Catherine K.L. Too

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. J. Cell. Biochem. 77:624–634, 2000.

Role of O-linked β-N-acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells

The mTOR alpha4 phosphoprotein is a prolactin (PRL)‐downregulated gene product that is found in the nucleus of PRL‐dependent rat Nb2 lymphoma cells. Alpha4 lacks a nuclear localization signal (NLS) and the mechanism of its nuclear targeting is unknown. Post‐translational modification by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O‐β‐N‐acetylglucosaminyltransferase (OGT) and O‐β‐N‐acetylglucosaminidase (O‐GlcNAcase) adds or remove O‐GlcNAc moieties, respectively. If O‐GlcNac moieties contribute to the nuclear targeting of alpha4, a decrease in O‐GlcNAcylation (e.g., by inhibition of OGT) may redistribute alpha4 to the cytosol. The present study showed that alpha4 and Sp1 were both O‐GlcNAcylated in quiescent and PRL‐treated Nb2 cells. PRL alone or PRL + streptozotocin (STZ; an O‐GlcNAcase inhibitor) significantly (P ≤ 0.05) increased the O‐GlcNAc/alpha4 ratio above that in control quiescent cells. However, PRL + alloxan (ALX; an OGT inhibitor) or ALX alone did not decrease O‐GlcNAcylation of alpha4 below that of controls and alpha4 remained nuclear. In comparison, PRL (±ALX/STZ) greatly increased Sp1 protein levels, caused a significant decrease in the GlcNAc/Sp1 ratio (P ≤ 0.05, n = 3) as compared to controls and partially redistributed Sp1 to the cytosol. Finally, a 50% downregulation of OGT gene expression by small interfering RNA (i.e., siOGT) partially redistributed both alpha4 and Sp1 to the cytosol. The alpha4 protein partner PP2Ac had no detectable O‐GlcNAc moieties and its nuclear distribution was not affected by siOGT. In summary, alpha4 and Sp1 contained O‐GlcNAc moieties, which contributed to their nuclear targeting in Nb2 cells.

Prolactin and Estrogen Up-Regulate Carboxypeptidase-D to Promote Nitric Oxide Production and Survival of MCF-7 Breast Cancer Cells

Carboxypeptidase-D (CPD) and carboxypeptidase-M (CPM) release C-terminal arginine (Arg) from polypeptides, and both are present in the plasma membrane. Cell-surface CPD increases intracellular Arg, which is converted to nitric oxide (NO). We have reported that prolactin (PRL) regulated CPD mRNA levels in MCF-7 breast cancer cells. This study examined PRL/17beta-estradiol (E2) regulation of CPD/CPM expression, and the role of CPD in NO production for survival of MCF-7 cells. We showed that PRL or E2 up-regulated CPD mRNA and protein expression. PRL/E2 increased CPD mRNA levels by 3- to 5-fold but had no effect on CPM. In Arg-free DMEM, exogenous L-Arg or substrate furylacryloyl-Ala-Arg (Fa-Ala-Arg) increased NO levels and cell survival, measured using 4,5-diaminofluorescein diacetate and the MTS assay, respectively. In the presence of Fa-Ala-Arg, NO production was enhanced by PRL and/or E2 but inhibited by CPD/CPM-specific inhibitor, 2-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MGTA). MGTA also decreased MCF-7 cell survival. In Arg-free medium, annexin-V staining showed that apoptotic MCF-7 cells (approximately 60%) were rescued by Fa-Ala-Arg (25%) or diethylamine/NO (10%). Finally, CPD or CPM gene expression was knocked down with small interfering (si) CPD or siCPM, respectively, with nontargeting siNT as controls. In Arg-free DMEM, the stimulatory effect of Fa-Ala-Arg on NO production was inhibited by siCPD only, showing that CPD depletion inhibited Fa-Ala-Arg cleavage. Furthermore, more than 60% of siCPD-transfectants were apoptotic, and L-Arg, not Fa-Ala-Arg, significantly decreased apoptosis to 32% (P<or=0.05). Thus, CPD gene knockdown did not affect L-Arg uptake, which protected cells from apoptosis. In summary, PRL/E2-induced cell-surface CPD released Arg from extracellular substrates, increased intracellular NO, promoted survival and inhibited apoptosis of MCF-7 cells.

