ABSTRACT

Bone metastases in breast cancer may be osteolytic, osteosclerotic, or a mixture of the two. Although stimulation of bone resorption by breast cancer cells has attracted some interest, the formation of osteosclerotic secondary tumours and the influence of human mammary carcinoma cells on osteoblasts (bone forming cells), both important in understanding breast cancer - bone interactions, have been largely neglected. We therefore examined the effects of conditioned medium (CM) from two cultured human breast cancer cell lines (MCF7 and ZR-75) and from primary cultures of breast carcinomas from two patients, on osteoblasts and recruitment of bone-resorbing cells (osteoclasts) in vitro. Osteoblast-like cells (BDC) were cultured from human trabecular bone explants. Osteoclast maturation was studied in fetal rat calvaria cultured on collagen gels. CM from the MCF-7 line and cells derived from one patient each inhibited BDC DNA synthesis, but stimulated osteoclast recruitment. In contrast, CM from the second patient's cells or ZR-75 enhanced DNA synthesis in BDC, but blocked osteoclast maturation. This suggests that human breast carcinomas secrete soluble factors which influence both osteoclasts and osteoblasts. A further unexpected implication is that mammary carcinoma cells may cause local osteosclerosis by directly stimulating osteoblasts, rather than through raised bone turnover in metastases.

SUMMARY

Single intravenous injections of ovine luteinizing hormone (LH) in adult hamsters and rats had no effect on fluid secretion by the testes, as measured by the gain in weight or water content during a 10-h period after ligation of the efferent ducts (EDL). Neither was there any obvious effect on the liberation of spermatozoa, as judged by the total number of sperm in the unligated and EDL testes and from the concentration of spermatozoa in the secreted fluid, calculated from the difference between the number of sperm in the EDL and unligated testes divided by the difference in weight.

In adult rats treated for 9 weeks with human chorionic gonadotrophin and/or pregnant mare serum gonadotrophin, there was a decrease in sperm production (measured as described above during a 20-h period after EDL) when the two hormones were given together. These treatments, continued for 3 or 9 weeks, had no effect on testicular weight, nor on the secretion of fluid, and the two hormones given together for 9 weeks caused a decrease in the concentration of sperm in collected rete testis fluid. The concentration of spermatozoa in collected rete testis fluid was about one quarter of the calculated concentration in the total secreted fluid.

Ovine follicle-stimulating hormone (FSH) injected daily for 3 days in 21-day-old rats stimulated fluid secretion slightly, and FSH +ovine growth hormone stimulated fluid secretion by the testes of adult rats with oestrogen implants. Ovine LH caused a slight decrease in fluid secretion in oestrogen-treated adults and also in the 21-day-old rats. Ovine prolactin and growth hormone had no significant effect on fluid secretion by the testis in these two groups of rats.

SUMMARY

Immunoassays specific for limited regions of bovine parathyroid hormone were developed in four ways.

With the heterogeneous antisera produced by immunizing with intact bovine parathyroid hormone (BPTH 1–84), the specificity of radioimmunoassays could be enhanced by presaturating either with an amino-terminal (BPTH 1–34) or carboxy-terminal (BPTH 53–84) fragment. Then, the antibodies which had not been neutralized reacted exclusively with the opposite end of the molecule, even using [¹²⁵I]BPTH 1–84 as tracer. With some antisera, the appropriate fragment and intact hormone reacted identically. However, with other antisera, the fragment reacted less well than the intact hormone, possibly because these antisera contain antibodies reacting with the middle of the molecule.

Using the labelled fragment ([¹²⁵I]BPTH 1–34) as tracer, with heterogeneous antisera, radioimmunoassays specific for the amino-terminal region were obtained. With one antiserum, BPTH 1–34 reacted identically with the intact hormone, but with another antiserum, the fragment was more reactive than the intact molecule.

A region-specific radioimmunoassay was also developed using antibodies produced by immunization with a fragment of the hormone. An antiserum raised against BPTH 1–34 had high affinity for the amino-terminal fragment, but reacted less well with the intact hormone.

Immunoradiometric assays, specific for the amino- or carboxy-terminal regions, were developed by using immunoadsorbents consisting of a fragment (either BPTH 1–34 or BPTH 53–84) coupled to cellulose. These were used to fractionate ¹²⁵I-labelled antibodies. With some of these selected antibodies, the appropriate fragment was of lower reactivity than the intact hormone. This may have been due to the presence of an incomplete antigenic site on the fragment, or to conformational differences between the fragment and the corresponding region of the intact hormone. With other selected antibodies the fragment and the intact molecule reacted identically.

Careful selection of antisera and of technique is necessary to obtain an assay in which a fragment and the intact hormone behave identically.

Abstract

It has been suggested, but not shown, that in the fetus placental lactogen (PL) may affect the regulation of the IGFs and fetal metabolism. To examine the effects of PL on the circulating concentrations of the IGFs, IGF-binding proteins (IGFBPs), glucose, free fatty acids (FFAs) and amino nitrogen (AN), we infused late gestation sheep fetuses with recombinant ovine PL (roPL). Five chronically-catheterised sheep fetuses were infused intravenously with three 24 h infusions of saline, roPL (100 μg bolus then 500 μg over 24 h) and then saline again.

Fetal roPL infusion increased plasma oPL from 0·4 ± 0·1 to 3·3 ± 0·5 nm (mean ± s.e.m.; P<0·05; factorial analysis of variance and Scheffé's test). Fetal plasma IGF-I, IGF-II, insulin, FFAs and blood glucose were unaffected by the roPL infusion. Fetal plasma IGFBP-3, as measured by Western ligand blotting, decreased by 30% during fetal roPL infusion while other fetal plasma IGFBPs were unaffected. Fetal roPL infusion decreased fetal blood AN from 7·3 ± 0·5 to 6·6 ± 0·2 mm (P<0·05). Maternal plasma IGF-I, IGF-II, IGFBPs, insulin, FFAs, blood glucose and AN were unaffected by the fetal roPL infusion. Saline infusion had no effect on any parameter.

The data suggest that PL is not a significant determinant of plasma IGFs in the late gestation sheep fetus although there may be an indirect effect via alterations in levels of IGFBP-3. The effect of fetal roPL infusion on fetal blood AN concentrations may suggest some role for PL in the regulation of fetal amino acid metabolism.

Journal of Endocrinology (1995) 144, 333–338