Prolactin/androgen-inducible carboxypeptidase-D increases with nitrotyrosine and Ki67 for breast cancer progression in vivo, and upregulates progression markers VEGF-C and Runx2 in vitro.

PURPOSE: Carboxypeptidase-D (CPD) cleaves C-terminal arginine (Arg) to produce nitric oxide (NO). Upregulation of CPD and NO by 17ß-estradiol, prolactin (PRL), and androgen increases survival of human breast cancer (BCa) cells in vitro. To demonstrate similar events in vivo, CPD, nitrotyrosine (NT, hallmark of NO action), androgen receptor (AR), prolactin receptor (PRLR), and phospho-Stat5a (for activated PRLR) levels were evaluated in benign and malignant human breast tissues, and correlated with cell proliferation (Ki67) and BCa progression (Cullin-3) biomarkers. METHODS: Paraffin-embedded breast tissues were analyzed by immunohistochemistry (IHC). BCa progression markers in human MCF-7 and T47D BCa cell lines treated with NO donor SIN-1 or PRL, ±CPD inhibitors were analyzed by RT-qPCR and immunoblotting. RESULTS: IHC showed progressive increases in CPD, NT, Ki67, and Cullin-3 from low levels in benign tissues to high levels in ductal carcinoma in situ, low-grade, high-grade, and triple-negative BCa. CPD and NT staining were closely associated, implicating CPD in NO production. Phospho-Stat5a increased significantly from benign to high-grade BCa and was mostly nuclear. AR and PRLR were abundant in benign breast and BCa, including triple-negative tumors. SIN-1 and PRL increased VEGF-C and Runx2, but not Cullin-3, in BCa cell lines. PRL induction of VEGF-C and Runx2 was inhibited partly by CPD inhibitors, implicating NO, produced by PRL-regulated CPD, in BCa progression. CONCLUSIONS: The CPD-Arg-NO pathway contributes to BCa progression in vitro and in vivo. PRL/androgen activation of the pathway support combined AR and PRLR blockade as an additional therapy for BCa.

Prolactin- and testosterone-induced carboxypeptidase-D correlates with increased nitrotyrosines and Ki67 in prostate cancer.

BACKGROUND: Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression. METHODS: Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67. RESULTS: RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased ∼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5. CONCLUSIONS: CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa.

Design, synthesis and bioevaluation of novel 6-(4-Hydroxypiperidino)naphthalen-2-ol-based potential Selective Estrogen Receptor Modulators for breast cancer.

In a study directed towards development of novel Selective Estrogen Receptor Modulators (SERMs), 1-(4-(2-(dialkylamino)ethoxy)benzyl)-6-(4-hydroxypiperidin-1-yl)-2-naphthol and corresponding aryl methyl ethers were synthesized and bioevaluated against the estrogen-responsive human MCF-7 breast cancer cell line. The phenolic analogs displayed little or no activity, but aryl methyl ether analogs showed significant cytotoxic potency. Also, representative compounds from the aryl methyl ether series showed significant binding and antagonistic activity against ERα. Two representative compounds were also evaluated for in vitro membrane permeability, plasma stability as well as in-vivo toxicity in mice. The compounds displayed well-acceptable drug-like in vitro membrane permeability as well as plasma stability and were well-tolerated in experimental mice at 300 mg/kg dose.

Carboxypeptidase-D is elevated in prostate cancer and its anti-apoptotic activity is abolished by combined androgen and prolactin receptor targeting.

