Rat Lymphoma Cell Line Research Articles

The adaptor function of SHP-2 downstream of the prolactin receptor is required for the recruitment of p29, a substrate of SHP-2

SHP-2, a cytosolic protein tyrosine phosphatase with two SH2 domains and multiple tyrosine phosphorylation sites, contributes to signal transduction as an enzyme and/or adaptor molecule. Here we demonstrate that prolactin (PRL) stimulation of the PRL-responsive Nb2 cells, a rat lymphoma cell line, and T47D cells, a human breast cancer cell line, lead to the complex formation of SHP-2 and growth factor receptor-bound protein-2 (grb2). Using transient co-overexpression studies of the prolactin receptor (PRLR) and several tyrosine to phenylalanine mutants of SHP-2, we show that grb2 associates with SHP-2 through the C-terminal tyrosine residues of SHP-2, Y 546 and Y 584. Furthermore, in this study, we found a highly phosphorylated, 29-kDa protein (p29), a substrate of SHP-2. The recruitment of p29 to SHP-2 requires the carboxy-terminal tyrosine residues of SHP-2 (Y 546 and Y 584). Together, our results indicate that SHP-2 may function as an adaptor molecule downstream of the PRLR and highlight a new recruitment mechanism of SHP-2 substrates.

Spontaneous apoptosis of circulating T-lymphocytes and its correlation to their prolactin receptor expression and prolactin plasma levels in patients with breast cancer

We have previously shown that a higher percentage of circulating CD3+ T lymphocytes undergo spontaneous apoptosis in cancer patients as compared with normal controls. Prolactin (PRL) has been reported to inhibit apoptosis in various cell types, including a Nb2 rat lymphoma cell line. In addition, there is evidence that the human PRL-antagonist hPRL-G129R induces apoptosis in breast cancer cell lines. We investigated a possible relationship between prolactin receptor (PRL-R) expression and apoptosis of CD3+ T lymphocytes, as well as PRL plasma levels, in patients with breast cancer. Peripheral blood mononuclear cells of patients (n = 11) and sex-matched normal controls (n = 12) were stained with AnnexinV, anti-Fas mAb (CD95), mouse antihuman PRL-R mAb B6.2, anti-CD3 mAb and respective isotype control mAbs. Multicolor flow cytometry was used to compare expression of these markers on T cell. In patients, 37 ± 19% (median ± SD) of CD3+ cells bound AnnexinV, marking early apoptosis of T lymphocytes compared with 17 ± 10% in controls (P <0.004). Furthermore, 82 ± 15% of the CD3+ T cells were Fas+ in patients, compared with 51 ± 9% in controls (P <0.0001). All CD3+ T lymphocytes were positive for PRL-R expression in breast cancer patients, as well as in normal control individuals. The mean fluorescence intensity of PRL-R on T lymphocytes of breast cancer patients was 106-172 (median 119) compared with 87-176 (median 123), suggesting no difference in PRL-R expression on T lymphocytes in patients versus controls. PRL plasma levels were comparable in patients and normal controls (4.8 ± 3.4 ng/ml versus 9.8 ± 4.6 ng/ml). In concordance with these findings, PRL was not able to inhibit the onset of apoptosis of Jurkat cells, a thymic lymphoma cell line, incubated with Fas cross-linking CH-11 mAb. These results indicate that PRL/PRL-R might not be involved in modulating Fas/Fas ligand interactions, which are, in part, responsible for apoptosis of T lymphocytes, leading to excessive turnover of T cells in the circulation of patients with breast cancer.

Adenosine acts as an inhibitor of lymphoma cell growth: a major role for the A3 adenosine receptor

In this study, we demonstrated several mechanisms exploring the inhibitory effect of low-dose adenosine on lymphoma cell growth. Adenosine, a purine nucleoside present in plasma and other extracellular fluids, acts as a regulatory molecule, by binding to G-protein associated cell-surface receptors, A1, A2 and A3. Recently we showed that low-dose adenosine released by muscle cells, inhibits tumour cell growth and thus attributes to the rarity of muscle metastases. In the present work, a cytostatic effect of adenosine on the proliferation of the Nb2-11C rat lymphoma cell line was demonstrated. This effect was mediated through the induction of cell cycle arrest in the G0/G1 phase and by decreasing the telomeric signal in these cells. Adenosine was found to exert its antiproliferative effect mainly through binding to its A3 receptor. The cytostatic anticancer activity, mediated through the A3 adenosine receptor, turns it into a potential target for the development of anticancer therapies.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells.

