hGH Expression Research Articles

Tumor Vascular Interaction in Melanomas and Neurogenesis: A Review

Background: Melanomas arise from multipotent neural crest derivative, the melanocytes. At the tumor-stroma interphase, melanoma cells show organized neural differentiation forming tumor-vascular complexes. The present study explores the sequence of neurogenesis from aurophilic radial glial like cells in relation to angiogenesis, utilizing the tumor-vascular-complex (TVC) as a 3D model. Methods: Serial frozen and paraffin sections stained with HE, reticulin-gold impregnation for aurophilia; dopa oxidase; immunopositivity for: neural differentiation (nestin, Glial Fibrillary Acidic Protein (GFAP), Neural Fibrillary Protein (NFP), synaptophysin); indoleamines: (serotonin and melatonin), catecholamines (dopamine (DA), Noradrenalin (NA), pigment; Dopa Oxidase (DO)); hormones: ((PRL), Prolactin; (HGH), Human Growth Hormone;) and mitosis. The pattern of neural differentiation in tumor-vascular-complexes (TVC) is assessed by positivity in layer1- layer 5 and cell counts in each layer of the perimantle zone (PMZ). Statistical Analysis: ANOVA: Kruskal-Wallis One Way Analysis of Variance; All Pairwise Multiple Comparison Procedures [Tukey Test]. [t-test or Mann-Whitney U- test]. Results: A TVC is formed during angiogenic tumor-vascular interaction. Nes +ve angiogenic tubes enter the tumor margins. Periluminar cells show aurophilia, and extend dendritic arbors into the outer layers of the mantle zone. Cells and dendritic processes in layer 1/ layer 2 show Nes, Auro and GFAP positivity. NFP and Syn positivity is seen in layer 4/ layer 5 with a transition zone between layer 2/ layer 3. As two layers accrue, a wave of mitotic activity is seen and cells acquire PRL and HGH and indoleamine positivity. Catecholamine positivity is in layer 4/ layer 5 thus establishing a polarity. Dopamine is positive in layer 3/ layer 4 coinciding with dopa-oxidase which peaks in layer 4 with NA, ACTH positivity in the outer layers is in association with pigmentation in layer 4/ layer 5. Discussion: Thus during tumor-vascular interaction, melanoma cells differentiate into aurophilic radial glial like cells, as during embryonic neurogenesis, Nes, a marker of multi-lineage progenitor cells, identifies them as MASC, which differentiate into neuronal cells. The angiogenic vessel confers polarity and an embryonal microenvironment in the perivascular mantle zone of the TVC, inducing aggressive melanoma cells to function as neuronal stem cells recapitulating neurogenesis of bio-aminergic cells. The cell cycle is orchestrated by the three pituitary hormones, PRL, HGH and ACTH. The expression of PRL and HGH is related to mitotic activity while ACTH and pigment indicate differentiated function.

The AlfpCre mouse revisited: Evidence for liver steatosis related to growth hormone deficiency