Testosterone and prolactin increase carboxypeptidase‐D and nitric oxide levels to promote survival of prostate cancer cells

Plasma‐membrane carboxypeptidase‐D (CPD) releases arginine from extracellular substrates. Arginine is converted intracellularly to nitric oxide (NO). This study determined the effects of testosterone (T) and prolactin (PRL) on CPD expression, and the role(s) of CPD in NO production and survival of prostate cancer (PCa) cells.

CAT-1-mediated arginine uptake and regulation of nitric oxide synthases for the survival of human breast cancer cell lines.

Growth of the human MCF‐7 breast cancer cell line is highly dependent on L‐arginine. We have reported that L‐arginine, released from extracellular substrates by prolactin (PRL)‐ and 17β‐estradiol (E2)‐induced carboxypeptidase‐D in the cell membrane, promotes nitric oxide (NO) production for MCF‐7 cell survival. Arginine uptake is mediated by members of the cationic amino acid transporter (CAT) family and may coincide with induction of nitric oxide synthase (NOS) for the production of NO. The present study investigated the CAT isoforms and PRL/E2 regulation of CAT and NOS in breast cancer cell lines. Using RT‐PCR analysis, CAT‐1, CAT‐2A, and CAT‐2B transcripts were detected in MCF‐7, T47D, and MDA‐MB‐231 cells. The CAT‐4 transcript was detected in MDA‐MB‐231 only. CAT‐3 was not detected in any of these cells. PRL and E2 did not significantly alter levels of CAT‐1 mRNA and protein, nor CAT‐2A and CAT‐2B mRNAs in MCF‐7 and T47D cells. PRL and E2 also had no effect on the overall uptake of L‐[2,3,4,5‐H3] arginine into these cells. However, confocal immunofluorescent microscopy showed that PRL and E2 upregulated eNOS and iNOS proteins, which distributed in the cytoplasm and/or nucleus of MCF‐7 cells. Knockdown of CAT‐1 gene expression using small interfering RNA significantly decreased L‐[2,3,4,5‐H3]‐arginine uptake, decreased viability and increased apoptosis of MCF‐7 and T47D cells. In summary, several CAT isoforms are expressed in breast cancer cells. The CAT‐1 isoform plays a role in arginine uptake and, together with PRL/E2‐induced NOS, contribute to NO production for the survival of MCF‐7 and T47D cells. J. Cell. Biochem. 112: 1084–1092, 2011.

Distribution of serotonin in the sea scallop Placopecten magellanicus

Summary Serotonin (5-HT) was detected in various body tissues of the scallop using both immunocy-tochemistry and high performance liquid chromatography with electrochemical detection (HPLC-ED). Much of the 5-HT in the body appears to originate from nerve cells in pedal and cerebral ganglia. Neuropilar regions of these ganglia are also abundant in 5-HT. Far fewer 5-HT containing cells reside within the parietovisceral ganglion, although the accessory lobe contains numerous such cells. The lateral lobes of the parietovisceral ganglion are richly innervated with 5-HT containing fibres. Immunoreactive fibres were also found in the heart, kidneys, labial palps, gills and various muscles. The digestive tract contained numerous immunoreactive cell bodies. Immunoreactive fibres were found principally around the collecting tubules of both the testes and the ovaries in the spring, but also around the germinal acini by autumn. HPLC-ED was used to quantify the amount of 5-HT present in the above-mentioned tissues in ...

