester and a diacylglyceride, were ineffective alone, with hormones or with the calcium ionophore. The following conclusions can be made: (1) cAMP, calcium and calmodulin play an important role in MMTV expression, (2) these second messengers all act post-transcriptionally, since they do not affect MMTV RNA, and (3) PKC does not appear to have a role in MMTV production in normal mammary epithelium.