With great interest we read the article by Mueller et al. on the development of steatosis and hepatocellular carcinoma in mice by disrupting hepatic growth hormone (GH) and glucocorticoid receptor signaling.1 They demonstrate that abrogation of hepatic signal transducer and activator of transcription 5 (Stat5) signaling caused liver steatosis by up-regulation of genes involved in lipid uptake and synthesis. However, we have concerns regarding the use of AlfpCre+ transgenic mice to inactivate Stat5 in the liver. While generating the AlfpCre transgene, the entire human GH (hGH) gene was inserted for its introns and polyadenylation signal.2 As a consequence, a polycistronic messenger RNA (mRNA) transcript encoding Cre Recombinase and hGH was transcribed in the liver, hypothalamus, and pituitary (data not shown). However, hGH was only translated in the hypothalamus and pituitary (Fig. 1A). It has previously been described that targeted expression of hGH in the hypothalamus reduces expression of hypothalamic GH releasing hormone (GHRH), leading to GH deficiency (GHD).3 This is also the case in AlfpCre+ mice in a C57Bl6/J background, as shown by quantitative real-time polymerase chain reaction (qRT-PCR). In the hypothalamus, expression levels of GHRH were reduced by 70% (P < 0.01) and mouse GH messenger RNA (mRNA) in the adenopituitary by 61% (P < 0.05). AlfpCre+ mice also showed hypoplasia of the pituitary (Fig. 1B). Hepatic insulin-like growth factor 1 (Igf1) mRNA and Igf1 plasma protein levels were decreased by 71% (P < 0.001) and 60% (P < 0.001), respectively, and reduced phosphorylation of Stat5 in AlfpCre+ livers was shown by western blotting (Fig. 1C), confirming GHD in AlfpCre+ mice. Consequently, AlfpCre+ mice exhibit postnatal growth retardation (Fig. 1D), a finding that was also observed in AlfpCre+/−c-JunF/F mice.4 Downstream of the impaired GH signaling, AlfpCre+ livers showed increased levels of triglycerides (TGs), free fatty acids (FFAs), and cholesterol, indicating liver steatosis (Fig. 1E). Finally, genes involved in lipid uptake and synthesis, including CD36, very low-density lipoprotein receptor, and peroxisome proliferator-activated receptor gamma, were significantly up-regulated by 2.52- (P < 0.01), 5.44- (P < 0.01), and 2.09-fold (P < 0.001), respectively, an expression profile indicating liver steatosis (Fig. 1F). Altogether, AlfpCre+ mice show liver steatosis related to GHD, and therefore care should be taken to use the right controls (AlfpCre+ unfloxed mice instead of AlfpCre− floxed mice) while conditionally inactivating or overexpressing genes in livers of mice. Vincent P.E.G. Pruniau, M.Sc. Els Louagie, M.Sc., Ph.D. Bas Brouwers, M.Sc. Jeroen Declercq, M.Sc., Ph.D. John W.M. Creemers, Ph.D. Laboratory for Biochemical Neuroendocrinology Department for Human Genetics KU Leuven Leuven, Belgium Els Louagie is currently affiliated with the Laboratory for Molecular Immunology and Inflammation Department of Rheumatology Ghent University Hospital Ghent, Belgium

Autocrine human growth hormone increases sensitivity of mammary carcinoma cell to arsenic trioxide-induced apoptosis

Human growth hormone (hGH) has been increasingly implicated in a variety of cancers; its up-regulation is observed in breast cancer and correlates with a poor outcome. Autocrine hGH promotes mammary carcinoma cell survival, proliferation, immortalization; it confers an invasive phenotype as a result of an epithelial–mesenchymal transition and contributes to chemoresistance and radioresistance. Arsenic trioxide (ATO) is being successfully used as a first and second line therapy for the treatment of patients with acute promyelocytic leukemia. It also inhibits tumor cell growth and induces apoptosis in a broad range of solid tumors. In the present study, we investigated the effect of hGH on sensitivity of a mammary adenocarcinoma cell to ATO, using a stable hGH-transfectant MCF-7 cell line, MCF7-hGH. Our results demonstrated for the first time that the overexpression of hGH increased sensitivity of the breast cancer cell line MCF-7 to ATO through apoptotic and anti-proliferative mechanisms. The effect of ATO on the transcriptional level of genes involved in survival (Bcl-2, Bax and Survivin), self-sufficiency in growth signals (c-Myc, ARF, Cdc25A, p53 and Bax), immortalization (hTERT) and invasion and metastasis (MMP-2 and MMP-9, uPA and uPAR and E-cadherin) was more pronounced in MCF7-hGH compared with its parental MCF-7 line. Our study may highlight the potential application of ATO for the treatment of patients with breast cancer, especially in those who have metastatic and chemoresistant tumor phenotype possibly due to the over expression of hGH.

Hormone Expression in Melanomas Related to Tumor Angiogenesis

Background: Vicarious production of pituitary hormones by malignant tumors is a well known phenomenon. Prolactin [PRL], human growth hormone [HGH], adenocorticotropic hormone [ACTH] and alpha-melanocyte stimulating hormone [α-MSH] expression has been reported in malignant melanoma tumor cell lines. The present study examines the pattern of expression of the hormones ACTH, PRL and HGH in melanoma with regard to tumor growth and differentiation in relation to angiogenesis. Methods: During angiogenesis the tumor cells encircle a central vessel in layers to form a tumor-vascular-complex [TVC] with a central vessel and a mantle of 5 to 6 layers. Hormone expression is assessed by immunopositivity for PRL, HGH, & ACTH in the tumor-vascular complexes, on serial frozen and paraffin sections and subjected to statistical analysis [t-test or Mann-Whitney U- test]. Results: As two layers accrue the tumor cells acquire PRL & HGH positivity and are associated with a wave of mitotic activity in these layers. Further addition of layers shows ACTH positivity in the outer layers in association with pigmentation. Discussion: There is a coordinated growth of melanoma cells around neovascular channels, the cell cycle of malignant melanocytes being orchestrated by the three pituitary hormones, PRL, HGH & ACTH. The expression of PRL & HGH is related to mitotic activity while ACTH & pigment indicate differentiated function.