BACKGROUND: Carboxypeptidase-D (CPD) cleaves C-terminal arginine for nitric oxide (NO) production. CPD and NO levels are upregulated by testosterone (T) and prolactin (PRL) to promote survival of prostate cancer (pCa) cells. This study evaluated CPD immunostaining and T/PRL regulation of CPD and NO levels in benign and malignant prostate tissues/cells to determine the role of CPD in pCa. METHODS: Immunohistochemistry (IHC) and tissue microarrays (TMA) were used to determine CPD immunostaining in prostate specimens. QPCR and immunoblotting were used to quantify CPD mRNA/protein expression in prostate cells. NO production was measured using 4,5-diaminofluorescein diacetate assay. RESULTS: CPD staining increased from 8.9 ± 3.8% (Mean ± SEM, n = 15) of benign epithelial cell area to 30.9 ± 2.9% (n = 30) of tumor cell area in one set of TMAs (P = 0.0008) and from 5.9 ± 0.9% (n = 45) of benign epithelial cell area to 18.8 ± 1.9% (n = 55) of tumor area in another (P < 0.0001). IHC of prostate tissues (≥50 mm(2)) confirmed increased CPD staining, from 13.1 ± 2.9% in benign (n = 16) to 29.5 ± 4.4% in pCa (n = 31, P = 0.0095). T and/or PRL increased CPD expression in several pCa but not benign cell lines. T and PRL acted synergistically to increase NO production, which was abolished only when receptor antagonists flutamide and Δ1-9-G129R-hPRL were used together. CONCLUSIONS: CPD immunostaining and T/PRL-stimulated CPD expression were higher in pCa than benign tissues/cells. Elevated CPD increased NO production, which was abolished when both AR and PRLR were inhibited. Our study implicates a critical role for the T/PRL-stimulated CPD-Arg-NO pathway in pCa progression, and suggests that AR+PRLR inhibition is a more effective treatment for pCa.

Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17ß-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.

Progestin-inducible EDD E3 ubiquitin ligase binds to α4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac.

Mammalian α4 phosphoprotein binds to the protein phosphatase 2A catalytic subunit (PP2Ac) to regulate PP2A activity, and to poly(A)-binding protein (PABP) and progestin-inducible EDD E3 ubiquitin ligase. This study showed induction of the EDD protein by progesterone, 17ß-estradiol and prolactin in breast cancer cells. Co-immunoprecipitation analyses, using lysates of COS-1 cells transfected with α4-deletion constructs, showed the α4 N-terminus binding to endogenous PP2Ac and PABP, and the C-terminus to EDD. Monoubiquitinated α4 in MCF-7 cells was unaffected by EDD-targeting siRNA (siEDD) nor by non-targetting siNT, thus, EDD does not ubiquitinate α4. PP2Ac is polyubiquitinated, and 36-kDa PP2Ac only was detected in siEDD- or siNT-transfected cells. However, treatment with proteasomal inhibitor MG132 showed polyubiquitinated-PP2Ac molecules (∼65-250kDa) abundantly in siNT controls but low in siEDD-transfectants, implicating PP2Ac as an EDD substrate. Finally, progesterone induction of EDD in MCF-7 cells correlated with decreased PP2Ac levels, further implicating hormone-inducible EDD in PP2Ac turnover.

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

BACKGROUND: 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. METHODS: LNCaP cells were treated with T and/or PRL. CPD expression was measured. Regulation by T (low doses) was determined using transfected cells overexpressing 5α-reductase type-1 (5αR1), which converts T to the more potent dihydrotestosterone. The effects of siRNAs targeting CPD (siCPDs) on NO production, cell viability, and apoptosis were determined using DAF2-DA, MTS, and Annexin-V assays. The effects of PRL/T on CPD/NO levels in PC-3, MDA-PCa-2b, and 22Rv1 cells were also evaluated. RESULTS: In LNCaP cells, 10 nM T and 10 ng/ml PRL-upregulated CPD mRNA/protein levels. In pTRE-transfectants, 1 nM T-upregulated CPD mRNA levels by ∼2-fold over controls, whereas 0.1 nM T caused similar upregulation in pTRE-5αR1-transfectants. In LNCaP cells cultured in arginine-free medium, addition of furylacryloyl-Ala-Arg (FAR; CPD substrate) increased NO levels. NO production, with FAR, was enhanced by PRL and/or T. siCPDs decreased NO production and cell viability, but increased apoptosis. QPCR analysis showed T/PRL-upregulation of CPD in 22Rv1, MDA-PCa-2b, and PC-3 cells. NO production was doubled by T/PRL in 22Rv1 cells, tripled by T in MDA-PCa-2b cells, and marginally increased by PRL in MDA-PCa-2b and PC-3 cells. CONCLUSIONS: T and PRL upregulate CPD and NO levels in PCa cells. CPD increases NO production to promote PCa cell survival.

