Nb2-11C Cells Research Articles

New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin

Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: Placental growth hormones have no lactogenic activity in humans

Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5 l of fermentation culture varied between 400 and 700 mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs’ secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the α-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10–11 at 4 °C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.

Characterization of 123I-iodo-α-methyltyrosine transport in rat lymphoma cells

123I-Iodo-α-methyltyrosine (IMT) transport into lymphomas has not been fully characterized. In rat Nb2-11C and Nb2-Sp lymphoma cell lines, linear uptake of 123I-IMT occurred rapidly within 5–10 min. Eadie–Hoftee plots of 123I-IMT uptake gave apparent K m's of 8.34±1.17 and 9.64±1.05 μM for Nb2-11C and Nb2-Sp cells, respectively, and involved the L and B 0,+ systems. In lymphoma-bearing rats, injected 123I-IMT accumulated rapidly in the primary tumors but gave a low tumor-to-background ratio of 2:1. 123I-IMT was transported rapidly into lymphoma cells both in vitro and in vivo, but low target-to-nontarget ratio may not make 123I-IMT practical for scanning in vivo.

Identification and nuclear localization of a novel prolactin and cytokine-responsive carboxypeptidase D.

A full-length, PRL-inducible complementary DNA (cDNA) encoding a novel, nuclear-targeted carboxypeptidase D isoform (designated CPD-N) was identified in the rat PRL-dependent Nb2-11C and PRL-independent Nb2-Sp lymphoma cell lines by differential display. The CPD-N cDNA (3751 bp) has 99% (3582/3583) homology with rat carboxypeptidase D (CPD; 4377 bp). In comparison to the rat CPD cDNA (ORF of 4134 bp; 180-kDa protein), CPD-N was shorter by approximately 600 bases but contained 148 unique bases at the 5'-end to give an ORF of 3399 bp. RT-PCR with primers specific to the 5'-end of CPD-N or to CPD showed that the CPD-N transcript was expressed in the Nb2-11C and Nb2-Sp cells but was not detected in rat brain or lung. Conversely, the CPD transcript was expressed in rat brain but was not detected in the two Nb2 cell lines. CPD-N expression (7.5-kb messenger RNA) was stimulated by PRL (10 ng/ml) and/or by interleukin-2 (24 U/ml) in Nb2-11C and Nb2-Sp cells. Most rat tissues expressed multiple CPD transcripts (7.5, 4.1, and 2 kb). Curiously, CPD transcripts were low or undetectable in male rat liver but readily detected in female liver, suggesting that sex-specific hormone levels may regulate its expression. Indeed, CPD expression in the PRL-responsive HepG2 hepatoma and MCF-7 breast cancer cell lines was low in control cells but was markedly stimulated by PRL after 3 h. Consistent with the shorter ORF of CPD-N, Western analysis detected proteins of smaller molecular sizes of 160 kDa (abundant) and 117 kDa (weak) in the Nb2-11C cells. The Nb2-Sp cells expressed a single and abundant 117-kDa protein, implicating differential protein processing in the two cell lines. Rat CPD has been reported to colocalize with the trans-Golgi network marker TGN38. Subcellular fractionation showed predominant nuclear localization of CPD-N and trace amounts were detected in the 100,000 x g microsomal fraction after PRL treatment (4 h); in contrast, TGN38 was found only in the microsomal fraction at this time. In cells treated with PRL for 24 h, immunofluorescent confocal microscopy showed nuclear and cytoplasmic distribution of CPD-N. Cytoplasmic CPD-N colocalized with TGN-38 whereas nuclear CPD-N had a mesh-like distribution and colocalized with nuclear lamin B.

Malignant Progression of Rat Nb2 Lymphoma Cells: Chromosomal Alterations and Metastatic Properties

