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Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes Fil: Menendez, Javier A.. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Peirce, Susan K.. Clemson University. Pearce Center Professional Communication; Estados Unidos Fil: Papadimitropoulou, Adriana. Biomedical Research Foundation Academy of Athens; Grecia Fil: Cuyàs, Elisabet. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Steen, Travis Vander. Mayo Foundation for Medical Education and Research. Mayo Clinic; Estados Unidos Fil: Verdura, Sara. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Vellón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Chen, Wen Y.. Clemson University; Estados Unidos Fil: Lupu, Ruth. Mayo Foundation for Medical Education and Research. Mayo Clinic; Estados Unidos. Clemson University; Estados Unidos. Mayo Clinic Cancer Center; Estados Unidos

Introduction: Sarcoidosis is a systemic granulomatous disease which may involve the liver in ∼11.5% of cases. Coronavirus disease 2019 (COVID-19) can induce a systemic, pro-inflammatory state but the development of systemic sarcoidosis after COVID-19 has not been described. We present the case of a newly diagnosed systemic sarcoidosis after COVID-19 infection. Case Description/Methods: A 50 year old male patient was evaluated for increased liver enzymes. He had a history of familial hypercholesterolemia, fatty liver disease, and diabetes mellitus. Seven months prior he was admitted to the hospital with shortness of breath, cough, lightheadedness, nausea and was diagnosed with COVID-19. After recovery, he developed pleurisy. CT scan showed mediastinal lymphadenopathy and scattered nodules. PET scan showed uptake concerning for reactive nodes, chronic granulomatous disease or lymphoproliferative disease. Subsequent lymph node aspirate found noncaseating granulomas consistent with sarcoidosis. He was referred to hepatology for abnormal liver enzymes (ALT 104, AST 81, Alkaline Phosphatase 216, Total Bilirubin 1.1). Tests for viral/autoimmune hepatitis, AMA, ANA, hemochromatosis, A1AT and ceruloplasmin were negative. Liver US showed steatosis and no evidence of obstruction. Transient elastography by FibroScan confirmed steatosis, CAP score of 347 and stiffness of 28kPa consistent with cirrhosis or infiltrating disease. Liver biopsy (Figure 1) revealed marked granulomatous inflammation with 10- 20% macro/micro-steatosis and stage 2-3 portal fibrosis. AFB stains were negative. Given the marked inflammation and fibrosis, the patient was started on prednisone 40mg daily with 4 week labs showing liver enzyme improvement (ALT 80, AST 31, Alk Phos 149, T Bili 0.6). Prednisone resulted in hyperglycemia requiring insulin, so azathioprine was started as steroid sparing treatment with budesonide added which he tolerated well. Discussion: Multisystem inflammatory syndrome in adults (MIS-A) is a rare complication of COVID-19. This case represents a unique type of MIS-A with hepatic sarcoidosis. The liver may be affected during COVID-19 with liver enzymes elevation reported in 20-30% of those with acute infection. After recovery, persistent elevation in hepatic transaminases and alkaline phosphatase should alert the clinicians to investigate for MIS-A triggered diseases such as sarcoidosis.

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-β-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan. Fil: De Lorenzo, Mariana S.. State University of New Jersey; Estados Unidos Fil: Chen, Wen. Clemson University; Estados Unidos Fil: Baljinnyam, Erdene. State University of New Jersey; Estados Unidos Fil: Carlini, María José. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: La Perle, Krista. Ohio State University; Estados Unidos Fil: Bishop, Sanford P.. State University of New Jersey; Estados Unidos Fil: Wagner, Thomas E.. Clemson University; Estados Unidos Fil: Rabson, Arnold B.. State University of New Jersey; Estados Unidos Fil: Vatner, Dorothy E.. State University of New Jersey; Estados Unidos Fil: Puricelli, Lydia Ines. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vatner, Stephen F.. State University of New Jersey; Estados Unidos

GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Furthermore, GHR and PRLR associate in a fashion augmented acutely by GH, even though GH primarily activates PRLR, rather than GHR, in these cells. To better understand PRLR's impact, we examined the effects of PRLR knockdown on GHR availability and GH sensitivity in T47D cells. T47D-ShPRLR cells, in which PRLR expression was reduced by stable short hairpin RNA (shRNA) expression, were compared with T47D-SCR control cells. PRLR knockdown decreased the rate of GHR proteolytic turnover, yielding GHR protein increase and ensuing sensitization of these cells to GHR signaling events including phosphorylation of GHR, Janus kinase 2, and signal transducer and activator of transcription 5 (STAT5). Unlike in T47D-SCR cells, acute GH signaling in T47D-ShPRLR cells was not blocked by the PRLR antagonist G129R but was inhibited by the GHR-specific antagonist, anti-GHR(ext-mAb). Thus, GH's use of GHR rather than PRLR was manifested when PRLR was reduced. In contrast to acute effects, GH incubation for 2 h or longer yielded diminished STAT5 phosphorylation in T47D-ShPRLR cells compared with T47D-SCR, a finding perhaps explained by markedly greater GH-induced GHR down-regulation in cells with diminished PRLR. However, when stimulated with repeated 1-h pulses of GH separated by 3-h washout periods to more faithfully mimic physiological GH pulsatility, T47D-ShPRLR cells exhibited greater transactivation of a STAT5-responsive luciferase reporter than did T47D-SCR cells. Our data suggest that PRLR's presence meaningfully affects GHR use in breast cancer cells.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are structurally similar cytokine receptor superfamily members that are highly conserved among species. GH has growth-promoting and metabolic effects in various tissues in vertebrates, including humans. PRL is essential for regulation of lactation in mammals. Recent studies indicate that breast tissue bears GHR and PRLR and that both GH and PRL may impact development or behavior of breast cancer cells. An important facet of human GH (hGH) and human PRL (hPRL) biology is that although hPRL interacts only with hPRLR, hGH binds well to both hGHR and hPRLR. Presently, we investigated potential signaling effects of both hormones in the estrogen receptor- and progesterone receptor-positive human T47D breast cancer cell line. We found that this cell type expresses ample GHR and PRLR and responds well to both hGH and hPRL, as evidenced by activation of the Janus kinase 2/signal transducer and activator of transcription 5 pathway. Immunoprecipitation studies revealed specific GHR-PRLR association in these cells that was acutely enhanced by GH treatment. Although GH caused formation of disulfide-linked and chemically cross-linked GHR dimers in T47D cells, GH preferentially induced tyrosine phosphorylation of PRLR rather than GHR. Notably, both a GHR-specific ligand antagonist (B2036) and a GHR-specific antagonist monoclonal antibody (anti-GHRext-mAb) failed to inhibit GH-induced signal transducer and activator of transcription 5 activation. In contrast, although the non-GHR-specific GH antagonist (G120R) and the PRL antagonist (G129R) individually only partially inhibited GH-induced activation, combined treatment with these two antagonists conferred greater inhibition than either alone. These data indicate that endogenous GHR and PRLR associate (possibly as a GHR-PRLR heterodimer) in human breast cancer cells and that GH signaling in these cells is largely mediated by the PRLR in the context of both PRLR-PRLR homodimers and GHR-PRLR heterodimers, broadening our understanding of how these related hormones and their related receptors may function in physiology and pathophysiology.