Design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators.

In an systematic attempt to develop novel Selective Estrogen Receptor Modulators (SERMs), chiral 1-((4-(2-(dialkylamino)ethoxy)phenyl)(2-hydroxynaphthalen-1-yl)methyl)piperidin-4-ols were designed based on an accepted pharmacophore model. Simpler prototypes, viz. racemic 1-((2-hydroxynaphthalen-1-yl)arylmethyl)piperidin-4-ols, were first synthesized to develop kinetic resolution to pure enantiomers. Simultaneously, a series of racemic 1-((4-(2-(dialkylamino)ethoxy)phenyl)(2-hydroxynaphthalen-1-yl)methyl)piperidin-4-ols were evaluated against estrogen-responsive human MCF-7 breast cancer cells, but the compounds were found to be moderately active. The lack of potency could be due to the molecular bulk resulting in inadequate fit at the receptor. Subsequently, the molecular motif was modified to achiral 1-(4-(2-(dialkylamino)ethoxy)benzyl)naphthalen-2-ols by removing the piperidinol moiety. Bioevaluation of this new series of compounds displayed significantly enhanced cytotoxicity against MCF-7 cells. A representative compound for this series showed estrogen receptor alpha binding activity and the action is that of an antagonist.

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 17ß-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-H(3)] 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-H(3)]-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.

alpha4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding protein.

Mammalian alpha4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. alpha4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using alpha4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of alpha4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with alpha4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between alpha4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. alpha4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD-alpha4 immunocomplex. Furthermore, deletion mutants of alpha4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of alpha4, independent of the alpha4-PP2Ac binding site. Therefore, in addition to PP2Ac, alpha4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression.

Overexpression of 5 alpha-reductase type 1 increases sensitivity of prostate cancer cells to low concentrations of testosterone.

INTRODUCTION: Conversion of testosterone to dihydrotestosterone (DHT) by the enzymes 5 alpha-reductase types 1 (5 alpha R1) and 2 (5 alpha R2) is important for normal and pathological growth of the prostate. The predominant isoenzyme in normal prostate is 5 alpha R2. However, prostate cancer (PCa) development is accompanied by a decrease in 5 alpha R2 and an increase in 5 alpha R1. The biological significance of increased 5 alpha R1 expression is not fully understood. Therefore, the aim of this study was to determine the effect of overexpression of 5 alpha R1 on growth and prostate-specific antigen (PSA) production in PCa cells. MATERIALS AND METHODS: LNGK-9 PCa cells, transiently transfected with pTRE-5 alpha R1 or pTRE alone, were cultured in the presence or absence of testosterone at varying concentrations. Cell growth and PSA secretion were measured after 4-6 days. Cyclin E1, Ki67, and PSA mRNA levels were evaluated using RT-PCR after 24 hr of treatment. RESULTS: 10 pM testosterone increased growth of pTRE-5 alpha R1 transfectants by 54.1% over cells grown in the absence of testosterone, compared to 25.0% in pTRE transfectants (P < 0.01). Likewise, PSA secretion was increased by 56-fold in pTRE-5 alpha R1 transfectants treated with 10 pM testosterone, compared to 26-fold in pTRE transfectants (P < 0.01). At concentrations of testosterone above 10 pM, the stimulatory effect on growth and PSA secretion was not distinguishable between pTRE-5 alpha R1 and pTRE transfectants. CONCLUSIONS: These results demonstrate that upregulation of 5 alpha R1 enhances the cellular response to low, but not high, concentrations of testosterone. This explains one mechanism by which castration-recurrent PCa can proliferate in the presence of castrate levels of circulating testosterone.