Cultured rat pre-T Nb2 lymphoma cell lines have provided a useful model for tumor progression of T-cell cancers. Comparative analysis of the non-metastatic, prolactin (PRL)-dependent parental Nb2-U17 line and its PRL-independent and/or metastatic sublines, can be used in a search for progression-related genomic alterations. In the present study, the PRL-dependent, cloned Nb2-11C and PRL-independent Nb2-Sp sublines were used to examine development of metastatic ability and PRL independence relative to chromosomal alterations. Metastatic ability was determined using Noble rats carrying subcutaneous tumor transplants; PRL dependence/autonomy was checked in culture. Nb2-11C tumor transplants quickly gave rise to morbidity, associated with metastases in kidney and liver. Transplants of the slower growing Nb2-Sp cells showed variable tumorigenicity as metastases developed in only 40% of the rats (in lungs, kidney, stomach). G-banded chromosome analysis showed the Nb2-11C culture had the karyotype of the parental Nb2-U17 line plus an extra chromosome 19, thus, indicating an association between the development of metastatic ability in Nb2-11C cells and trisomy 19. The Nb2-Sp subline was not clonal. Its stemline showed two alterations in the parental karyotype: acquisition of an add(7)(q10) and loss of the extra chromosome add(15)(p12). Additional abnormalities, add(6)(q11) and trisomy 19, occurred in 15% and 5% of the Nb2-Sp population, respectively. Passaging of the Nb2-Sp subline in vivo resulted in generation and/or outgrowth of new sublines, a major one of which showed an apparent transient growth requirement for lactogens. Possible mechanisms underlying the PRL independence and in vivo properties of the Nb2-Sp cells are discussed.

Prolactin induces expression of FGF-2 and a novel FGF-responsive NonO/p54 nrb-related mRNA in rat lymphoma cells

The rat Nb2-11C lymphoma cell line expresses high affinity prolactin (PRL) receptors, and requires lactogenic hormones for survival and proliferation. We have applied differential display to identify genes which are differentially induced in Nb2-11C cells following PRL stimulation, or which are constitutively expressed in the PRL-independent Nb2-Sp cells. In the present study we characterized a clone (22c.2) which was expressed in Nb2-Sp cells, and in Nb2-11C cells given PRL for 3 h but not in untreated cells. The 279 bp cDNA had 95% homology with the 3′ end of the murine 2.6 kb FGF-inducible gene 14 (FIN14). When clone 22c.2 was used to screen a Nb2-Sp cDNA library to obtain a longer cDNA, a unique 1039 bp clone PNR (Prolactin-responsive/NonO-Related) was isolated, subcloned and sequenced. The deduced amino acid sequence encoded by the PNR open reading frame had significant homology with a family of RNA- and DNA-binding proteins which include the human polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF), the murine non-POU-domain-containing octamer-binding protein (NonO) and the human NonO homologue p54 nrb. Nb2-11C cells expressed three PNR-related mRNA transcripts of 2.5, 3.0 and >10 kb. Expression of the 2.5 and 3.0 kb transcripts were increased at least 4-fold within 3 h of PRL treatment. PNR expression was also significantly stimulated within 3 h by addition of FGF-2 to either Nb2-11C or Nb2-Sp cells, although alone FGF-2 was not mitogenic for either cell line. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the expression of both FGF-2 and FGF receptor mRNA in Nb2 cells, raising the possibility of an autocrine or paracrine function for FGF-2 in lymphoma cells. Furthermore, PRL rapidly stimulated the expression of FGF-2 mRNA in a time- and dose-dependent manner in both Nb2-11C and Nb2-Sp cells. FGF-2 expression was increased within 1 h and was maintained at a high level for at least 10 h following treatment with 2 ng/ml PRL. Western blotting with anti-FGF2 antisera demonstrated PRL stimulation of intracellular accumulation, but not secretion of immunoreactive FGF-2. The observation of PRL-responsive expression of FGF-2 in Nb2 cells suggests a previously unrecognized pathway for PRL action in lymphoid cells.

Differential expression of elongation factor-2, α4 phosphoprotein and Cdc5-like protein in prolactin-dependent/independent rat lymphoid cells