GH binds dimerized GH receptors (GHRs) to form a trimolecular complex and induces downstream signaling events. The mechanism by which GH binding converts the inactive predimerized GHR to its active signaling conformation is uncertain. GH has no axis of symmetry. Its interaction with GHR is mediated by two asymmetric binding sites on GH, each with distinct affinity. Site 1 is of high affinity and is thought to mediate the first binding step. Mutation of binding site 2 (as in the human GH mutant, G120R) disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist; it binds only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH. We previously demonstrated that prolactin receptor signaling was achieved by a dimeric version of a prolactin antagonist. We now employ assays of cellular signaling and receptor conformational changes to examine whether GH molecules harboring two site 1 regions can trigger GHR activation. We used recombinantly produced GH-GH and G120R-G120R dimers in which monomers in tandem are connected by a short linker peptide. Rabbit GHR-expressing human fibrosarcoma cells (C14) were treated with GH, G120R, GH-GH, or G120R-G120R. As expected, GH and GH-GH, but not G120R, induced GHR disulfide linkage, as assessed by anti-GHR blotting of cell extracts resolved by SDS-PAGE under nonreducing conditions. Disulfide linkage of GHRs reflects attainment of the active signaling conformation. Likewise, GH and GH-GH, but not G120R, caused Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) activation. Notably, G120R-G120R, despite its lack of an intact site 2 in either dimer partner, also promoted GHR disulfide linkage and JAK2 and STAT5 activation, albeit less potently than either GH or GH-GH. Time-course responses of the three agonists were similar in terms of JAK2 and STAT5 activation. Pretreatment of cells with our conformation-sensitive inhibitory monoclonal antibody, anti-GHR ext-mAb, prevented ligand-induced receptor activation for all three agonists. GHR was also rendered less immunoprecipitable by anti-GHR ext-mAb after treatment with these agonists. These results are important in that they indicate that a ligand with two intact binding sites 1 causes GHR to adopt similar conformational changes as does GH and thus triggers activation of JAK2 and downstream signaling. Furthermore, we infer that there is substantial flexibility in the GHR extracellular domain, such that it productively accommodates GH dimers that are much larger than GH.

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

Prolactin (PRL) and GH have two distinct binding sites (site 1 with high affinity; site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands, human (h) PRL and hGH, and their antagonists (hPRL-G129R and hGH-G120R) to 1) bind to PRLRs; 2) induce conformational changes in PRLRs; 3) activate signaling pathways associated with the PRLR; and 4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated Janus family of tyrosine kinases 2/signal transducer and activator of transcription 5, Ras/Raf/MAPK kinase/Erk, and phosphatidylinositol 3-kinase/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be site 1 plus site 2 or two site 1’s, to elicit receptor-mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GH receptor) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multifunctional therapeutics that target this class of cytokine receptors.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

To gain insight into the molecular mechanisms involved in human prolactin receptor antagonist (hPRL-G129R)-induced apoptosis, we used real-time reverse transcription-polymerase chain reaction to measure bax and bcl-2 gene expression in 11 human breast cancer cell lines following treatment with hPRL and hPRL-G129R. We also measured bax and bcl-2 gene expression in the mammary glands of transgenic mice expressing hPRL or hPRL-G129R. A time-course study of hPRL and antagonist treatment in T-47D cells indicated changing bax/bcl-2 mRNA ratios beginning at 24 h. We found that bax/bcl-2 mRNA ratios were significantly elevated in seven of the 11 hPRL-G129R-treated cell lines, as well as in the hPRL-G129R transgenic mice. To confirm these results, Bax and Bcl-2 proteins were analysed by Western blot methods in mammary gland tissue homogenates of transgenic mice. Bax/Bcl-2 ratios were highest in the 6-month group of hPRL-G129R transgenics, and lowest in the 6-month group of hPRL transgenics. We expanded our findings by examining the release of a downstream Bax-induced protein, cytochrome c, a hallmark protein of apoptosis, in transgenic mice. Again, cytochrome c levels were highest in the 6-month hPRL-G129R transgenic group. Thus, hPRL-G129R-induced breast cancer cell and/or mammary gland apoptosis is mediated, at least in part, through the regulation of Bax and Bcl-2 gene expression.