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.

Direct evidence that lactogenic hormones induce homodimerization of membrane-anchored prolactin receptor in intact Nb 2-11C rat lymphoma cells

The ability of full-size prolactin receptor (PRLR) from Nb 2 rat lymphoma cell line to undergo lactogenic hormone-induced dimerization in intact cells or in a partially purified microsomal fraction was tested. The stoichiometry of ovine placental lactogen (oPL) binding to PRLR was documented by SDS-PAGE of the covalently cross-linked complexes between [ 125I]oPL and intact Nb 2-11C cells. The molecular masses of the specific bands were 82 and 141 kDa, corresponding to PRLR:oPL and (PRLR) 2:oPL complexes. These results provide direct evidence for the occurrence of hormone-induced receptor dimerization in intact cells. Gel-filtration studies revealed that under non-denaturing conditions, the purified receptor forms high-molecular-mass aggregates (190 and 540 kDa) composed of receptor dimers and oligomers. Since this aggregation was not dependent on the presence of lactogenic hormone, it is possible that the receptor in the intact cells may already exist as a non-covalent dimer or oligomer and that hormone-induced dimerization stabilizes the complex or changes its conformation.

Cross-resistance and collateral sensitivity to natural product drugs in cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cells.

The cytotoxicity of mitotic spindle poisons, vinca alkaloids and the anthracycline, adriamycin, against cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cell lines was investigated. Interestingly, it was found that all cell lines were more sensitive to the mitotic spindle poisons, vincristine and vinblastine. Adriamycin was the least effective and taxol had intermediate activity. The Walker rat lymphoma cell line resistant to cisplatin (WR) exhibited the multiple drug resistance phenotype since it showed collateral resistance to all drugs (ranging from twofold to taxol, colcemid and colchicine and sixfold to the vinca alkaloids). Verapamil potentiated the cytotoxic activity of adriamycin and vincristine in a striking fashion with the Walker cells. P-glycoprotein was found to be present in the plasma membranes of the Walker cells with approximately a 2.5-fold increase in the WR as compared to the sensitive (WS) cells. Glutathione levels were elevated in all of the cisplatin-resistant cell lines when compared to the cisplatin-sensitive parental cell lines. A profound effect of buthionine sulfoximine pretreatment on adriamycin cytotoxicity was observed. Glutathione S-transferase (pi) was present in all the human cell lines but the WS cells had markedly lower levels (almost negligible) when compared to the WR cells. These observations imply that cisplatin-resistant cells may be more sensitive to mitotic spindle poisons and vinca alkaloids, irrespective of the mechanism of platinum resistance, and that the cytotoxicity of vinca alkaloids could be further modulated by verapamil, irrespective of the presence or absence of P-glycoprotein.

Multiple, large deletions in rat mitochondrial DNA: evidence for a major hot spot

This study identified 33 different deletions in mitochondrial DNA from four aging Fischer-344 rat brains and from a cultured rat lymphoma cell line (Nb2 cells). The deletions were located in the longer arc between the heavy and light strand origins of replication. PCR products that spanned across the deleted regions were sequenced, and deletions ranging between 6548 bp and 9977 bp in length were identified. Short direct repeats of ≤ 8 bp were present at the end points of all but one of the deletions. The remaining deletion contained, instead, a near-perfect direct repeat ( 9 10 bp) within two base pairs of its end points. In 24 of the deletions, a sequence equivalent to one member of the paired direct repeats was lost with the deleted segment. In the remaining nine, either more or less of the base pairs of a single repeat were lost. Twelve of the 33 different deletions terminated on one side at a common locus (major hot spot) of 5 bp in length, located at the 5′ end of the tRNA Thr gene. The opposite ends of these 12 deletions were at different sites. The hot spot was located in a region of the mtDNA with strong potential for secondary structure and was flanked by a pair of AT-rich sequences. The utilization of the hot spot as an end point for deletions appeared to be widespread in that it was represented in 1 3 − 1 2 of the deletions characterized in each of the five mtDNA sources examined. In addition, several minor hot spots, where one end of two or three different deletions coincided, were also identified.