The differential display of mRNAs technique was used to identify genes which are differentially expressed in the prolactin (PRL)-dependent Nb2-11C and PRL-independent Nb2-Sp rat lymphoma cell lines. The technique was validated by the differential display of the proto-oncogene c- myc in PRL-treated, but not in untreated, Nb2-11C cells. Nine DNA bands, isolated from the 6% display gels and confirmed by reverse Northerns to be differentially expressed, were cloned and gave rise to ten unique clones. DNA sequencing showed that these clones had limited homologies to known genes or uncharacterized expressed sequence tags. Using one of these clones (6ac1.12) as a probe in Northern analysis, a transcript of approximately 4 kb was detected which was elevated in PRL-treated Nb2-11C cells and in mid-log phase growing Nb2-Sp cells. Similar to c- myc, expression of the 4 kb transcript increased in Nb2-11C cells given PRL for 3 h (±the phorbol ester TPA) but not in cells given TPA alone. The 4 kb transcript also increased with increasing Nb2-11C cell densities. By screening an Nb2-Sp cDNA library with 6ac1.12 as probe, three unique genes were isolated and identified as elongation factor 2 (EF-2), α4 phosphoprotein and a Cdc5-like protein. Each of the three genes were PRL responsive in Nb2-11C cells and expressed constitutively in Nb2-Sp cells. The expression of EF-2 or α4, but not the Cdc-like protein, was dependent on cell densities. EF-2 regulates protein synthesis while the α4 and Cdc5-like phosphoproteins have been implicated in IgG receptor-mediated and mitogen-activated signaling, respectively. The identification that these genes are PRL-responsive and/or differentially expressed in the Nb2-11C and Nb2-Sp cell lines may permit insights into the molecular changes that are involved in regulating the transition to growth factor independence in lymphoid tumors.

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.

Lactogenic hormones rapidly activate p21 ras /mitogen-activated protein kinase in Nb2-11C rat lymphoma cells

Lactogenic hormone-dependent Nb2-11C cells proliferate in response to prolactin (PRL) or human growth hormone (hGH). We have investigated the activation of p21( ras ) and mitogen-activated protein kinase (MAP-kinase) by hGH in lactogen-dependent Nb2-11C and in autonomous hormone-independent Nb2-SP rat lymphoma cells. Exposure of Nb2-11C cells to hGH resulted in a dose-dependent activation of p21( ras ) and of MAP-kinase. Activation occurs at physiological hGH concentration and with a rapid onset (∼1 min) reaching maximal level at 10-20 min. In contrast, in Nb2-SP autonomous lactogen-independent cells, p21( ras ) and MAP-kinase are constitutively activated and a challenge with lactogenic hormone had a modest additional activating effect. TPA, an activator of protein kinase C, enhanced p21( ras ) and MAP-kinase activity in Nb2-11C cells but failed to induce proliferation. The mechanism of activation of p21( ras ) in Nb2-11C cells by lactogenic hormones involves both an increased binding of guanine nucleotides to p21( ras ) as well as an increase in GTP/GDP+GTP ratio. In summary, we have demonstrated here that activation of the p21( ras )/MAP-kinase pathway follows PRL receptor activation but is not sufficient for the lactogenic hormone-dependent mitogenesis.

Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen.

Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation. However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity. The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity. Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells). Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure. Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL. Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties. It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors. Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.

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.

The Role of G-Proteins in the Mitogenesis of Rat Lactogen-Dependent and Lactogen-Independent NB2 Lymphoma Cells*

The expression of guanine nucleotide-binding proteins (G-proteins) was compared in two clonal lines of rat Nb2 node lymphoma cells, the lactogen-dependent Nb2-11C line and the lactogen-independent Nb2-Sp (spontaneous) line. Both cell lines expressed mRNA transcripts for the G-protein species Gs alpha [1.85 kilobases (kb)], Gi2 alpha (2.35 kb), Go alpha (4.1-4.5 kb), and Gi3 alpha (3.5 kb). Gi1 alpha was not detected. ADP ribosylation in the presence of activated cholera or pertussis toxins and [32P]NAD demonstrated the presence of G-proteins in the membrane fractions of both lines. The cholera toxin substrates consisted of two proteins (mol wt, 46.5 and 43.5 kD), while a single protein (mol wt, 41.5 kD) was ADP ribosylated by pertussis toxin. Surprisingly, the cholera toxin-sensitive proteins (Gs) were at least 20-fold less abundant in the Nb2-Sp cells than in the Nb2-11C cells. Since Gs and Gi2 are associated with the adenylate cyclase system and the regulation of intracellular cAMP, the effects of the cAMP analog, (Bu)2cAMP (dbcAMP), on Nb2-11C and Nb2-Sp cell growth were examined. dbcAMP (100 microM) completely inhibited the growth of lactogen-dependent Nb2-11C cells. The inhibitory effect of dbcAMP was exerted at an early point in the cell cycle, as it also inhibited PRL-stimulated c-myc expression measured 3 h after addition of the mitogen. In contrast, dbcAMP had only minor inhibitory effects on lactogen-independent Nb2-Sp cells, increasing their doubling time from 20 to 30 h and slightly reducing their density at confluence. The inhibitory effect of dbcAMP on both cell lines was reversible. Nb2-11C cells resumed growth after a lag period of approximately 3 days. The recovered cells did not arise from selection of a cAMP-resistant subpopulation, since both they and normal untreated Nb2-11C cells remained equally sensitive to dbcAMP. Similarly, Nb2-Sp cells resumed their normal doubling time upon removal of dbcAMP. The observation that the lactogen-independent Nb2-Sp cell line contained 20-fold less cholera toxin-sensitive Gs protein provides circumstantial evidence that dysfunction of the adenylate cyclase system may be implicated in the autonomous growth of these cells. This possibility is strengthened by the observation that Nb2-Sp cells are markedly less sensitive than the Nb2-11C clone to the growth inhibitory effects of an exogenous cAMP analog.