P4-01-11: Autocrine Human Growth Hormone (hGH) Reduces the Sensitivity of Breast Cancer Cells to Treatment with Tamoxifen and Fulvestrant.

Abstract Despite recent advances in breast cancer treatment regimes, intrinsic or development of resistance to conventional breast cancer therapies is still a significant clinical challenge. Expression of growth factors by tumour cells has been proposed as one mechanism for developing resistance to radiotherapy and chemotherapeutic agents. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development in human breast cancer. Recent studies have demonstrated that humans with growth hormone receptor deficiency are protected from developing cancer. Studies investigating human growth hormone (hGH) expression in human breast cancer have demonstrated that hGH expression is associated with specific histopathological features and survival outcomes for patients. Increased hGH expression is significantly associated with lymph node metastasis, tumour stage, proliferative index, worse relapse free survival and reduced overall survival in mammary carcinoma. In recent studies we have demonstrated that autocrine hGH promotes cell proliferation, cell survival and oncogenicity of human mammary carcinoma cells, enhancing anchorage independent cell growth, and supporting tumour formation in immunodeficient mice. Autocrine hGH also promotes mammary carcinoma cell migration/invasion and epithelial to mesenchymal transition and tumour vascularisation in xenograft studies. In this study we investigated whether autocrine hGH promotes resistance to anti-estrogen therapeutic drugs, sensitivity to tamoxifen and fulvestrent using the human breast cancer cell lines MCF-7, T47D and BT474. Stable transfection with an expression vector containing the hGH gene enhanced MCF-7, T47D and BT474 cell viability, total cell number and anchorage independent growth following treatment with tamoxifen or fulvestrant when compared to control transfected cells. Conversely, treatment of BT474 cells with an hGH receptor antagonist (B2036), increased BT474 cell sensitivity to treatment with tamoxifen or fulvestrant. Using an estrogen response element (ERE) luciferase assay, we observed that autocrine hGH enhanced basal estrogen receptor (ER)-mediated transcriptional activity. Our results demonstrate that forced expression of hGH in mammary carcinoma cells enhances ER-mediated signal tranduction and promotes resistance to tamoxifen and fulvestrant, while functional antagnonism of hGH increased cell sensitivity. Consequently, antagonism of the hGH receptor may help maintain sensitivity to anti-estrogen therapy and improve the prognosis of patients with hormone sensitive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-11.

P3-03-06: Regulation of microRNA Expression by Autocrine Human Growth Hormone in Breast Cancer Cells.