Monoclonal antibodies to human growth hormone modulate its biological properties

Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2 cells, a rat lymphoma cell line which proliferates in the presence of lactogenic hormones. Experiments involving previous binding of the hormone to receptors before adding 125I-mAbs indicated that the hGH domain defined by overlapped epitopes AE5, AC8 and F11 is uncovered in hGH when it is bound to the cell membranes. To reveal any alteration in the hGH molecule induced by the mAbs, preformed 125I-mAb:hGH complexes were added to the cell membranes. Data showed that 125I-mAb AE5:hGH complexes bound better to the receptors than free hormone. On the contrary, hGH previously bound to 125I-mAb AC8 or 125I-mAb F11 was poorly recognized by Nb2 receptors. Furthermore, both mAbs AC8 and F11 strongly inhibited 125I-hGH binding to Nb2 cell membranes and hGH-induced Nb2 cell proliferation whereas mAb AE5 enhanced both hormone binding and hGH mitogenic effect. Additionally, since mAb AC8 is directed towards an epitope shared by hGH and human placental lactogen (hPL), it was also shown that this mAb could impair hPL biological activity even though it recognizes the hPL region left exposed in hPL:Nb2 cell receptor complexes. Data presented in this work suggest that mAbs directed to the hGH or hPL regions unmasked after binding to Nb2 cell receptors produce allosteric alterations in the binding properties of these hormones leading to either enhancement or decrease of their biological activities.

Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS

Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1–3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

Regulation of heat-shock protein (hsp70) gene expression by hGH and IL2 in rat Nb2 lymphoma cells

A comparative study of hGH and IL2 post-signaling effects, as examined by RNA expression (Nb29) and protein levels of the heatshock protein hsp70, was performed in a hormone-dependent rat lymphoma cell line, Nb2-llC. Optimal doses of hGH or IL2 increased Nb29 expression in a dose-dependent manner. Addition of both mitogens to cell cultures affected Nb29 expression and mitogenesis synergystically, indicating a possible interaction between the post-receptoral mechanisms of these mitogens. Pretreatment of the cells with cholera toxin (CT) inhibited Nb29 expression, protein levels and mitogenesis of hGH- or IL2-induced cells up to 50%, indicating the involvement of Gs-proteins in the post-signaling processes of both hGH and IL2. Incubation of cell cultures with low concentrations of pertussis toxin (IAP) (0.01 ng/ml) markedly increased Nb29 expression in hGH but not in IL2-induced cells, suggesting specific involvement of the Gi-protein in post-signaling processes of hGH-induced cells. Addition of the PKC activator 12- O-tetra-decanoyl phorbol ester (TPA) to control cell cultures markedly increased the expression of Nb29 RNA levels but not mitogenesis, indicating that induction of these proteins in the cells is not sufficient for cell proliferation. Furthermore, incubation of hGH- or IL2-induced cells with the potent PKC inhibitor staurosporin (ST) decreased the levels of Nb29 in both hGH- and IL2-induced cells, although the effect of the mitogens differed significantly in their inhibition slopes. These results indicate that activation of PKC is one of the signaling pathways differentially involved in hGH and IL2 stimulation of cells. A model for post-receptoral processes is therefore proposed in which hGH and IL2 counteract in more than one signaling pathway, leading to the final stimulation and mitogenesis of Nb2 cells.

Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein, Gi alpha.

In a previous report we showed that glucocorticoid inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Gi alpha levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Gi alpha. In order to establish the nature of the coupling between cytosolic Gi alpha and cytosolic PLC we examined the effects of G-protein activators, and inhibitors on cytosolic PLC activity from rat splenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. 1) Neither GTP nor its nonhydrolyzable analogue, GTP gamma S, at 100 microM had any effect on the calcium stimulated as well as the basal PLC activity. 2) However, affinity purified antibodies to Gi alpha 1 and Gi alpha 2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50-70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. 3) Administration of Gi alpha 1 antisense oligonucleotides to splenocytes for 48 h produced 25-40% decrease in cytosolic Gi alpha 1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25-50% compared to control conditions. This suggest that cytosolic Gi alpha is associated with the activation of splenocyte soluble PLC. 4) Pertussis toxin administered in vivo significantly reduced cytosolic Gi alpha immunoreactivity and soluble PLC activity when PI was used as substrate, providing additional evidence that cytosolic Gi alpha is associated with the activation of soluble PLC. 5) Another agent that has been used extensively to define G-protein coupled processes is NaF/AlCl3. NaF (5 mM; with or without AlCl3) inhibited soluble PLC activity with PIP2 as substrate, in contrast to the stimulatory effect that has been reported in the activation of membrane PLC. 6) Because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperizine (50 microM, TFP), an inhibitor of protein phosphatase 2B; TFP (50 microM) significantly inhibited soluble PLC activity when PI was used as substrate. These results suggest a direct involvement of cytosolic Gi alpha in the activation of soluble PLC from splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Gi alpha coupled PLC is addressed in the second paper.