To gain insight into the molecular basis of human prolactin (hPRL) antagonist induced apoptosis, we compared the differential gene expression profile of four human breast cancer cell lines following treatment with hPRL and its antagonist (hPRL-G129R). Among the genes identified, the bcl-2 gene was of particular interest. We found that bcl-2 mRNA was up regulated in three of the four cell lines that were treated with hPRL. To further confirm these results, real time RT-PCR and ELISA analyses were used to detect bcl-2 mRNA and Bcl-2 protein, respectively, in 11 different breast cancer cell lines after hPRL or hPRL-G129R treatment. Our data suggests that Bcl-2 is up-regulated in response to hPRL stimulation and is competitively inhibited by hPRL-G129R in the majority of the cell lines tested. Thus, we propose that the anti-apoptotic role of hPRL in breast cancer is mediated, at least in part, through regulation of Bcl-2.

Genetic education of dendritic cells (DCs) with tumor-associated antigens is an encouraging development in DC-mediated tumor immunotherapy. In this study, to increase the transgene expression by DCs using nonviral vectors, a cytoplasmic T7 vector (T7T7/T7Luc) was used to transfect bone marrow-derived DCs with the firefly luciferase gene as a reporter and as a model tumor antigen. As a result, the luciferase activity of T7T7/T7Luc-transfected DCs was more than four times greater than that of DCs transfected with pCMVLuc, a commonly used nonviral vector. Furthermore, the luciferase activity was increased three times more when dendritic progenitor cells rather than mature DCs were transfected. In vivo tumor studies showed that T7T7/ T7Luc-transfected DCs, which express high levels of luciferase (model tumor antigen), stimulated a stronger immune response than did pCMVLuc-transfected DCs, which express relatively low levels of luciferase, as indicated by the cytotoxic T lymphocyte assay. T7T7/T7Luc transfected DCs, when injected into recipient mice, evoked an antigen-specific immune response that can effectively eradicate implanted metastasis and prevent new tumor development by murine melanoma cells genetically modified to express luciferase. Therefore, the T7 system is a powerful nonviral vector that can be used to genetically educate DCs with tumor-associated antigens for tumor immunotherapy.

The sections in this article are: 1 Studies Using Growth Hormone Fragments 2 Multiple Activities of Growth Hormone 3 Crystal Structure 4 Studies Regarding Disulfide Bonds 5 Homologue/Alanine Scanning as A Means of Defining Biologically Active Domains 6 The Third α-Helix 6.1 Mutagenesis of the Growth Hormone Gene Encoding α-Helix 3 and Transgenic Mouse Studies 6.2 Importance of the Amphiphilicity of the Third α-Helix 6.3 Designing a Growth Hormone Analogue with a Perfect Amphiphilic α-Helix 6.4 Systematic Substitution Mutations in the Hydrophilic Region of the Third α-Helix of Bovine Growth Hormone 7 Growth Hormone Antagonists 7.1 Pegylated Growth Hormone Antagonists as Therapeutic Agents 8 Finding of One Growth Hormone/Two Growth Hormone-Binding Proteins 9 Mutagenesis Studies on the Prolactin Gene 10 Concluding Remarks

Long-term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co-implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin-like growth factor-1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell-derived mini-organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.

Bovine growth hormone (bGH) transgenic (Tg) mice have been shown to possess enhanced growth phenotypes and exhibit severe glomerulosclerosis. One amino acid substitution in GH, i.e. G119R in bGH or G120R in human (h) GH, results in GH antagonists (GHAs). GHA-Tg mice exhibit dwarf phenotypes and normal kidneys. In order to investigate the possibility of GHAs as pharmaceutical agents for the treatment of human diseases with excessive GH levels, we cross bred mice that express bGH with those that express hGHA. Double positive Tg mice were identified that express both genes although at different levels. Kidney histological studies revealed that the double positive Tg mice with high GHA/GH expression ratios possessed normal or near normal kidneys, whereas those with low GHA/GH ratios exhibited glomerulosclerosis similar to GH-Tg mice. Thus, co-expression of GH and GHA genes in vivo results in animal phenotypes and kidney histopathologies which are a reflection of the relative expression levels of each gene.

Inhibition of diabetic nephropathy by a GH antagonist: A molecular analysis. Streptozotocin-treated C57B1/SJL mice developed glomerular hypertrophy and light microscopic lesions mimicking human diabetic glomerulosclerosis. In contrast, there were no glomerular hypertrophy and lesions in diabetic mice transgenic (TG) for a mutated growth hormone (bGH-G119K) that competes with native endogenous GH and results in dwarfism. We examined the molecular events underlying these findings. The non-transgenic (non-TG) diabetic mouse glomeruli had an increase in mRNA coding for α 1IV collagen, laminin B1, TGF- β 1, 72kDa collage-nase, and TIMP-3. In contrast, glomerular type IV collagen and laminin B1 mRNA levels were normal in diabetic TG dwarf mice. However, the 72kDa gelatinase, TIMP-3, and TGF- β 1 mRNAs were elevated in the diabetic dwarfs. Type IV collagen and laminin accumulated in the glomeruli of diabetic non-TG, but not of diabetic dwarf mice, by immunofluorescence microscopy, confirming the mRNA data. GH binding protein mRNA levels were comparable in glomeruli from dwarf and non-TG mice, both diabetic and non-diabetic. We did not detect GH receptor mRNA in glomeruli. These data suggest that diabetic glomerulosclerosis is associated with an increase in type IV collagen and laminin synthesis, and that these changes do not occur in mice transgenic for bGH119K, a functional antagonist of GH. The increase of 72kDa gelatinase, TIMP-3, and TGF- β 1 mRNAs, independent of GH, suggested that these changes induced by hyperglycemia were not sufficient for the induction of glomerulosclerosis.