Abstract Introduction: MicroRNAs (miRNAs) are a class of small non-protein coding RNAs approximately 20–24 nucleotides in length which regulate the expression of genes by pairing with the 3’ untranslated regions of target mRNA molecules and inhibiting translation or promoting mRNA degradation. Mature miRNAs are processed from longer transcripts by the miRNA biogenesis machinery, which includes key enzymes Dicer, Drosha and AGO-2. Altered miRNA expression has been implicated in many pathologies, including breast cancer. In general it is the deregulated expression of individual miRNAs that has been implicated in breast cancer pathogenesis. However, global downregulation of all or the majority of miRNAs has also been observed and can lead to a more invasive phenotype and increased proliferation in various cancers. Global miRNA downregulation has been associated with changes in expression levels of proteins involved in miRNA biogenesis. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development. Studies investigating hGH expression in human breast cancer have demonstrated that hGH expression is positively correlated with lymph node metastasis, tumour stage, HER-2 status and proliferative index. In mammary carcinoma cells autocrine hGH promotes cell proliferation, survival, migration/invasion and epithelial-to-mesenchymal transition as well as tumour formation in a xenograft model. In the current study we demonstrate that autocrine hGH regulates miRNA biogenesis in breast cancer cells. Methods: Forced expression of hGH was established in the mammary carcinoma cell lines MCF-7 and T47D by stable transfection. miRNA expression was determined using miRNA microarray, quantitative real-time PCR (qPCR) arrays and qPCR assays. The expression levels of genes and proteins involved in miRNA biogenesis were assessed using real-time qPCR assays and Western blotting. Results: More than 90% of miRNAs assayed were downregulated in hGH-transfected MCF-7 and T47D cells, compared to control transfected cells, as demonstrated by miRNA microarray and qPCR array. Changes in miRNA expression determined by microarray and qPCR array were verified using miRNA-specific qPCR assays. In MCF-7 cells, autocrine hGH did not significantly affect the mRNA expression levels of miRNA machinery components Drosha, DGCR8, PACT, TARBP, EXP-5, Dicer and AGO2 when compared to control transfected cells. Whereas in T47D cells autocrine hGH increased mRNA expression of TARBP, PACT and EXP-5 by 2.0, 2.3 and 1.8 fold respectively, compared to control transfected cells. Western blot analysis demonstrated that, autocrine hGH decreased protein levels of Dicer in MCF-7 cells, whereas Drosha and AGO2 were unchanged compared to control transfected cells. In T47D cells autocrine hGH decreased protein levels of Dicer, Drosha and AGO2 when compared to control transfected cells. Conclusion: Our findings demonstrate that autocrine hGH stimulates global downregulation of miRNA expression in breast cancer cells. This may be one mechanism whereby autocrine hGH promotes tumour progression. Our results also indicate that autocrine hGH-mediated global downregulation of miRNA expression may occur through regulation of proteins involved in the miRNA biogenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-06.

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.

Tumor Expression of Human Growth Hormone and Human Prolactin Predict a Worse Survival Outcome in Patients with Mammary or Endometrial Carcinoma

Evidence suggests that human GH (hGH) and human prolactin (hPRL) possess an autocrine or paracrine oncogenic role in mammary and endometrial carcinoma. However, especially for hGH, the prognostic relevance of tumor expression of these hormones is not well defined. We investigated the potential association of tumor mRNA and protein expression of hGH and hPRL with the clinicopathological features of mammary and endometrial carcinoma. The prognostic relevance of the individual or combined expression of hGH and hPRL in mammary and endometrial carcinoma was also determined. The expression of hGH and hPRL was analyzed in histopathological samples of mammary and endometrial carcinoma, and the respective normal tissues, by in situ hybridization and immunohistochemistry. Kaplan-Meier and Cox regression analysis was performed to examine the association of tumor hGH and hPRL expression with relapse-free survival and overall survival of patients. hGH expression was significantly associated with lymph node metastasis, tumor stage, human epidermal growth factor receptor-2 status, and proliferative index in mammary carcinoma and with International Federation of Gynecology and Obstetrics grade, myometrial invasion, and ovarian metastases in endometrial carcinoma. hPRL expression was associated with lymph node metastasis, tumor grade, and tumor stage in mammary carcinoma and with International Federation of Gynecology and Obstetrics stage and myometrial invasion in endometrial carcinoma. Both hGH and hPRL expression, individually and combined, are associated with worse relapse-free survival and overall survival in patients with mammary or endometrial carcinoma. Tumor expression of both hGH or hPRL in mammary or endometrial carcinoma is associated with a large and significant difference in survival outcome for patients with these tumors.

Autocrine Proliferative Effects of hGH Are Maintained in Primary Cultures of Human Mammary Carcinoma Cells

Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines. We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC. Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined. One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not. Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.

