Serum specifically potentiates the mitogenic response of Nb2 lymphoma cells to rat prolactin

Previous observations in our laboratory indicated that rat serum samples being bioassayed for PRL with the use of Nb2 lymphoma cells produced mitogenic responses greater than maximally effective doses of purified rat PRL. The present studies were conducted to confirm these observations and to determine the possible serum factor or factors responsible for the potentiated response of serum. Twenty five rat serum pools prepared from blood samples obtained from lactating, ovariectomized, and steroid-treated rats were assayed in duplicate aliquots of 0.3-50 microliter against NIDDK-RP-1 rat PRL (11 IU/mg) by the Nb2 bioassay in Fischer's Medium containing 10% horse serum and by conventional double antibody RIA. The mitogenic response of 18 of the 25 pools were clearly greater than the response to standard rat PRL when included in the bioassay at 10-50 microliter/ml of cells. When less than 10 microliter serum pools were assayed, parallelism to the standard was observed; but the concentrations of PRL determined by bioassay were 82 +/- (SE) 4% of the levels determined by RIA in samples from ovariectomized, steroid-treated rats stored for two or four weeks and 62 +/- 2% in samples from lactating rats stored for 3-6 months. Horse serum and hypophysectomized rat serum also potentiated the mitogenic responses to 1 or 5 ng standard rat PRL when added at 10-150 microliter/ml of culture medium that already contained 100 microliter (10%) horse serum. No potentiation of the RIA was observed with rat or horse serum. Insulin, proinsulin, or C peptide of proinsulin (0.02 ng-2 micrograms/ml); fibroblast growth factor, nerve growth factor, epidermal growth factor, insulin-like growth factors I or II (0.002-200 ng/ml), T cell growth factor (TCGF) (0.0002-20 half-maximal units/ml) or platelet-derived growth factor (0.002-10 half-maximal units) did not potentiate the responses to 1 or 5 ng/ml standard rat PRL. Of the growth factors tested, only TCGF was mitogenic to Nb2 cells when placed alone in the medium, but this mitogenic effect of TCGF was not potentiated by horse serum. We conclude that serum can potentiate the mitogenic effect of PRL on Nb2 cells grown in Fischer's medium.     

Biological activities of human growth hormone and its derivatives estimated by measuring DNA synthesis in Nb2 node rat lymphoma cells

Nb2 cell is a rat lymphoma cell line that responds to lactogens such as prolactin and human growth hormone (hGH) with an increased rate of proliferation. We explored the relationship between the biochemical events induced by hGH and its derivatives and their receptor binding activities. hGH stimulated RNA, DNA and protein synthesis of Nb2 cells as a function of time. Stimulation of RNA and protein was maximal at 2-3 h and 12 h, respectively, after the addition of hGH. DNA synthesis, measured by the rate of [3H]thymidine incorporation, reached a maximum after 18-h incubation with hGH. Stimulation of DNA synthesis was elicited by hGH in a dose-dependent manner between 0.45 and 45 pmol/l. The activity of the 20 K hGH variant in stimulating DNA synthesis was approx 30% of that of hGH. In contrast, S1-hGH, which lacks a sequence of ten amino acids (140-149) of hGH, showed a 3.2-fold greater activity than hGH. F1 (amino-terminal sequence 1-134 of hGH) was only 0.06% as active as hGH, and the activity of F2 (C-terminal 42 amino acid residue of hGH) was less than 0.01%. Both fragment 1-15 and 32-46 were without effect. The relative potencies of these hGH derivatives in stimulating DNA synthesis were similar to their relative abilities to inhibit [125I]hGH binding to lactogenic receptors on Nb2 cell. Nb2 cells provide a suitable model to study the relationship between receptor binding and the biochemical events induced by lactogens.     