It has previously been shown that a human growth hormone (hGH) analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C. , Wagner, T. E., and Kopchick, J. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T. E., and Kopchick, J. J. (1991) Endocrinology 129, 1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner, T. E., and Kopchick, J. J. (1994) J. Biol. Chem. 269, 15892-15897). In this study, we report the ability of hGH and hGH-G120R to be internalized by GH receptor expressing cells. Additionally, results of chemical cross-linking experiments revealed that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH-binding protein (bp). The molecular mass of the complex was determined to be approximately 280 kDa which is consistent with multiple receptors interacting with the ligand. The predominant radiolabeled band detected was a complex of approximately 140 kDa which probably represents one GH molecule bound to one GH receptor. The cross-linked complexes were not detected in the presence of excess unlabeled hGH or hGH-G120R and were not observed in cells which do not express detectable levels of GH receptors. Also, GH induced tyrosine phosphorylation of a complex of proteins of approximately 95 kDa in these cells whereas hGH-G120R did not. Thus, we have separated the hGH or hGH-G120R/GHR binding and internalization capabilities from the ability to stimulate tyrosine phosphorylation of intracellular proteins.

The objectives of this study were to determine growth rates, feed intakes, feed efficiencies, and chemical composition of mice from three transgenic lines in 10-d periods from weaning to near maturity. Lines M4, M11, and G119K express bovine somatotropin (bST) mutations E117L, L121P-E126G, and G119K and display phenotypes of large, near normal, and small body size, respectively. M4 mice were 28% larger at 28 d and 84% larger at 68 d than non-transgenic control (NTC) mice. M11 mice were the same size at 28 d as NTC but were 25% larger at 68 d. G119K mice were 34% and 25% smaller than NTC at 28 and 68 d, respectively. Growth rates of G119K mice and NTC were similar, whereas growth rates of M11 and M4 mice were increased (P .05). Chemical composition was also altered by expression of bST analogs in transgenic mice. G119K and M4 mice had increased body fat percentages and decreased body protein percentages in comparison to M11 and NTC mice (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.

An analog of bovine growth hormone (bGH-M8: [Leu117, Arg119, Asp122]-bGH) with an idealized amphiphilic third alpha-helix has been proposed to be a functional antagonist of GH. In accordance with this proposition, bGH-M8 profoundly inhibited bGH-stimulated lipolysis by chicken adipose tissue in vitro. bGH-M8 alone was a weak agonist in the lipolytic assay (1.9% the potency of bGH). The present evidence indicates that bGH-M8 is a competitive antagonist of the lipolytic action of GH based upon the following results: (i) increasing concentrations of bGH-M8 (antagonist) produce progressively greater inhibition of GH-stimulated lipolysis; (ii) increasing concentrations of bGH (agonist) are capable of overcoming this antagonism; and (iii) Schild plot analysis (slope = -0.94) suggests a receptor antagonist with an equilibrium dissociation constant (KB) of 4.54 nM. In contrast to the antagonistic effects of bGH-M8 on bGH-stimulated lipolysis, bGH-M8 retained full insulin-like ("antilipolytic") activity (i.e., inhibition of glucagon-induced lipolysis). bGH-M8 and bGH were similarly potent in eliciting antilipolytic effects in vitro. Moreover, the antilipolytic effects of bGH-M8 and bGH were additive. Therefore, the third alpha-helix (particularly residues 117, 119, and 122) of bGH contains major structural determinants for the lipolytic effects of GH. The ability of bGH-M8 to act as an antagonist for at least one action of GH (lipolysis) while being a full agonist for another (antilipolysis) suggests that different domains of GH are responsible for its various biologic activities, possibly involving different binding sites and/or signal transduction mechanisms.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

To prolong the circulation half-life of human prolactin (hPRL), human GH (hGH), and their competitive antagonists, hPRL-G129R and hGH-G120R, we examined the effects of fusing a serum albumin-binding peptide (SA20) to their amino- or carboxyl-terminus. Fusion of the SA20 peptide to the amino-terminus of the ligands was less detrimental upon their ability to induce or inhibit signal transduction and cell proliferation in vitro than fusion to the carboxyl-terminus. Pharmacokinetic (PK) studies in mice revealed that the half-life of SA20-hPRL and SA20-hGH was prolonged and their clearance was reduced in comparison with hPRL and hGH. Pharmacodynamic (PD) studies in 8-week-old female mice revealed that lobuloalveolar development in mammary glands was greater in all three groups (daily, every 2 days, or every third day over a 12-day period) of mice treated with SA20-hPRL (4 mg/kg) compared with hPRL (3.59 mg/kg). Similarly, daily administration (i.p.) of SA20-hGH (8 mg/kg) or hGH (7.15 mg/kg) to 23-day-old female mice over a 40-day period revealed the superiority of SA20-hGH over hGH as measured by weight gain, body length, and lobuloalveolar development in the mammary glands. These findings indicate that SA20 modification of hPRL, hGH, and their respective antagonists improves their PK/PD properties.

