L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphoma cells

Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL-dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitric oxide (NO) was recently identified as a downstream regulator of PRL action, and as an inhibitor of apoptosis in immune cells. In the present study, the role of NO in PRL-regulated Nb2 cell function was investigated. Nb2 cells expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The eNOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells but decreased by at least 3-fold at 3-24 h following PRL treatment. Downregulation of eNOS was not accompanied by a corresponding decrease in the eNOS protein, the level of which remained constant for at least 24 h after PRL treatment. PRL had no effect on the phosphorylation state or subcellular redistribution of the eNOS enzyme, or on production of NO by Nb2 cells. However, increasing concentrations of L-arginine (NOS substrate) alone increased NO production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanced Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.125 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survival and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone significantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Expression of the anti-apoptotic gene bcl-2, which was stimulated within 1 h by PRL, was upregulated by L-arginine or DEA/NO alone at 2 h and 8 h, respectively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prolactin-independent, Bcl-2-mediated pathway.     

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.     

Expression of biologically active rat prolactin in mammalian COS-1 cells

Rat prolactin (PRL) cDNA was constructed in mammalian expression vector, pSVL. Transient expression of rat PRL was performed in COS-1 cells by the DEAE-dextran method. The production of recombinant rat PRL started within 48 h from the cells and reached the level of 1.0-1.5 micrograms/ml/5 x 10(5) cells. The molecular size of recombinant rat PRL was the same as that of standard rat PRL (Mw: 23,000), suggesting successful removal of the signal peptide. The radioimmunoassay and isoelectric focusing analysis showed that recombinant rat PRL has almost the same immunological and biochemical characteristics as those of standard rat PRL. As biological tests, receptor-binding activity, Nb 2 node lymphoma cell growth activity, and mammary gland stimulating activity were examined. The radioreceptor assay showed that recombinant rat PRL has binding activity to mammary microsomal membrane similar to that of standard rat PRL. Recombinant rat PRL also stimulated the growth of Nb 2 lymphoma cells as standard rat PRL. Finally it was shown that recombinant rat PRL promotes the synthesis of the secretory materials in the lumen of mouse mammary gland with the same potency as that of standard rat PRL. In conclusion, recombinant rat PRL, which was produced in mammalian cells in the present experiment, has immunological, biochemical and biological characteristics similar to those of standard PRL, and has full bioactivity.     

Butyrate-induced reversal of dexamethasone resistance in autonomous rat Nb2 lymphoma cells

The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL i n a time- and concentration-dependent manner. Eval uati on of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sequence of prolactin effects on phospholipid synthesis in Nb2 node lymphoma cells

In Nb2 node rat lymphoma cells, the effects of prolactin (PRL) on the rates of incorporation of several precursors into neutral lipids, phospholipids and proteins were determined. The onset of the PRL stimulation of radiolabeled-precursor incorporation into lipids occurred between 1 and 4 hours after PRL addition to Nb2 cells; precursors employed included [14C]-acetate, [3H]-glycerol, [32P]O4, [3H]-choline, [3H]-ethanolamine, [3H]-serine and [3H]-myoinositol. No effects were observed during the initial 60 min of culture with PRL. The effects on precursor incorporation that occur after 1 hr of PRL exposure are likely related to the stimulation of cell growth by PRL. In cells that were prelabeled with the radiolabeled precursors and subsequently incubated with PRL, PRL had no effect on the metabolism of the radiolabeled phospholipids or the accumulation of phospholipid products until several hours after hormone addition. We would conclude from these studies that the initial (60 min) effect of PRL on Nb2 node lymphoma cells does not likely use a signal transduction mechanism that involves products derived from the cellular phospholipids.     

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.     