Glycosylated human growth hormone variant

The human growth hormone variant (hGH-V) gene is expressed by the syncytiotrophoblastic layer of the human placenta in two forms: hGH-V mRNA encoding a 22 kD protein, and hGH-V2 mRNA which retains intron 4 and is expected to encode a 26 kD protein. There is a predicted N-linked glycosylation site in hGH-V at amino acid 140 that is absent in both hGH-V2 and in the highly homologous normal pituitary GH (hGH-N). Cell lines transfected with the hGH-N gene secrete 22 kD GH and the 20 kD product of an alternatively spliced mRNA, while cell lines transfected with the hGH-V gene secrete three proteins of 22, 24, and 26 kD. To determine whether any of these hGH-V isoforms are glycosylated, the cell lines were grown in the absence and presence of tunicamycin. In addition, conditioned medium from metabolically labelled hGH-V transfected cells was separately digested with peptide:N-glycosidase F and endoglycosidase H. The 26 and 24 kD bands were both absent from the media after tunicamycin treatment and were both sensitive to peptide:N-glycosidase F treatment. Endoglycosidase H digestion resulted in the selective loss of the 24 kD band. These results indicate that hGH-V is partially modified posttranslationally by N-linked glycosylation in a fibroblastic cell line.     

Human chorionic gonadotropin stimulates proliferation of Nb 2 rat lymphoma cells

The effects of an onco-fetal hormone, hCG, were tested on replication of Nb 2 node rat lymphoma cells, which have previously been shown to be responsive to lactogenic hormone stimulation. Cells were maintained in suspension culture in Ham's F-10 medium containing horse serum (15%) and fetal calf serum (2.5%). Forty-eight hours before experiments, medium was replaced with horse serum (10%) only. In the absence of added hCG, cell doubling time was about 24 h. Purified hCG preparations (CR 119) and the Second International Standard for hCG (WHO) stimulated lymphoma cell replication after 72 h of exposure to the cells. The stimulation was dose dependent, beginning at 100 pg/ml and peaking at 10 ng/ml (140% vs. controls, P less than 0.001). Specific hCG antiserum (SB6) did not alter cell replication, but when added simultaneously with hCG, completely blocked the stimulation induced by hCG (10 ng/ml). No effect on cell proliferation was seen when the beta-subunit of hCG, the common glycoprotein alpha-subunit, human LH, FSH, or placental lactogen was added to the cells. These results indicate that hCG can stimulate proliferation of rat lymphoma cells in vitro. If hCG affects human tumors in a similar fashion, the ectopic production of the hormone by tumors may stimulate growth of the neoplasm.     

Presence of a nonlactogenic factor in human serum which synergistically enhances prolactin-stimulated growth of Nb2 rat lymphoma cells in vitro

A nonlactogenic factor(s) which synergistically enhanced the effect of ovine PRL in stimulating the growth of Nb2 rat lymphoma cells in suspension culture was found in human serum. The identity of the factor is unknown, but it is heat stable, with a mol wt apparently in excess of 8000 daltons. It possesses no growth-stimulating activity in the absence of PRL. Insulin acts in a similar manner to this factor in the Nb2 bioassay, suggesting that the factor may be somatomedin-like, possibly related to the previously described synlactin.     

Enhancement of human growth hormone-stimulated mitogenesis of Nb2 node lymphoma cells by 12-O-tetradecanoyl-phorbol-13-acetate

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) enhanced human (h) GH- and ovine PRL-stimulated mitogenesis of the Nb2-11C clone of rat lymphoma cells. Maximal enhancement of 25% in the proliferation rate was achieved with 20 nM TPA. The enhancing effect was found at all levels of hGH (0.031-2.0 ng/ml), but was more pronounced at lower hormone concentrations. TPA alone had no effect on cell proliferation, and its activity was absolutely dependent on the simultaneous presence of the lactogenic hormones. We have analyzed the changes that occurred in the distribution of cells in different phases of the cell cycle during the first 22 h after exposure to hGH and measured the proliferation rate through 3 days. We have found that the mitogenic effect of hGH resulted from 1) an increase in the rate of G0/G1----S transition, 2) a decrease in the lag period required for entry into the S phase, and 3) an increase in the number of cells entering this transition. TPA enhanced all three effects. Binding of [125I]hGH was not affected by prior exposure to TPA, suggesting that the effect of TPA is at a postreceptor level. Proliferation of an autonomous clone of Nb2 cells that does not require lactogenic hormones for growth was not stimulated by TPA, although these cells bound [3H]TPA to the same extent as the PRL-dependent Nb2-11C clone.     