Previously, prolactin receptor antagonist (G129R)- based fusion proteins were developed including G129R fusions with an angiogenesis inhibitor (endostatin), an immune system modulator (interleukin 2), and a modified truncated cytotoxin (PE38KDEL). Each fusion protein was designed to target the PRLR-positive cells via the G129R moiety and at the same time attack a hallmark common to cancer cells via the second moiety. In this study, we tested the efficacy of the three fusion proteins as a combination therapy in an aggressive but clinically relevant mouse tumor model. To test the feasibility and to optimize a treatment regimen, allografts of a mammary carcinoma cell line (McNeuA) derived from an MMTV-neu transgenic mouse were first used. Growth of the allografts was significantly retarded by regimens which combined all three fusion proteins. In addition, a significant increase in cytotoxic CD8+ T cells was observed within the tumors of the combination treated groups. After establishing the dosing regimen, two doses of cocktail treatment (low and high doses administered twice weekly) along with individual component controls were administered to female MMTV-neu transgenic mice after surgical removal of a naturally occurring tumor. The average tumor recurrence time was significantly delayed in both low and high combination treatment groups in comparison to the no treatment control group (34, 50 and 18 days, respectively). The total number of lung metastases was also significantly decreased in both combination treatment groups. In conclusion, using G129R-based fusion proteins to target mammary carcinomas and to tackle multiple hallmarks of cancer at the same time was an effective strategy for treating HER2-postive mammary cancer in this mouse tumor model.

MMTV/neu transgenic mouse line is a well-documented model for studying HER2/neu-related breast cancer. Approximately 80% of these mice develop mammary tumors by 11 months of age, whereas a small percentage appears to have naturally acquired resistance to HER2/neu tumorigenesis. To identify factors responsible for tumor resistance in these transgenic mice, comparative genetic profiling was used to screen alterations in gene expression in the mammary gland. A novel gene, the RAS association domain (RalGDS/AF-6) family 3 (Rassf3), which belongs to a family of RAS effectors and tumor suppressor genes, was identified. Data indicated 1) that Rassf3 is overexpressed in mammary gland of tumor-resistant MMTV/neu mice compared to tumor-susceptible MMTV/neu littermates or non-transgenic mice, and 2) Rassf3 is significantly up-regulated in neu-specific mouse mammary tumors compared to adjacent normal tissues. In vitro overexpression of RASSF3 inhibited cell proliferation in HER2/neu positive human and mouse breast cancer cell lines, possibly through induction of apoptosis. A novel MMTV/Rassf3-neu bi-transgenic mouse line, overexpressing Rassf3 and neu genes in mammary glands, was established. Mammary tumor incidence in bi-transgenic mice was delayed compared to their MMTV/neu+/- littermates. These data suggest that Rassf3 may influence mammary tumor incidence in MMTV/neu transgenic mice.

Bovine GH (bGH) analogs with single amino acid substitutions at positions 117 (bGH-E117L), 119 (bGH-G119R), and 122 (bGH-A122D) were generated. These analogs bind to mouse liver membrane preparations with affinities similar to native bGH. However, transgenic mice which express the analogs demonstrate different phenotypes ranging from dwarfism to gigantism. For example, expression of bGH or bGH-E117L result in large transgenic mice. In contrast, transgenic mice with a growth phenotype similar to nontransgenic animals result from expression of bGH-A122D. Surprisingly, transgenic mice with relatively high serum levels of bGH-G119R possessed a dwarf phenotype. Together these results suggest that Gly 119 and Ala 122 are involved in growth-promoting activity of GH.

To investigate the relationship between the secondary structure of the third alpha-helix (amino acids 109-126) of bovine growth hormone (bGH) and the biological activity of the molecule, proline or glycine residues have been used as substitutes for native amino acids at positions 114, 118, 121, and 126, respectively. Mutations at the positions 114, 118, and 121 resulted in a dramatic decrease in bGH secretion by transiently transfected mouse L cells whereas the substitution of glycine for glutamate at position 126 (bGH-E126G) did not affect secretion. Immunofluorescence staining revealed that those nonsecretory bGH mutations possessed a different intracellular location as compared with wild-type bGH or the mutated secretory forms of bGH. Similar results were seen in the distribution of these mutated bGH molecules in transfected rat GH-3 cells. Transgenic mice that express wild-type bGH or bGH-E126G grew to approximately 1.6 times the mass of nontransgenic littermates. Transgenic mice that express two nonsecretory forms of mutated bGHs were found to lack the enhanced mouse growth phenotype in spite of elevated levels of serum bGH. These results suggest that the secondary structure in the third alpha-helix of bGH may be important for efficient intracellular targeting in vitro and in growth promotion in transgenic mice.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Human prolactin (hPRL) promotes the proliferation and differentiation of mammary epithelial cells during mammary gland development and has been linked to breast tumor development. The receptor for hPRL (hPRL-R) is elevated in a majority of human breast tumors, suggesting the overexpression of hPRL-R makes cancer cells highly sensitive to the mitogenic and anti-apoptotic activity of hPRL. These findings provide the rationale for the development of hPRL-R targeting breast cancer therapeutics. Previously, an hPRL-R antagonist, G129R, was developed that competitively binds to the hPRL-R resulting in growth inhibition and the induction of apoptosis in certain types of breast cancer cells. To further increase the potency of G129R, we fused G129R to a truncated form of Pseudomonas exotoxin A (PE(40)) that lacks the cell recognition domain of the toxin but retains the domains necessary for PE(40)_ to translocate into the cytosol and inhibit protein synthesis. We postulated that the fusion of G129R with PE(40)-KDEL would (1) deliver the recombinant toxin to breast cancer cells where hPRL-R is overexpressed; (2) block hPRL signaling via its G129R moiety; and (3) inhibit protein synthesis via its PE(40)-KDEL moiety. We demonstrate that the fusion toxin can competitively bind to hPRL-Rs on T-47D human breast cancer cells and inhibit STAT5 phosphorylation induced by hPRL. In addition, we show that G129R-PE(40)-KDEL is selectively cytotoxic to breast cancer cell lines expressing the hPRL-R and that cell death is associated with the inhibition of protein synthesis and does not involve caspase mediated apoptosis.