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

Levels of 5alpha-reductase type 1 and type 2 are increased in localized high grade compared to low grade prostate cancer.

PURPOSE: In the prostate testosterone is converted to dihydrotestosterone by 5alpha-reductase type 1 and/or 2. Although 5alpha-reductase type 2 is predominant in normal prostates, type 1 is increased in cancer vs benign tissue. It is unclear whether 5alpha-reductase type 1/2 levels correlate with cancer grade. We compared the relative expression of 5alpha-reductase type 1 and 2 in localized high and low grade prostate cancer. MATERIALS AND METHODS: Immunostaining for 5alpha-reductase type 1/2 was evaluated in 64 prostate tissues from untreated men with localized prostate cancer. The percent of tumor area with moderate-high intensity staining was estimated for each Gleason pattern in the tissues. Adjacent benign tissue was evaluated in 26 prostate cancer specimens. RESULTS: Moderate-high staining for 5alpha-reductase type 1 increased from 18.8% +/- 2.9% (mean +/- SEM) in 34 Gleason pattern 3 cancers to 31.0% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.016). Staining for 5alpha-reductase type 2 increased from 22.9% +/- 3.0% in 34 Gleason pattern 3 cancers to 39.2% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.002). Compared to benign prostatic hyperplasia tissues staining for 5alpha-reductase type 1 was greater than 3-fold higher and staining for 5alpha-reductase type 2 was significantly lower in benign tissue adjacent to cancer (p = 0.006 and 0.0236, respectively). CONCLUSIONS: Levels of 5alpha-reductase type 1 and 2 are increased in localized high vs low grade prostate cancer. Levels of 5alpha-reductase type 1 are higher in benign tissue adjacent to cancer than in benign prostatic hyperplasia. These results raise the possibility that increased 5alpha-reductase type 1 in localized high grade cancers may contribute to the decreased effectiveness of the 5alpha-reductase type 2 selective inhibitor finasteride against high grade prostate cancer in the Prostate Cancer Prevention Trial.

Type 1 and type 2 5alpha-reductase expression in the development and progression of prostate cancer.

OBJECTIVES: Both normal and pathological growth of the prostate is dependent on dihydrotestosterone (DHT) synthesis, which is catalysed by two 5alpha-reductase (5alphaR) isoenzymes, 5alphaR1 and 5alphaR2, of which only 5alphaR2 has traditionally been viewed as important in the prostate. The objective of this study was to evaluate the role of both isoenzymes during development/progression of prostate cancer. METHODS: A thorough literature search was performed with the MEDLINE database to identify studies that have assessed expression of 5alphaR1/2 in prostate tissue. RESULTS: DHT suppression data for the 5alphaR2-specific inhibitor, finasteride, and the dual 5alphaR1/2 inhibitor, dutasteride, show that both isoenzymes are active in benign prostate. Furthermore, immunostaining studies have shown that 5alphaR1 expression increases and 5alphaR2 expression decreases in prostatic intraepithelial neoplasia (PIN) and prostate cancer, compared with nonmalignant prostate tissue. Both isoenzymes appear increased in high-grade compared with low-grade localised cancer. Dual inhibition of both isoenzymes with dutasteride may, therefore, be effective in preventing or delaying the growth of prostate cancer. The 4-yr REduction by DUtasteride of prostate Cancer Events (REDUCE) trial is underway to test this hypothesis. Androgen-withdrawal therapy can reverse prostate tumour growth by reducing circulating testosterone. However, 5alphaR-catalysed DHT synthesis within the prostate can continue and most tumours eventually develop resistance to androgen-deprivation therapy. Full assessment of the role of a 5alphaR inhibitor in this scenario is warranted. CONCLUSIONS: The consensus of evidence to date shows that 5alphaR1 is present in the prostate, and that levels are higher in malignant compared with benign prostate hyperplasia tissue.