Growth and protein phosphorylation in the Nb2 lymphoma: effect of prolactin, cAMP, and agents that activate adenylate cyclase

The Nb2 T lymphoma is unique in that these lymphocytes proliferate in response to prolactin as well as in response to interleukin-2. In this study, we have examined the responsiveness of the adenylate cyclase system in Nb2 cells and the role of this signaling system in regulating proliferation and protein phosphorylation. An analog of cAMP inhibited prolactin-stimulated proliferation and blocked a prolactin-induced decrease in protein phosphorylation. Forskolin, a potent activator of adenylate cyclase in T lymphocytes, did not elevate cAMP levels in Nb2 cells and was not an effective inhibitor of prolactin-induced proliferation. In fact, one preparation of forskolin stimulated proliferation of quiescent Nb2 cells. Like forskolin, prostaglandin E2 did not stimulate cAMP production in Nb2 cells even though it increased cAMP in a preparation of rat peripheral blood lymphocytes. Cholera toxin appeared to ADP-ribosylate a stimulatory guanine nucleotide-binding protein in Nb2 cells, but the toxin did not increase intracellular levels of cAMP nor was it a potent anti-mitogenic agent. Pertussis toxin, an agent that can increase cAMP production through suppression of the inhibitory guanine nucleotide-binding protein, exerted only minor anti-proliferative actions on prolactin-stimulated Nb2 cells. These data suggest that cAMP inhibits Nb2 cell proliferation and prolactin-induced changes in protein phosphorylation but that the adenylate cyclase system in our clone of Nb2 cells responds poorly to agents that normally increase cAMP.     

Interleukin 2 and a lactogen regulate proliferation and protein phosphorylation in Nb2 cells.

Cell proliferation and protein phosphorylation in response to activation of lactogenic and interleukin 2 (IL-2) receptors were studied in Nb2 cells, a rat T-lymphocyte cell line. Human growth hormone (hGH) and rat IL-2 stimulated Nb2-cell proliferation to approximately the same degree, and the actions of both mitogens were potentiated by phorbol 12-myristate 13-acetate (PMA). A monoclonal antibody specific for the rat IL-2 receptor inhibited the mitogenic actions of rat IL-2, but not those of hGH. Exposure of Nb2 cells to either mitogen for 2-3 h increased phosphorylation of an 18,600-Da protein and decreased phosphorylation of a 15,600-Da protein. PMA also inhibited phosphorylation of the latter protein, but, by itself, PMA did not stimulate phosphorylation of the 18,600-Da protein. Overall, the results suggest that hGH and IL-2 act through separate receptors to stimulate proliferation of Nb2 cells, and that some of the actions of both mitogens may be mediated, in part, through regulation of protein phosphorylation.     

Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb(2) cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor.

The Nb2 cell line is a pre-T rat lymphoma that is dependent on prolactin (PRL) for mitogenesis. Two forms of PRL receptor (PRL-R), which differ in the length of their cytoplasmic domains have been identified in different tissues and species. In the present study we have cloned the cDNA and characterized the mitogenic form of PRL-R in Nb2 cells. Polymerase chain reaction amplification of first strand cDNA prepared from Nb2-11C (PRL-dependent) and Nb2-Sp (PRL-independent) cell lines was performed using oligonucleotide primers specific for the binding domain, the short form of the PRL-R, and the cytoplasmic domain of the long form of the PRL-R. These studies indicate that both cell lines express a novel form of PRL-R. A cDNA was isolated from an Nb2-Sp cDNA library, which contains 1446 base pairs identical to the nucleotide sequence of the long form of the rat PRL-R. However, the cDNA sequence is missing 594 base pairs in the cytoplasmic domain compared with the long form of the PRL-R. The cDNA encodes a protein of 393 amino acids, lacking 198 amino acids in the cytoplasmic domain. Scatchard analysis of 125I-labeled ovine prolactin (oPRL) binding to microsomes prepared from transiently transfected COS-7 cells with either PRL-R long form cDNA or Nb2 PRL-R cDNA indicates that the long form of PRL-R binds oPRL with high affinity (K alpha = 8.8 x 10(9) M-1), while the Nb2 PRL-R showed a 3.3-fold increased affinity for PRL (K alpha = 29.1 x 10(9) M-1). In addition, immunoblot analysis of these microsomes using 125I-labeled monoclonal antibody (U6) to the PRL-R demonstrates a Mr of approximately 82,000 for the long form and approximately 62,000 for the Nb2 form of PRL-R. Polymerase chain reaction amplification of genomic DNA prepared from PRL-dependent and -independent cell lines suggests that this form of PRL-R results from a deletion in the PRL-R gene. The identification of a modified long form of PRL-R in the Nb2 cell line should help localize domains of the PRL-R involved in signal transduction and further the investigation of prolactin's role in immune cell proliferation.     