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP2) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP2 when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb β-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.

Traditional Chinese herbal medicines (TCM) that for centuries have been used in disease prevention and treatment are finding use as alternatives to Western cancer therapies. From a panel of TCM, we chose four compounds representing two functional classes of botanicals, the purified plant flavins scutellarin (a circulatory stimulant) and baicalin (antipyretic), and two extracts purified from Salvia miltiorrhiza (SM-470, circulatory stimulant) and Camellia sinensis (Cam-300, antipyretic), and examined their anti-proliferation effects on the human breast cancer cell lines MCF-7 and T-47D. All four compounds inhibited MCF-7 and T-47D cell proliferation, baicalin being the most potent inhibitor. Moreover, the combination of compounds from different classes offers enhanced potential therapeutic benefits; the combination of SM-470 with scutellarin, Cam-300 or baicalin, augmented the inhibition of cell proliferation. A synergistic inhibitory effect on MCF-7 cell proliferation was also observed when SM-470 and baicalin were applied together. In contrast, inhibition of T-47D cell proliferation using the same combination was dependent on baicalin only. The anti-proliferative effects of these compounds can be extended to other cancer types; the human head and neck cancer epithelial cell lines CAL-27 and FaDu were also sensitive to the four drugs. Overall, SM-470, Cam-300, scutellarin and baicalin inhibited the proliferation of human breast cancer cells and CAL-27 and FaDu cells with different potency. Baicalin and SM-470 in combination produced additive effects, suggesting these compounds may function by different mechanisms. T-47D, MCF-7, and FaDu cells may be useful in exploring the cellular and molecular mechanisms of action of baicalin and SM-470.

Traditional Chinese herbal medicines (TCM) that for centuries have been used in disease prevention and treatment are finding use as alternatives to Western cancer therapies. From a panel of TCM, we chose four compounds representing two functional classes of botanicals, the purified plant flavins scutellarin (a circulatory stimulant) and baicalin (antipyretic), and two extracts purified from Salvia miltiorrhiza (SM-470, circulatory stimulant) and Camellia sinensis (Cam-300, antipyretic), and examined their anti-proliferation effects on the human breast cancer cell lines MCF-7 and T-47D. All four compounds inhibited MCF-7 and T-47D cell proliferation, baicalin being the most potent inhibitor. Moreover, the combination of compounds from different classes offers enhanced potential therapeutic benefits; the combination of SM-470 with scutellarin, Cam-300 or baicalin, augmented the inhibition of cell proliferation. A synergistic inhibitory effect on MCF-7 cell proliferation was also observed when SM-470 and baicalin were applied together. In contrast, inhibition of T-47D cell proliferation using the same combination was dependent on baicalin only. The anti-proliferative effects of these compounds can be extended to other cancer types; the human head and neck cancer epithelial cell lines CAL-27 and FaDu were also sensitive to the four drugs. Overall, SM-470, Cam-300, scutellarin and baicalin inhibited the proliferation of human breast cancer cells and CAL-27 and FaDu cells with different potency. Baicalin and SM-470 in combination produced additive effects, suggesting these compounds may function by different mechanisms. T-47D, MCF-7, and FaDu cells may be useful in exploring the cellular and molecular mechanisms of action of baicalin and SM-470.

In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583-3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813-818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent.

We compared the differential gene expression profile of 4 human breast cancer cell lines following treatment with hPRL or its antagonist. The Bcl-2 gene was of particular interest, and we found that this gene was upregulated in 3 of the 4 PRL-treated cell lines. Real time RT PCR analysis was used to quantitate Bax and Bcl-2 mRNA levels in 11 human breast cancer cell lines, following treatment with PRL or G129R. Bcl-2 levels increased in response to PRL treatment and Bax to G129R treatment, in a majority of cell lines tested. Findings were supported by an in vivo study using 3 age groups of PRL or G129R transgenic mice; the Bax/Bcl-2 mRNA ratio was increased in the C129R group, findings that were expanded by examining the release of a downstream Bax-induced protein, cytochrome c. These were highest in the 6 month G129R group, concomitant with the highest Bax/Bcl-2 mRNA ratio. Thus, Bax is up-regulated in response to G129R stimulation.

Previously we demonstrated that a mutated human prolactin (hPRL) with a single amino acid substitution at position 129 (hPRL-G129R) was able to inhibit human breast cancer cell proliferation via the induction of apoptosis. In this study, we report the in vivo anti-tumor effects of hPRL-G129R in nude mice bearing human breast cancer xenografts (T-47D and MCF-7). In an effort to prolong the half-life of the proteins, hPRL or hPRL-G129R were formulated with either growth factor reduced Matrigel or into slow-releasing pellets (custom made 5 mg/5 day release). Initially, nude mice inoculated (s.c.) with T-47D human breast cancer cells were treated with either hPRL or hPRL-G129R formulated with Matrigel. At the end of the 7-week study, it was found that hPRL significantly stimulated the in vivo growth of T-47D xenografts (mean tumor volume, 202 +/- 62 mm(3) as compared to 124 +/- 31 mm(3) in control mice), whereas hPRL-G129R inhibited the tumor growth (mean tumor volume, 79+/-32 mm3). The inhibitory effects of hPRL-G129R were further confirmed in a second experiment using nude mice bearing MCF-7 human breast cancer xenografts and treated with slow-releasing pellets containing hPRL-G129R. Based on these results, we believe that hPRL-G129R can be used to improve the outcome of human breast cancer treatment in the near future.