Overexpression of the mTOR alpha4 phosphoprotein activates protein phosphatase 2A and increases Stat1α binding to PIAS1

Alpha4 phosphoprotein in the mTOR pathway is a prolactin (PRL)-downregulated gene product that interacts with the catalytic subunit of serine/threonine protein phosphatase 2A (PP2Ac) in rat Nb2 lymphoma cells. Transient overexpression of alpha4 in COS-1 cells inhibited PRL-inducible interferon-regulatory-1 (IRF-1) promoter activity, but the mechanism underlying this inhibition was not known. The present study showed a stable alpha4-PP2Ac complex that was not dissociated by rapamycin in COS-1 cells. Transient overexpression of alpha4 in COS-1 cells had no effect on endogenous PP2Ac protein levels but significantly increased PP2Ac carboxymethylation and PP2A activity as compared to controls. The increased PP2A activity was accompanied by decreased phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) but had no effect on Stat phosphorylation. However, overexpressed alpha4 decreased arginine methylation of Stat1alpha and increased Stat1alpha binding to the Stat1alpha-specific inhibitor, PIAS1. In summary, ectopic alpha4 increased PP2A activity in COS-1 cells and this was accompanied by Stat1alpha hypomethylation and increased Stat1alpha-PIAS1 association. These events would inhibit Stat action and ultimately inhibit PRL-inducible IRF-1 promoter activity.

Detection of FMRFamide-like immunoreactivities in the sea scallopPlacopecten magellanicus by immunohistochemistry and Western blot analysis

FMRFamide-like immunoreactivity was detected histochemically in the sea scallopPlacopecten magellanicus. Most immunoreactivity was concentrated in the cerebral, pedal, and parietovisceral ganglia, particularly in the cortical cell bodies and in their fibers which extend into the central neuropile. Whole-mount immunofluorescence studies were used to localize concentrations of immunoreactive cells on the dorsal and ventral surfaces of each ganglion. Immunoreactivity was also detected in nerves emanating from the ganglia. Strong immunoreactivity was localized in peripheral organs, including the gut and gills of juvenile and adult scallops. Weak immunoreactivity was detected in the gonads, heart, and adductor muscle of the adults. A broad FMRFamide-like immunoreactive band of 2.5–8.2 kDa was detected by Western blotting of acetone extracts of the parietovisceral ganglia. In the presence of protease inhibitors, two FMRFamide-like immunoreactive bands (7.2–8.2 kDa and >17 kDa) were obtained. Neither of these bands comigrated with the FMRFamide standard. It is concluded that peptides of the FMRFamide family are probably regulators of numerous central and peripheral functions inP. magellanicus.

Induction of Sp1 activity by prolactin and interleukin-2 in Nb2 T-cells: Differential association of Sp1-DNA complexes with Stats

The induction of the transcription factor Sp1 by prolactin (PRL) and interleukin-2 (IL-2) was investigated in the PRL- and IL-2 responsive rat Nb2 T-cell line. Western analysis showed a rapid increase in Sp1 synthesis in Nb2 cells in response to PRL or IL-2. Elevation of Sp1 protein levels occurred within 15 min following PRL or IL-2 stimulation, reached a maximum by 1 h and was inhibited by cycloheximide, indicating de novo protein synthesis. Interestingly, dilution of confluent, growth-arrested Nb2 cells to low density also caused a rapid elevation in Sp1 suggesting that growth arrest may down-regulate Sp1 synthesis. Electrophoretic mobility shift assays using an Sp1 consensus oligonucleotide as probe showed a rapid but transient formation of a single PRL-inducible complex at 30 min. In contrast, three IL-2-inducible complexes were formed at 30 min and persisted to at least 60 min. Mobility shift interference assays using specific Stat antibodies failed to detect Stat1alpha, Stat3 or Stat5 in the 30 min PRL-inducible complex. In contrast, the IL-2 induced complexes contained Stat3 alone at 30 min and both Stat3 and Stat5 at 60 min. The PRL- and IL-2-inducible complexes did not contain the tumor suppressor protein, p53. The time dependent association of the Stat proteins with the IL-2-inducible complexes, but not with the PRL-inducible complex, suggests that the two mitogens may selectively utilize specific promoter elements for transcriptional activation of PRL- and IL-2-responsive genes. Alternatively, the two mitogens may be activating different genes with Sp1-binding promoter elements for their mitogenic action in Nb2 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), alpha4 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 alpha4, but not the Cdc-like protein, was dependent on cell densities. EF-2 regulates protein synthesis while the alpha4 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.