Stem cell antigen‐1 positive cell‐based systemic human growth hormone gene transfer strategy increases endosteal bone resorption and bone loss in mice

The present study assesses the effect of the stem cell antigen-1 positive (Sca-1(+) ) cell-based human growth hormone (hGH) ex vivo gene transfer strategy on endosteal bone mass in the mouse. Sublethally irradiated recipient mice were transplanted with Sca-1(+) cells transduced with lentiviral vectors expressing hGH or β-galactosidase control genes. Bone parameters were assessed by micro-computed tomography and histomorphometry. This hGH strategy drastically increased hGH mRNA levels in bone marrow cells and serum insulin-like growth factor-I (IGF-I) (by nearly 50%, p < 0.002) in hGH recipient mice. Femoral trabecular bone volume of the hGH mice was significantly reduced by 35% (p < 0.002). The hGH mice also had decreased trabecular number (by 26%; p < 0.0001), increased trabecular separation (by 38%; p < 0.0002) and reduced trabecular connectivity density (by 64%; p < 0.001), as well as significantly more osteoclasts (2.5-fold; p < 0.05) and greater osteoclastic surface per bone surface (2.6-fold; p < 0.01). Targeted expression of hGH in cells of marrow cavity through the Sca-1(+) cell-based gene transfer strategy increased circulating IGF-I and decreased endosteal bone mass through an increase in resorption in recipient mice. These results indicate that high local levels of hGH or IGF-I in the bone marrow microenvironment enhanced resorption, which is consistent with previous findings in transgenic mice with targeted bone IGF-I expression showing that high local IGF-I expression increased bone remodeling, favoring a net bone loss. Thus, GH and/or IGF-I would not be an appropriate transgene for use in this Sca-1(+) cell-based gene transfer strategy to promote endosteal bone formation. Published 2011 John Wiley & Sons, Ltd.

Characterization of Expression in Mice of a Transgene Containing 3.3 kb of the Human Lactase-Phlorizin Hydrolase (LPH) 5′ Flanking Sequence

The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7days and declines significantly by 28days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

Influence of pH on recombinant human growth hormone production by Pichia pastoris

AbstractBACKGROUND: Effect of pH on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut+ was investigated at pH = 4.2, 5.0, 5.5, and 6.0.RESULTS: The highest cell concentration was obtained at pH = 6.0 with 53 g L−1, while the highest rhGH concentration was attained at pH = 5.0 as 0.27 g L−1. Total protease secretion increased with increase in pH and with the cultivation time. Oxygen uptake rate increased with increasing pH up to pH = 6.0, having the maximum value, 37 mmol m−3 s−1, at pH = 5.5. KLa values were similar at all the conditions, having a maximum value of 0.14 s−1 at pH = 5.0. Taking the final rhGH concentration into account, the most favourable pH was 5.0; where AOX1 expression level showed a similar trend to AOX activity profiles, having the highest value of 9.4 × 1010 copy mg−1 CDW at t = 15 h; in parallel to AOX1 expression profile, hGH expression level increased until t = 15 h, with the highest value of 4.0 × 1010 copy mg−1 CDW, where a sharp increase in rhGH concentration was obtained. The expression levels of pep4, prb1 and prc1 genes, responsible from the production of proteinase A, proteinase B and, carboxypeptidase Y, were parallel to each other.CONCLUSION: Since it was shown that pH is a crucial operating parameter in fermentation processes using P. pastoris, keeping pH constant at its determined optimum value, pH = 5.0, during the bioprocess is vital in terms of recombinant protein production. Copyright © 2010 Society of Chemical Industry

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Delta, vps22Delta, and vps34Delta, were determined on account of their hGH secretion efficiency. The mutant vps10Delta was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

144. Sustained Hgh Expression after Electrotransfer of Naked DNA into Dwarf “Little” Mouse Skeletal Muscle

144. Sustained Hgh Expression after Electrotransfer of Naked DNA into Dwarf “Little” Mouse Skeletal Muscle

Autocrine Human Growth Hormone Promotes Tumor Angiogenesis in Mammary Carcinoma

Accumulating literature implicates pathological angiogenesis and lymphangiogenesis as playing key roles in tumor progression. Autocrine human growth hormone (hGH) is a wild-type orthotopically expressed oncogene for the human mammary epithelial cell. Herein we demonstrate that autocrine hGH expression in the human mammary carcinoma cell line MCF-7 stimulated the survival, proliferation, migration, and invasion of a human microvascular endothelial cell line (HMEC-1). Autocrine/paracrine hGH secreted from mammary carcinoma cells also promoted HMEC-1 in vitro tube formation as a consequence of increased vascular endothelial growth factor-A (VEGF-A) expression. Semiquantitative RT-PCR analysis demonstrated that HMEC-1 cells express both hGH and the hGH receptor (hGHR). Functional antagonism of HMEC-1-derived hGH reduced HMEC-1 survival, proliferation, migration/invasion, and tube formation in vitro. Autocrine/paracrine hGH secreted by mammary carcinoma cells increased tumor blood and lymphatic microvessel density in a xenograft model of human mammary carcinoma. Autocrine hGH is therefore a potential master regulator of tumor neovascularization, coordinating two critical processes in mammary neoplastic progression, angiogenesis and lymphangiogenesis. Consideration of hGH antagonism to inhibit angiogenic processes in mammary carcinoma is therefore warranted.