Human prolactin (hPRL) has been shown to be one of the important survival/growth factors that promotes the proliferation of breast cancer cells in an autocrine/paracrine manner. In our recent studies, we demonstrated that a hPRL antagonist with a single amino acid substitution mutation (hPRL-G129R) was able to inhibit breast cancer cell proliferation via induction of apoptosis (1). In this study three independent yet related experiments were carried out regarding the effects of hPRL-G129R in breast cancer cells. We investigated the possible mechanism(s) of hPRL-G129R induced apoptosis in breast cancer cells. It is well documented that transforming growth factors (TGF) in conjunction with hormones such as estrogen and PRL play a major role in modulating the proliferation and apoptosis of mammary cells. We first investigated the relationships between hPRL/hPRL-G129R and TGFs. We show that hPRL is able to down-regulate TGF beta 1 (apoptotic factor) secretion and up-regulate TGF alpha (survival factor) secretion in a dose-dependent manner in T-47D cells. More importantly the hPRL antagonist up-regulates TGF beta 1 and down-regulates TGF alpha secretion. When hPRL-G129R was applied together with hPRL, it blocked the effects of hPRL. Secondly, we tested the possible involvement of caspases in hPRL-G129R induced apoptosis. We have shown that caspase-3 is activated by hPRL-G129R at a concentration of 250 ng/ml in T-47D breast cancer cells. Thirdly, we explored the additive effects of an anti-neoplastic drug, cisplatin, with the hPRL-G129R in T47D breast cancer cells. We show that cisplatin and hPRL-G129R when applied together resulted in about 40% growth inhibition in T-47D cells.

We have previously demonstrated that a hPRL antagonist (hPRL-G129R) was able to inhibit PRL induced breast cancer cell proliferation through induction of apoptosis. In the present study, we test the hypothesis that the inhibitory effect of hPRL-G129R in breast cancer cells occurs, at least in part, through the inhibition of oncogene STAT3 activation. We first demonstrated that STAT5 and STAT3 could be activated by either hGH or hPRL in T-47D breast cancer cells. Although the patterns of STAT5 activation by hGH and hPRL are similar, we observed a nearly 10-fold greater efficacy of hPRL in STAT3 activation as compared to that of hGH. More importantly, we have demonstrated that activation of STAT3 by hPRL could be inhibited by hPRL-G129R. Since T-47D cells coexpress GHR and PRLR, an attempt was made to dissect the molecular events mediated through hGHR or hPRLR using mouse L-cells expressing a single population of receptors (hGHR or hPRLR). To our surprise, only STAT5, not STAT3 phosphorylation was observed in these L-cells. In conclusion, our results suggest that: a) STAT3 is preferably activated through hPRLR in T-47D cells; b) hPRL-G129R is effective in inhibiting STAT3 phosphorylation; and c) the mechanism of STAT3 activation is different from that of STAT5.

There were two specific tasks listed in the proposal (finishing cell proliferation as says and summarizing data and manuscript preparation) for the final year of this three-year project. Since we have completed a majority of the tasks related to this project in terms of development of a PRL antagonist, hPRL-G129R. Our focus of research in the past 12 month has been on (1) to further confirm the anti-tumor effects of hPRL-G129R in vivo; (2) to develop hPRL-Gl29R based, breast cancer specific therapeutics; and (3) to reach a conclusion on hPRL-BP project. We have demonstrated that hPRL-G129R is able to inhibit the growth of two human breast cancer cell xenografts in nude mice (manuscript attached) . In terms of designing hPRL-G129R based therapeutics, we have designed and demonstrated efficacy of dual functional protein (G129R-1L2, manuscript attached) . We have also generated preliminary results regarding G129R-PE40 (abstracts attached) . We further confirmed relationship between hPRL, hPRL-G129R and bcl-2 in multiple breast cancer cells (manuscript attached) . On the other hand, we have exhausted our attempts in terms of producing hPRL-BP. In summary, we have demonstrated that hPRL-G129R is a true PRL receptor antagonist. It's anti-breast tumor effects were confirmed using both in vitro and in vivo assays. We are currently conducting pre-clinical studies of hPRL-G129R and hope to launch clinical phase studies.

Retinal neovascularization is the major cause of untreatable blindness. The role of growth hormone (GH) in ischemia-associated retinal neovascularization was studied in transgenic mice expressing a GH antagonist gene and in normal mice given an inhibitor of GH secretion (MK678). Retinal neovascularization was inhibited in these mice in inverse proportion to serum levels of GH and a downstream effector, insulin-like growth factor–I (IGF-I). Inhibition was reversed with exogenous IGF-I administration. GH inhibition did not diminish hypoxia-stimulated retinal vascular endothelial growth factor (VEGF) or VEGF receptor expression. These data suggest that systemic inhibition of GH or IGF-I, or both, may have therapeutic potential in preventing some forms of retinopathy.