Prolactin and the Immune System

Prolactin (PRL) Is involved In a wide range of physiological effects in several species and its immunoregulatory role has already been well documented. The PRL receptor has been cloned from various species. There are at least two receptor isoforms (short and long) in rats and mice, which differ only in their cytoplasmic domains, generated by alternative splicing of a single gene, although in human only the long form exists. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we detected transcripts encoding both forms of PRL receptor in all lymphoid tissues examined in human, mouse, and rat, but in mouse and rat the ratio between the two forms was variable from animal to animal. Concerning the transcript encoding the PRL itself, a clear signal was always found in human lymphocytes and occasionally in rat thymus. We also developed a quantitative PCR (Q-PCR) in order to measure the absolute number of transcripts in thymus and spleen from rats at two stages of estrous cycle. The level of expression of the two forms was about equal. Finally, we identified the tyrosine kinase JAK2, which is constitutively associated with the PRLR, using the Nb2 rat lymphoma cell line as a model system with which to study the action of PRL on cell mitogenesis. We also showed that, after stimulation by PRL, the dimerization process is a prerequisite step for the phosphorylatlon of the PRLR and JAK2, which represents the earliest event in the signal transduction pathway.

Purification from Human Pregnancy Serum of a Low Molecular Weight Mitogen Similar to Placental Lactogen and Growth Hormone

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.

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.

An Epstein-Barr Virus-Negative Burkitt Lymphoma Cell Line (sfRamos) Secretes a Prolactin-Like Protein during Continuous Growth in Serum-Free Medium

Pituitary human PRL (hPRL) antiserum inhibits growth of B-lymphoblastoid cells in vitro, but the mechanism of inhibition is unclear. In this study the mechanism of inhibition of human B-cell growth by anti-hPRL was explored with an Epstein-Barr virus nuclear antigen (EBNA)-negative Burkitt lymphoma cell line (sfRamos) that proliferates continuously in serum-free medium with human transferrin as the only protein supplement. The data show that antiserum immunoglobulin fraction G (IgG) to pituitary hPRL, but not nonimmune serum IgG, completely inhibited the growth of sfRamos cells. In addition, anti-hPRL IgG identified a single band (29 kDa) in sfRamos spent medium, but not in fresh serum-free medium or in human transferrin, as demonstrated by sodium dodecyl sulfate-reducing polyacrylamide gel electrophoresis and Western immunoblot analysis. Polyacrylamide gel electrophoresis and Western analysis of a mixture containing sfRamos spent medium and excess pituitary hPRL established that the sfRamos 29-kDa PRL-like protein (PLP29) and pituitary hPRL (23 kDa) were electrophoretically distinct. Finally, sfRamos spent medium, but not fresh serum-free medium, was mitogenic for sfRamos and Nb2, a PRL-sensitive node rat lymphoma cell line. These findings demonstrate that PLP29 is biologically and immunologically like pituitary hPRL, but is electrophoretically different from this hormone. We suggest that PLP29 is secreted as an autocrine growth factor by sfRamos Burkitt lymphoma cells during continuous serum-free growth.