Overexpression of the mTOR alpha4 phosphoprotein activates protein phosphatase 2A and increases Stat1alpha 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.

Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells.

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.

Role of O-linked beta-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 beta-N-acetylglucosamine (O-GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O-beta-N-acetylglucosaminyltransferase (OGT) and O-beta-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

Characterization of a novel, cytokine-inducible carboxypeptidase D isoform in haematopoietic tumour cells.

CPD-N is a cytokine-inducible CPD (carboxypeptidase-D) isoform identified in rat Nb2 T-lymphoma cells. The prototypic CPD (180 kDa) has three CP domains, whereas CPD-N (160 kDa) has an incomplete N-terminal domain I but intact domains II and III. CPD processes polypeptides in the TGN (trans-Golgi network) but the Nb2 CPD-N is nuclear. The present study identified a cryptic exon 1', downstream of exon 1 of the rat CPD gene, as an alternative transcription start site that encodes the N-terminus of CPD-N. Western-blot analysis showed exclusive synthesis of the 160 kDa CPD-N in rat Nb2 and Nb2-Sp lymphoma cells. Several haematopoietic cell lines including human K562 myeloma, Jurkat T-lymphoma and murine CTLL-2 cytotoxic T-cells express a 160 kDa CPD-immunoreactive protein, whereas mEL4 T-lymphoma cells express the 180 kDa CPD. The CPD-immunoreactive protein in hK562 cells is also nuclear and cytokine-inducible. In contrast, MCF-7 breast cancer cells express only the 180 kDa CPD, which is mainly in the TGN. CPD/CPD-N assays using substrate dansyl-L-alanyl-L-arginine show approx. 98% of CPD-N activity in the Nb2 nucleus, whereas MCF-7 CPD activity is enriched in the post-nuclear 10000 g pellet. The K(m) for CPD-N and CPD are 132+/-30 and 63+/-9 microM respectively. Specific activity/K(m) ratios show that dansyl-L-alanyl-L-arginine is a better substrate for CPD-N than for CPD. CPD-N has an optimal pH of 5.6 (due to domain II), whereas CPD has activity peaks at pH 5.6 (domain II) and pH 6.5-7.0 (domain I). CPD and CPD-N are inhibited non-competitively by zinc chelator 1,10-phenanthroline and competitively by peptidomimetic inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The Nb2 CPD-N co-immunoprecipitated with phosphatase PP2A (protein phosphatase 2A) and alpha4 phosphoprotein. In summary, a cytokine-inducible CPD-N is selectively expressed in several haematopoietic tumour cells. Nuclear CPD-N is enzymatically active and interacts with known partners of CPD.

Characterization of (123)I-iodo-alpha-methyltyrosine transport in rat lymphoma cells.

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

Coexpression and regulation of the FGF-2 and FGF antisense genes in leukemic cells.

Fibroblast growth factor-2 (FGF-2) is a growth and survival factor whose expression is elevated in many hematopoietic malignancies. A natural antisense RNA (FGF-AS) has been implicated in the posttranscriptional regulation of FGF-2 mRNA expression. We demonstrate for the first time that FGF sense and antisense RNAs are coordinately expressed and translated in hematopoietic cells and tissues. Cytokine stimulation of growth-arrested K562 cells elicited a rapid transient increase in FGF-AS mRNA expression followed by a slower but sustained increase in FGF-2 mRNA. This was accompanied by a marked increase in the expression and nuclear translocation of FGF-2 and the FGF-AS encoded protein, GFG/NUDT6. These findings suggest a role for both FGF-2 and GFG proteins in the cell survival and proliferation of lymphoid and myeloid tumor cells.