Elevated GH levels are frequently seen in poorly controlled type I diabetics and have been implicated in diabetic complications. Studies of GH and GH antagonist (GHA) transgenic mice with streptozotocin (STZ)-induced diabetes have revealed that GH has a permissive effect for diabetic nephropathy, and that expression of a GHA gene protected mice against diabetic kidney lesions. To investigate whether kidney GH receptor (GHR) and/or GH-binding protein may play a role in diabetic nephropathy, we evaluated GH-specific binding and messenger RNA levels for GHR/GH-binding protein in mouse livers and kidneys from bovine (b) GH or bGHA transgenic (Tg) mice and their nontransgenic (NTg) littermates with or without STZ-induced diabetes. We found that liver-specific GH binding is significantly higher in both bGH- and bGHA-Tg mice compared to that in their NTg controls. In contrast, kidney GH binding is significantly lower in bGH-Tg mice compared to that in NTg littermates. These results indicate that regulation of mouse GHR expression is tissue specific. STZ-induced diabetes decreased GH-specific binding in both liver and kidney of NTg and GHA-Tg mice, but not in bGH-Tg mice. The lowered GHR binding in diabetic NTg and GHA-Tg mice suggests the involvement of insulin in the regulation of GHR expression. The down-regulation of kidney GHR in GHA-Tg mice in combination with the presence of GHA may partially explain the protective mechanism of GHA against diabetic kidney lesions.

Adenylyl cyclase (AC) catalyzes the conversion of ATP to the second messenger cAMP, which in turn activates protein kinase A (PKA). AC/cAMP/PKA signaling pathway is known to mediate tumor growth but the results of previous studies have been controversial. More importantly, little is known about how specific AC isoforms affect tumor growth. Based on our previous data showing that knocking out the isoform AC type 5 in mice (AC5KO) extends longevity and that the major cause of premature mortality in mice is neoplastic diseases, we hypothesized that AC5KO mice can be protected against tumor growth. We previously demonstrated that AC5KO mice are protected against oxidative stress and obesity, which should also provide protection against neoplastic disease. In order to elucidate whether AC5 plays a role in the tumor microenvironment, we initially investigated the growth of B16F10 melanoma in this model. We have found that AC5KO mice exhibit protection against B16F10 melanoma tumor growth (p Tumor latency, wet tumor weights and tumor volume were also significantly reduced in AC5KO mice (p Obesity is a risk factor for the development of breast cancer. In accord with the fact that AC5KO are protected against obesity, we have found that circulating levels of leptin were significantly reduced in AC5KO: 1.99±0.28 vs. WT: 3.23±0.35 ng/ml (p Citation Format: Mariana S. De Lorenzo, Wen Y. Chen, Erdene Baljinnyam, Krista La Perle, Sanford P. Bishop, Thomas E. Wagner, Patricio Abarzua, Arnold B. Rabson, Dorothy E. Vatner, Lydia I. Puricelli, Stephen F. Vatner. Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-337. doi:10.1158/1538-7445.AM2013-LB-337

Transgenic mice expressing growth hormone genes have been shown to have kidney lesions resembling those found in human diabetic patients. However, transgenic mice expressing a growth hormone antagonist gene have normal kidneys. In this study, streptozotocin was used to induce diabetes in growth hormone or growth hormone antagonist transgenic mice and total glycated hemoglobin levels were determined. We found streptozotocin treatment resulted in a significant increase in glycated hemoglobin levels in these animals. Despite comparable levels of glycemia and glycated hemoglobin, severe glomerulosclerosis was found in diabetic and nondiabetic growth hormone transgenic mice; moderate glomerulosclerosis was seen in diabetic nontransgenic mice; and normal glomeruli were seen in diabetic and non-diabetic growth hormone antagonist transgenic mice as well as non-diabetic nontransgenic littermates. These results suggest that growth hormone is playing a role in diabetic nephropathy, and elevated levels of growth hormone can directly affect the kidneys independent of the levels of glucose and glycated hemoglobin.

Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kilodaltons. With the aid of computer molecular simulation, an hGH analog was created by altering an hGH gene to reflect the change of one amino acid (glycine [G] 120 to arginine [R]) within the third alpha-helix of the hGH molecule. This hGH analog, named hGHG120R, was found to be an hGH antagonist. It may have important implications in treating human conditions in which hGH levels are abnormally high, as found in type I diabetics. Several hundred milligrams of purified hGHG120R were needed to determine the biological activity of the antagonist in animal models. A multistep downstream process was developed to purify hGHG120R from cultured mouse L cells transfected with the hGHG120R gene. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, reversed phase high performance liquid chromatography, phase separation, and lyophiliation. This work discusses the rationale for the design of the process and experimental results on the purification of hGHG120R using the process. (c) 1995 John Wiley & Sons, Inc.

Porcine (p) growth hormone receptor (GHR) complementary DNA (cDNA) has been cloned and the primary amino acid structure was deduced from the nucleotide sequence. A comparison of pGHR to other GHRs revealed an approximately 70% similarity in amino acid sequence (Cioffi et al., 1990). Hybridization of this receptor cDNA to RNA samples isolated from various porcine tissues revealed a single RNA band of 4.2 kb. The full-length pGHR cDNA was subcloned into an eukaryotic expression vector, transcription of which was directed by the mouse metallothionein-I transcriptional regulatory sequence. Stable mouse L cell lines which express the pGHR cDNA were established. Approximately 80% of the cell lines were found to possess pGHR mRNA (approximately 2 kb) which corresponds to the length of the cloned pGHR cDNA. Binding studies showed that the stable cell lines were capable of specifically binding 125I-labeled pGH with a dissociation constant (Kd) of approximately 1.0 nM. The apparent molecular mass of the receptor, as determined by cross-linking studies, was found to be 118 kDa. Also, the receptor-ligand complex could be internalized. These results suggest that an active form of pGHR had been cloned and stably expressed in mouse L cells.