Studies on the possible role of protein kinase C in the prolactin regulation of cell replication in Nb2 node lymphoma cells

The results of several recent studies have indicated that protein kinase C (PKC) may be involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The PKC activator 12-O-tetradeconylphorbol-13-acetate (TPA) at certain concentrations has been shown to potentiate the mitogenic effect of PRL, whereas at higher concentrations, TPA inhibits the PRL response. Several inhibitors of PKC have also been shown to impair the PRL stimulation of metabolic process in the Nb2 cells. These studies provide further evidence for the likely involvement of PKC in the PRL stimulation of mitogenesis in the Nb2 cells. A transient, time-dependent accumulation of PKC in the particulate fraction of the Nb2 cells is observed in response to PRL. TPA is also shown to elicit a similar effect, albeit at a much earlier time and with a greater magnitude. On long-term exposure (3 days), high concentrations of TPA down-regulate the PKC enzyme; this down-regulation likely accounts for the inhibitory effect of high concentrations of TPA on the PRL stimulation of cell division. In further studies, the PKC inhibitors H-7 and gossypol were shown to inhibit the PRL stimulation of cell division in a concentration-dependent fashion.     

Heterogeneity in buffalo pituitary prolactin

The Ellis procedure of serial extraction of gonadotropins and growthhormone (GH) followed by alkaline ethanol extraction was adopted to processfreshly frozen buffalo pituitaries. The procedure after slight modificationwas found very useful as more than 2 mg of GH free immunoreactive prolactin(PRL) could be isolated from each gram of wet pituitary tissue. Further, thebiochemical purity and immunobiological potency of the extracted PRL,designated as P-I, was comparable with that of the highly purified samplesof homologous and heterologous PRLs. No non-PRL protein was detectable inP-I. Micro-heterogeneity with regard to size, charge, co- andpost-translational modifications was also investigated under differentconditions of extraction and at different stages of purification.Immunological and biological potencies were compared in homologouscompetitive enzyme linked immunosorbent assay (ELISA) developed for buffaloPRL and in rat Nb2 lymphoma proliferation assay respectively. Structuralheterogeneity was observed in all the preparations checked including freshpituitary homogenate and highly purified hormone. Nevertheless a 25 Kspecies corresponding to the hormone monomer was always the only paramountform comprising more than 90% of the total PRL protein in all thesamples including P-I. Similar size forms were observed in all preparationsand were found to be equivalents of monomers, dimers, covalent-andnon-covalent multimers, disulphide bridged forms and cleaved fragments.Other sibling species identified were glycosylated PRL, charge isoforms andforms that perhaps differed in their extractability from the pituitarytissue. Strong apparent size heterogeneity was displayed by the monomericbuffalo PRL. In light of these observations and the information on thestructural and functional significance and the consequences of polymericforms, the use of a heterogeneous PRL (P-I) as a reference hormone isrecommended for a valid assay.     

Ascorbic acid recycling in Nb2 lymphoma cells: implications for tumor progression