Indication of different lactogen and somatogen binding sites in the human growth hormone molecule as probed with monoclonal antibodies

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction has been investigated using as probes monoclonal antibodies (Mabs) to hGH of defined epitope specificity profile. Seven high affinity Mabs were studied for their ability (i) to inhibit the binding of 125I-hGH to Nb2-SP rat lymphoma cells and to IM-9 human lymphocytes possessing lactogen and somatogen type receptors, respectively; and (ii) to interfere with the hormone (hGH or Met 8Leu hGH)-induced hGH)-induced proliferation in Nb2-11C lymphoma cells. The ability of these Mabs to inhibit the 125I-hGH binding and the hormone-induced proliferation in Nb2-11C cells was negatively correlated with the ability of these Mabs to cross-react with Met 14 hGH. Furthermore, Mabs Nos. 3 and 7, which cross-reacted minimally (0.2–0.4%) with Met 8Leu hGH, were unable to interfere with the mitogenic activity of Met 8Leu hGH in Nb2-11C cells. These results indicate that the first 13 amino acids of the N- terminal region of hGH are necessary for its lactogen activity. The inhibition of 125I-hGH binding to IM-9 cells by these Mabs was similar to those observed in Nb2-SP cells, except for Mabs Nos. 19 and 1. These Mabs inhibited more strongly the binding of 125I-hGH to IM-9 than to NB2 cells and recognized antigenic epitopes close to the C- terminal part of the molecule. These results suggest that the somatogen receptor binding site of hGH may be located on two sites, one at the N- terminal and the other one close to the C- terminal , while the lactogen receptor is mainly confined to the N- terminal part.

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 Met 14 hGH, 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.

A serum-free medium for culturing lactogen dependent and autonomous NB2 node lymphoma cells.

A serum-free, hormone-free medium (SF2) was designed for the Nb2 rat lymphoma bioassay for lactogens as batches of horse serum (HS), which were commonly used, were found to be inconsistent in their suitability and to contain factors modulating the PRL-induced growth response of clone Nb2-11C. In a 3-day incubation with less than 500 pg/ml human GH (hGH), SF2 was better than the traditional medium in supporting Nb2-11C growth, although the comparative efficiency of SF2 decreased at higher hGH levels. Known growth factors (epidermal growth factor, fibroblast growth factor, platelet derived-growth factor, recombinant somatomedin-C, multiplication-stimulating activity) and insulin had no consistent effect on the cell growth in SF2 either in the presence or absence of hGH. Corticosterone (12.4-150 nM) was toxic to the Nb2-11C cells. SF2 could support the growth of Nb2-11C cells for at least 30 passages in the presence of 5 ng/ml hGH, and that of 2 spontaneously proliferating cell lines (Nb2-SP and Nb2-HSP) for the same length of time in the absence of lactogen. However, in all cases the growth rate in SF2 was lower than that seen in the presence of 10% HS. Long-term culture of Nb2-SP and Nb2-HSP cells in SF2 led to an increase of the growth rate with time. There was a change in the responsiveness of Nb2-SP cells to lactogens after long-term culture in SF2 which was only apparent in the presence of HS. After 10 passages in SF2, Nb2-11C cells showed no apparent changes in lactogen-induced growth response, cell phenotype, cell size, or binding capacity for [125I]hGH.

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]wyo-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.