The replication of Nb 2 Node rat lymphoma cells in suspension culture is specifically stimulated by lactogenic hormones. Human (hPRL), ovine, bovine, and rat PRLs stimulated replication in a dose-dependent manner in the concentration range of 10 pg/ml to 1 ng/ml. Human, ovine, and bovine placental lactogens were similarly active. In addition, cell replication was stimulated by human GH (hGH), which is known to have lactogenic activity. Other hormones and growth factors examined were inactive. The growth stimulatory effects of hPRL and hGH were completely inhibited when excess anti-hPRL and anti-hGH, respectively, were added to the medium. A bioassay based on the response of the Nb 2 Node lymphoma cells to lactogenic hormones has been developed. Human serum stimulated cell replication. The effect was completely abolished if excess antibodies to both hPRL and hGH were present. The stimulation obtained with a number of human serum samples correlated very well with the sum of the hPRL and hGH concentrations in the sera, as determined by RIA (r = 0.95; P < 0.001). The concentrations of either hPRL or hGH in human serum could be individually determined by specifically blocking the growth stimulatory effect of the other hormone by adding excess anti-hGH or anti-hPRl. The sensitivity of this bioassay for PRL and hGH in serum exceeds that of RIAs.

The specific binding of 125

Chick embryo fibroblasts transformed by Rous sarcoma virus have an increased content of two membrane proteins of molecular weights 78,000 and 95,000. The increased content of the 95,000-dalton protein and the principal increase in the content of the 78,000-dalton protein are not an early consequence of cell transformation but instead are secondary to the rapid depletion of glucose from the growth medium of transformed cells. When glucose is maintained at high levels in the growth medium of transformed cells, the synthesis of the 95,000-dalton protein is arrested and that of the 78,000-dalton protein is markedly suppressed. Upon removal of glucose from the growth medium of normal cells, these proteins increase to levels comparable to those of transformed cells.Because the amount of these two proteins is influenced by the presence or absence of glucose, we suggest they be referred to as "glucose-regulated proteins," GRP-78 and GRP-95. These proteins may have an important role in regulating the utilization of glucose in cultured cells.Major differences in the composition of cell membrane proteins have been observed for fibroblastic cells transformed by various oncogenic viruses (1-5). For example, chick embryo fibroblasts (CEF) transformed by various strains of Rous sarcoma viruses (RSV) show a substantial decrease in the content of a major cell surface glycoprotein (CSP) with a molecular weight of about 220,000 (2, 3) and a significant increase in two other plasma membrane-associated proteins of molecular weights approximately 90,000 and 75,000 (4, 5). Because alterations of membrane protein composition are associated with the process of malignant transformation, these proteins could have a causative role in transformation. The 220,000 CSP is involved in determining the altered adhesive properties and morphology of transformed chick and other cells (6, 7) but apparently does not have a role in loss of growth control. The biological significance of the changes in the 90,000 and 75,000 membrane proteins and the mechanism by which these alterations take place are unknown.This report presents evidence to suggest that the major increase in the 75,000 and 90,000 cell membrane proteins is not an early consequence of cell transformation but is secondary to rapid depletion of glucose from the medium by RSV-transformed cells. When a high level of glucose is maintained in the growth medium, transformed CEF exhibit low levels of these two proteins. Furthermore, when glucose is removed from the growth medium, normal nontransformed CEF increase their content of these two proteins. Because the synthesis of these two proteins is controlled by glucose, we suggest these proteins be designated "glucose-regulated proteins" (GRP). METHODSCells. CEF were cultivated, propagated, and infected with Schmidt-Ruppin (SR) and Bryan high-titer (BH) strains of RSV as described (8, 9). Subconfluent cells were used in this study.Preparation of Membranes of CEF. Membrane ghosts of normal and transformed CEF were prepared by using a twophas...

A radioreceptor assay with a sensitivity of 5 nanograms per milliliter has been developed for mammalian and avian pituitary prolactin, placental lactogenic hormones, and humnan growth hormone, using a membrane receptor preparation isolated from rabbit mammary glands. Prolactin preparations inhibited the binding of [(125)I]prolactin to receptors in direct proportion to the biological potency of these preparations. Thus, the radioreceptor assay provides a convenient and simnple assay for hormones which have lactogenic activity.

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The possible role of the conceptus in stimulating the onset of maternal behavior through its secretion of placental lactogens and their passage into the brain was investigated in female rats. In the first study, significant mitogenic activity in the Nb₂ lymphoma cell bioassay was detected in cerebrospinal fluid (CSF) samples collected by push-pull perfusion from rats on days 12–21 of pregnancy, coincident with the establishment of placental function. In contrast, mitogenic activity was absent from CSF in lactating and gonadectomized, virgin females. In a second study the mitogenic activity in day 12 pregnant samples was neutralized 71% with antibodies to rat placental lactogen-I (rPL-I) and > 90% with a combination of antibodies to rPL-I plus rPL-II. In contrast, activity on day 21 of pregnancy, 1 day prepartum, was reduced by antibodies to rPL-II (>85%), but not by antibodies to rPL-I, indicating that the predominant lactogen in the CSF prepartum is rPL-II. The behavioral actions of placental secretions were assessed in the third experiment by infusing recombinant rPL-I and purified rPL-II directly into the medial preoptic area of the brain of steroid-primed, nulliparous rats. Latencies to respond maternally to foster young were significantly reduced in rPL-I- and rPL-II-treated rats (2- to 3-day latencies) when compared with latencies in control females (5- to 6-day latencies). Thus, the conceptus through its secretion of rPLs which apparently gain access to the CSF helps to prime the pregnant female’s brain to respond maternally at the end of gestation. This endocrine communication between the developing conceptus and pregnant female appears to be an important part of the biological system which helps to establish successful maternal care.

Abstract1,25-Dihydroxyvitamin D 3 and vitamin D 3 analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-L-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiationinduced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.