The 20,000 Da variant of human growth hormone does not bind to growth hormone receptors in human liver

The 20,000 Da variant of human growth hormone (hGH) (20K) exhibited no specific binding to hGH receptors in human liver plasma membranes. This contrasts with the 22,000 Da form of human growth hormone (22K), which bound with high affinity to the same hepatic receptor preparation. Since the liver is considered a major target organ for the somatogenic pathway of growth hormone action, this finding implies that in humans the 20K form plays little role in that pathway. The homologous hormone-receptor system examined here yielded results that differ from heterologous receptor binding experiments in animals. The differences are likely explained by the presence in non-primate mammals of more than one type of growth hormone receptor with varied specificities. In man, the 20K form of growth hormone may have a biological role distinct from that of the main 22K form of growth hormone.     

Skeletal muscle growth defect in human growth hormone transgenic rat is accompanied by phenotypic changes in progenitor cells

Growth hormone (GH) is known to have a pivotal role in the maintenance of skeletal muscle mass. Sarcopenia, the loss of skeletal muscle mass, is a common phenomenon in aging, and it is widely accepted that sarcopenia is largely attributed to age-related decline in GH secretion. In the present study, we tested if human growth hormone transgenic rats (GH-TG rats) whose plasma GH levels are maintained relatively low could be an appropriate model for sarcopenia. Analyses of GH-TG rats revealed that they exhibit skeletal muscle growth defect as well as atrophy of myofibers. The number of myofibers in tibialis anterior muscle was comparable to that of WT rats, while the proportion of type I slow myofibers in tibialis anterior muscle was increased in GH-TG rats after 5 months. Neither increased expression of ubiquitin ligases, MuRF1 and MAFbx, nor indication of apoptotic cell death was observed. Notably, myogenic differentiation potential of skeletal muscle progenitor cells in GH-TG rats was lower than WT rats, and this was accompanied by increased adipogenic potential. These results indicate that GH-TG rats could be a useful model to elucidate the mechanism of sarcopenia induced by reduced GH action and raised the possibility that decreased GH action may cause an alteration of differentiation potential of skeletal muscle progenitor cells.     

Stimulation of growth of Nb2 lymphoma cells by interleukin-2 in serum-free and serum-containing media

The human recombinant alanine-125 analogue of interleukin-2 (IL-2) causes a dose-dependent mitogenic response in rat lymphoma Nb2-11C cloned cells when tested in serum-containing medium and serum-free medium. IL-2 and hGH elicit their growth stimulation through different receptors since IL-2 does not compete with 125I-hGH for binding to Nb2 cells and Met14hGH, an antagonist of hGH, inhibits the hGH-stimulated growth of Nb2 cells but not that caused by IL-2. The tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiates the action of hGH on Nb2 cells grown in both serum-containing and in serum-free medium. TPA to a variable degree also potentiates IL-2-stimulated growth of Nb2 cells when cultured in medium containing serum but has no effect on cells grown in serum-free medium. In conclusion, IL-2 is a potent mitogen for Nb2-11C cells.     

Human growth hormone-stimulated mitogenesis of Nb2 node lymphoma cells is not mediated by an immediate acceleration of phosphoinositide metabolism

Lactogenic hormones stimulate the mitogenesis of Nb2-11C rat lymphoma cells and this stimulation is enhanced by 12-O-tetradecanoyl phorbol ester (TPA). The effect of human growth hormone (hGH) and TPA on phosphoinositide metabolism in Nb2-11C cells was tested by measuring the incorporation of [3H]myo-inositol or 32P or measuring the breakdown of polyphosphoinositides in cells prelabeled with [3H]myo-inositol, 32P or [3H]arachidonic acid. We found that none of these processes is immediately stimulated by either hGH and/or TPA. Our results indicate that the overall phosphoinositide turnover is slow or confined to small fractions only. On the other hand, the phosphorylation-dephosphorylation cycle of polyphosphoinositides is quite fast and remarkably exceeds the rate of incorporation of [3H]myo-inositol. Our present results indicate, therefore, that the mitogenic effect of hGH and its enhancement by TPA is not mediated by an immediate increase in phosphoinositide metabolism.     

Interaction of human growth hormone with isolated rat liver cells.