Analysis of cultured rat “Nb2 lymphoma” cell lines, showing different degrees of malignant progression, can lead to identification of phenotypic changes associated with this phenomenon in T-cell cancers. In the present study we have compared the metastatic sublines, Nb2-11 and Nb2-SFJCD1, with regard to ascorbate and glutathione recycling, important processes in cellular protection from oxidative stresses. Whereas the Nb2-11 subline is prolactin (PRL)-dependent, the genetically related Nb2-SFJCD1 subline is growth factor-independent and shows more chromosomal alterations, indicative of more advanced progression. The Nb2-SFJCD1 cells, compared to the Nb2-11 cells, were less sensitive to toxic effects of dehydroascorbate, a potentially toxic oxidation product of ascorbate. Results were consistent with a significantly higher production of reducing equivalents (e.g., NADPH, GSH) and an accelerated reduction of dehydroascorbate by homogenates of Nb2-SFJCD1 cells. However, the increased resistance was apparently not directly related to the cellular uptake and reduction of dehydroascorbate by whole cells, which was similar in both cell lines. Observations indicate that Nb2 lymphoma cells, in their progression to malignancy, can acquire an enhanced capability to protect themselves from oxidative damage assisting them in withstanding the oxidative stress that anti-neoplastic drugs can cause. The adaptation may also be a mechanism that is utilized by tumor cells in suppressing apoptosis and other protective cellular functions facilitating, or potentiating, a tumor cell’s ability to become more metastatic. However, the mechanism leading to this augmented capacity of Nb2 lymphoma cells to resist oxidative stress in not known and is the subject for further study.     

Prolactin stimulates activation of c-jun N-terminal kinase (JNK)

In recent years the mitogen-activated protein (MAP) kinase family has expanded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the extracellular regulated kinase (ERK) family. These kinases are activated by a variety of growth factors, as well as extra- and intracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation with 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal activation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL receptor (rPRLR), as well as the long form of the human PRLR (hPRLR). The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; however, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR was maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and declined after that time. Dose response studies indicated that activation of JNK was maximal after 30 min at a concentration of 10 nM, and the amount of activated JNK declined at the highest concentration of oPRL, 100 nM. Immunoblot analysis with an antibody that recognizes the activated (phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenovirus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to determine the biological effect of blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant inhibited cellular proliferation and induced DNA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or suppression of apoptosis in Nb2 cells.     

3-phenylacetylamino-2,6-piperidinedione inhibition of rat Nb2 lymphoma cell mitogenesis

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1-12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P less than 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.     

PAL31, a nuclear protein required for progression to the S phase

PAL31 is a nuclear protein expressed by various cell types. In the present study, the expression and function of PAL31 were examined in the cytokine-regulated growth of T and B cell lines. Treatment of the cells with mitogens [ovine PRL, recombinant rat placental lactogen-I (PL-I) and human IL-3] caused a dose-dependent increase in the expression of PAL31 mRNA in the PRL-dependent cell line Nb2, and IL-3 dependent cell line BaF3. A time-course study on synchronized Nb2 cells revealed that the expression of PAL31 is specific to the late G1 and S phases. Immunocytological studies revealed that PAL31 accumulates in the nuclei at the S phase. Furthermore, the antisense oligonucleotide for PAL31 severely inhibited the proliferation of Nb2 cells by inhibiting cells progressing to the S phase. Thus, PAL31 is a nuclear protein associated with cell cycle progression.     

Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG

Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.     

Alanine-scanning mutagenesis of human prolactin: importance of the 58-74 region for bioactivity.

We have generated 10 alanine mutants of human PRL (hPRL), a member of the PRL/GH family, to investigate the involvement of the highly conserved 58-74 region in the biological behavior of the protein. When treated with polyclonal anti-hPRL antibodies, all mutants were immunologically indistinguishable from the unmodified hPRL, and circular dichroism analyses indicated that their alpha-helix content was similar to that of the unmodified hormone. Mutations C58A, K69A, and, to a lesser extent, P66A affected drastically the ability of hPRL first to bind to the lactogenic receptor and second to stimulate the proliferation of Nb2 lymphoma cells, proving the importance of the 58-74 peptide segment for hPRL bioactivity. Binding affinities of these mutants to the Nb2 lactogenic receptor have been compared to lactogenic binding data previously obtained for several mutants of hGH. The comparison reveals that the residues involved in the biological properties of the two proteins are not at topologically equivalent positions. Hence, we suggest that the binding of these hormones to the lactogenic receptors occurs through a different molecular mechanism having distinct requirements at the residue level.