Lactogen-induced protein phosphorylation in Nb2 cells

Lactogenic hormones are potent mitogenic agents in the Nb2 rat lymphoma cell line. Because selective phosphorylation/dephosphorylation of specific proteins by many polypeptide hormones appears to be important in regulating cell growth, the effect of lactogenic hormones on protein phosphorylation was studied in Nb2 cells. Human growth hormone (hGH) promoted the phosphorylation of many proteins (2- to 3-fold) and an M r 29,000 species (pp29) (⩾ 10-fold). In growth-arrested cells phosphorylation of pp29 peaked 4 h after addition of hGH whereas that of other phosphoproteins increased steadily. Ovine prolactin, another potent mitogenic hormone, caused a concentration-dependent increase in pp29 phosphorylation with an EC 50 = 10–20 p m. Other agents which are not mitogenic in this cell line, i.e., 12- O-tetradecanoyl-phorbol-13-acetate (200 n m), 8-Br-cAMP (1 m m), and 8-Br-cGMP (1 m m), did not affect pp29 phosphorylation. Dexamethasone (100 n m), which inhibits lactogen-stimulated growth, reduced hGH-stimulated pp29 phosphorylation to control levels. Fractionation of detergent-treated extracts of hGH-treated cells showed that pp29 is associated with the ribosomal fraction. Taken together, these results show that pp29 phosphorylation is selective and is related to cell growth.

Phosphatidylethanolamine turnover is an early event in the response of NB2 lymphoma cells to prolactin

The effect of prolactin on phospholipid metabolism in the prolactin-dependent rat lymphoma cell line Nb2 was investigated in cells prelabeled with [3H]arachidonic acid or [3H]ethanolamine. Prolactin (20 ng/ml) caused (a) a 20-60% loss of radiolabeled phosphatidylethanolamine within 0.5 to 2 min, (b) a loss of [3H]ethanolamine-labeled phosphatidylethanolamine from crude membranes, (c) a rapid accumulation of [3H]phosphoethanolamine and [3H]ethanolamine, and (d) a transient increase (15 s to 2 min) in prostaglandin F2 alpha and E2. Arachidonic acid (1-2 micrograms/ml) induced Nb2 cell growth but prostaglandin F2 alpha, E2, ethanolamine, and phosphoethanolamine did not. Prostaglandin E2 inhibited while prostaglandin F2 alpha enhanced growth in the presence of prolactin or arachidonic acid. These results suggest that stimulation of Nb2 cell growth by prolactin is linked to activation of a phosphatidylethanolamine-specific phospholipase C. Arachidonic acid and prostaglandin F2 alpha may participate in regulating the mitogenic action of prolactin.

Fetal control of maternal prolactin production and bioactivity in utero

Amniotic fluid prolactin is a product of maternal decidualized endometrium that is derived by translocation of the hormone across the reflected fetal membranes. Amniotic fluids from 26 second-trimester (14 to 23 weeks) and 75 third-trimester (29 to 40 weeks) normal singleton pregnancies were evaluated for prolactin content by radioimmunoassay and bioassay with the Nb2 rat lymphoma cell line. The relative bioactivity was calculated as the ratio of bioassay to radioimmunoassay for each fluid. Data segregated by gestational age and fetal genetic sex identified a highly significant difference (p = 0.0004) in amniotic fluid prolactin radioimmunoassay concentrations (mean +/- SEM) that surround male (682 +/- 49, n = 42) versus female (440 +/- 39, n = 33) fetuses of third-trimester age. Paired bioassay values were significantly lower (p = 0.002) than radioimmunoassay values among males (626 +/- 52) but equivalent (p = 0.1066) among females (464 +/- 44). The bioassay/radioimmunoassay ratios of third-trimester fetal female-associated amniotic fluid prolactin were significantly higher (p = 0.0004) than those of third-trimester males and second-trimester males and females. The results suggest a fetal gender-related factor is associated with both the production and the biologic activity of the maternally derived hormone. Thus the fetus appears to have some control over the dynamics of uterine prolactin production.

Autocrine stimulation of Nb2 cell proliferation by secreted, but not intracellular, prolactin.

We have introduced expression constructs for mouse PRL (mPRL) or a nonsecreted form of mPRL into the PRL-responsive Nb2 rat lymphoma cell line. Cell lines resulting from transfection of Nb2 cells with the wild type mPRL construct synthesize and secrete mPRL. These cells are able to grow independently of added lactogens, and conditioned media and cell extracts from these cultures stimulate the growth of Nb2 cells. In contrast, cells synthesizing the nonsecreted mPRL do not proliferate in the absence of added lactogenic hormones, and conditioned media from these cell cultures do not have PRL-like activity in the Nb2 cell growth assay. PRL protein is detected in these nonsecreting cell lines; however, extracts from these lines are generally unable to stimulate Nb2 cell proliferation. These results indicate that cells can respond in an autocrine fashion to PRL, but that an intracellular form of PRL is unable to activate Nb2 cell growth.

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.