The interaction of 125I-labeled human growth hormone (hGH) with isolated rat liver cells is a specific, time dependent and saturable process. In male rats, one cell binds a maximum of 2000 hormone molecules; the dissociation constant of the cell-hGH interaction is about 3 X 10(-10)M. Liver cells of female rats bind 5 to 10 times more hGH than do those of male rats at equivalent hormone concentrations. Binding of 125I-labeled hGH to liver cells is readily inhibited by native hGH; 50% inhibition occurs at about 2 X 10(-9)M hGH irrespective of sex. In male rats, bovine growth hormone (bGH) is almost as potent as hGH in inhibiting 125I-labeled hGH binding; no displacement occurs with ovine prolactin (oPRL) except at very high (greater than 10(-6)M) concentrations. In female rats, bGH competes less effectively, and oPRL, more effectively, than they do in males; in addition, oPRL demonstrates a higher apparent affinity for the hGH binding sites (4 X 10(-9)M) than does bGH (1 X 10(-8M). These findings suggest that in female rats hGH, unlike bGH, interacts with additional, "lactogenic" binding sites that are distinct from the "growth hormone" binding sites. The 125I-labeled hGH eluted from liver cells as well as that which remains in the incubation medium retains full biological activity, as judged on its ability to bind specifically to liver membranes. Treatment of liver cells by phospholipase A causes a 5-fold increase in cell binding capacity. Liver cells bind about the same amount of hGH as do crude particulate fractions from these cells; this suggests that in the intact cell, binding occurs at relatively accessible sites, presumably localized in the plasma membrane.     

Growth hormone specific stimulation of mitosis by size-fractionated serum from patients with growth hormone insufficiency: a study on the rat lymphoma cell line, Nb2

The aim was to investigate the bioactivity of a high-molecular weight human growth hormone, identified following molecular sieve chromatography of serum. Nine patients with pituitary disease and GH insufficiency were studied. All patients had non-detectable levels of immunoreactive GH, less than 0.2 micrograms/l, in diurnal serum profiles. GH bioactivity was determined before and after size-fractionation of serum. The bioassay is based on the finding that a rat lymphoma cell line, Nb2, proliferates in the presence of lactogens. GH and PRL immunoreactivities were measured by radioimmunoassays. Pronounced GH immunoreactivity was found in fractions of sera from 7 out of the 9 patients and of 2 of 4 control sera, particularly in fractions corresponding to the elution volume of high-molecular weight proteins (greater than 160 kD). PRL immunoreactivity was only detected in fractions corresponding to the elution volume of monomeric PRL. Unfractionated serum had a dose-dependent mitogenic effect on the Nb2 cells. GH-antibodies could not inhibit this effect. Fractions of serum obtained from the patients stimulated Nb2 cell division as well. The mitogenic effect of serum fractions could be inhibited by GH-antibodies. Thus, high-molecular weight GH circulating in patients with GH insufficiency were shown to exert a GH-specific bioactivity in vitro after size-fractionation.     

Identification of placental human growth hormone as the growth hormone-V gene expression product

A GH variant of placental origin, placental GH, has recently been shown to replace pituitary GH in maternal serum during pregnancy. Besides, the GH variant (GH-V) gene has been demonstrated to be expressed in the placenta. The similarities between their known properties strongly suggest that the placental GH and the GH-V protein are the same molecular species. Here we provide final evidence that this is indeed the case by sequence analysis of both the 22K and 25K forms. Furthermore, the 25K form is shown to be glycosylated, while the 22K form is not. Both size variants of placental GH are, thus, likely to reflect the partial glycosylation of a unique peptidic chain.     

Expression of the growth hormone variant gene in human placenta

Besides the hGH-N gene, which codes for the pituitary 22 and 20K GH variants, the human genome contains a second GH gene, namely the GH-V, which has been thought to be silent. We recently discovered a placental variant of human growth hormone (hPGH), which appears in maternal serum at mid-pregnancy and which rises in concentration thereafter to term. As hPGH and GH-V proteins display very similar characteristics, including a high affinity for hepatic GH receptors, they could be identical. To verify this hypothesis, we sought hGH-V mRNA in placenta. Hybridization experiments were performed between dot-blotted mRNA originating either from placenta or from one pituitary hGH secreting adenoma and synthetic polynucleotide probes corresponding to specific portions of the hGH-V or hGH-N gene sequences. The results indicate that the V gene is indeed expressed in the placenta and, at a very low level, in the pituitary adenoma. Therefore hPGH is most likely the expression product of the hGH-V gene.