Distinct Functions of CCAAT Enhancer-binding Protein Isoforms in the Regulation of Manganese Superoxide Dismutase during Interleukin-1β Stimulation

The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1beta is mediated through a complex intronic enhancer element. To identify and characterize the transcription factors required for Mn-SOD enhancer function, a yeast one-hybrid assay was utilized, and two CCAAT enhancer-binding protein (C/EBP) members, C/EBP beta and C/EBP delta, were identified. These two transcription factors responded to IL-1beta treatment with distinct expression profiles, different temporal yet inducible interactions with the endogenous Mn-SOD enhancer, and also opposite effects on Mn-SOD transcription. C/EBP beta is expressed as three isoforms, LAP* (liver-activating protein), LAP, and LIP (liver-inhibitory protein). Our functional analysis demonstrated that only the full-length C/EBP beta/LAP* served as a true activator for Mn-SOD, whereas LAP, LIP, and C/EBP delta functioned as potential repressors. Finally, our systematic mutagenesis of the unique N-terminal 21 amino acids further solidified the importance of LAP* in the induction of Mn-SOD and emphasized the crucial role of this isoform. Our data demonstrating the physiological relevance of the N-terminal peptide also provide a rationale for revisiting the role of LAP* in the regulation of other genes and in pathways such as lipogenesis and development.

Autocrine Human Growth Hormone Stimulates Oncogenicity of Endometrial Carcinoma Cells

Recent published data have demonstrated elevated levels of human GH (hGH) in endometriosis and endometrial adenocarcinoma. Herein, we demonstrate that autocrine production of hGH can enhance the in vitro and in vivo oncogenic potential of endometrial carcinoma cells. Forced expression of hGH in endometrial carcinoma cell lines RL95-2 and AN3 resulted in an increased total cell number through enhanced cell cycle progression and decreased apoptotic cell death. In addition, autocrine hGH expression in endometrial carcinoma cells promoted anchorage-independent growth and increased cell migration/invasion in vitro. In a xenograft model of human endometrial carcinoma, autocrine hGH enhanced tumor size and progression. Changes in endometrial carcinoma cell gene expression stimulated by autocrine hGH was consistent with the altered in vitro and in vivo behavior. Functional antagonism of hGH in wild-type RL95-2 cells significantly reduced cell proliferation, cell survival, and anchorage-independent cell growth. These studies demonstrate a functional role for autocrine hGH in the development and progression of endometrial carcinoma and indicate potential therapeutic relevance of hGH antagonism in the treatment of endometrial carcinoma.

Effects of transplanted myoblasts transfected with human growth hormone gene on improvement of ventricular function of rats

Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone (hGH) has been shown to promote angiogenesis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy. Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay (RIA) was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion: pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group: received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor VIII. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of vascular endothelial growth factor (VEGF), bax and Bcl-2. hGH expression in myocardium was examined by Western blot. Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening (FS) and ejection fraction (EF) in pLghGHSN group were elevated significantly compared with pLgGFPSN group and control group after therapy (FS: 36.9+/-5.3 vs 29.5+/-3.5, 21.8+/-2.9; EF: 56.9+/-4.3 vs 47.1+/-3.6, 38.4+/-4.8, P<0.05). Left ventricular end-diastolic dimension (LVEDD) and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy (LVEDD: 5.9+/-0.3 vs 6.8+/-0.2, 8.6+/-0.3; heart infracted size: (34.5+/-4.2)% vs (40.0+/-3.9)%, (46.1+/-3.8)%, P<0.05); Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group (P<0.05). The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group (P<0.05). The expression of hGH gene in myocardium tissue can be detected by Western blot assay in pLghGHSN group. Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.

Stable expression of human growth hormone over 50 generations in transgenic insect larvae

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.

Construction of a protease‐deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease‐sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.