Combination Of Epidermal Growth Factor Research Articles

Lysosome‑targeted drug combination induces multiple organelle dysfunctions and non‑canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer-related mortality and has the lowest 5-year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC-3, KP-4, PANC-1 and MIA PaCa-2, were examined. It was observed that FTY720 enhanced the lapatinib-induced cytotoxic effect and caused non-canonical and lysosome-dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome-targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpene SO1861.

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma; however, a number of protein‐based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome‐inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC‐3 and MIA PaCa‐2 revealed no significant differences to off‐target cells in the half maximal inhibitory concentration (IC 50) for the fusion protein. In contrast, combination with SO1861 decreased the IC 50 for BxPC‐3 cells from 100 to 0.17 nm, whereas control cells remained unaffected. Monotherapy of BxPC‐3 xenografts in CD‐1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm3) when compared to placebo; however, combined treatment with SO1861 resulted in a more than 13‐fold better efficacy (3.0 mm3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit, and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1<sup>+</sup> Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature β cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to β cells that could eventually be used for the treatment of diabetes.

Influence of Epidermal Growth Factor with Cysteamine on in -Vitro Buffalo Embryo Development

FOR IMPROVING embryo development in buffalo, two experiments were conducted. The first one was carried out to evaluate the different concentrations (0, 5, 25, 50 ng/ml) of epidermal growth factors (EGF) on developmental competence of buffalo oocytes. The selected oocytes were cultured in the four concentrations of EGF. The embryo cleavage rate was significantly higher in oocytes exposed to 5, 25, 50 ng/ml EGF than control. There were no significant difference in four groups (0, 5, 25, 50 ng/ml EGF) in the rate of morula. But, better cleavage and blastocyst rates were observed at 5 ng/ml EGF. In the second experiment, the additive effect of 5 ng/ml EGF with 50 µM cysteamine on maturation and embryo development was studied. Oocytes were collected, matured and cultured in three groups. In the first group, media supplemented with 5 ng/ml EGF + 50 µM cysteamine combination. In the second group, media supplemented with EGF. The third group was supplemented with cysteamine. There was a significant increase in cleavage rate in combination group than EGF (P< 0.05) and cysteamine (P< 0.01) groups. But there was no significant difference in cleavage rate between EGF and cysteamine. The morula percentage was nearly similar in the three groups. But blastocyst rate was significantly (P<0.05) higher in combination group than cysteamine. Thus better cleavage and blastocyst rate were observed at combination group. It is concluded that, the addition of 5 ng/ml EGF in buffalo oocytes during in vitro culture was the best concentration for embryo developmental competence. Higher cleavage and blastocyst rate were achieved by combination of epidermal growth factor and antioxidant.

Effect of Growth Factor Supplementation on the Hair Cell Specific Markers of Cells Harvested from Basilar Membrane.

Loss of auditory hair cells is a major cause of deafness. The presence of auditory progenitor cells in the inner ear raises the hope for mammalian inner ear cell regeneration. In this study, we aimed to investigate the effect of growth factor supplementations, namely a combination of epidermal growth factor (EGF), insulin-like growth factor (IGF), and beta (β)-fibroblast growth factor (βFGF), on the expression of hair cell-specific markers by cells harvested from the cochlear membrane. This would provide an insight into the capability of these cells to differentiate into hair cells. EGF, IGF, and βFGF were supplemented into the culture medium. The cells were evaluated by morphology, growth kinetic, gene expression, and protein expression. The cultured cells of mouse basilar membrane were spindle shaped. Growth factors-enriched medium promotes a significantly higher proliferative activity than the basic culture medium but did not alter the cell morphology. Growth factors-enriched medium did not show any significant differences in the protein expression of the hair cell-specific markers myosin VIIa and calretinin and the stem-cell marker nestin. Gene expression analysis showed that the expression of the hair cell-specific genes myosin VIIa and calretinin as well as the stem cell genes nestin, Rex1, and Sox2 was reduced after the cells were passaged in the growth factor-supplemented medium. Cells in the basic medium expressed a significantly higher level of hair cell-specific genes at certain passages. Growth factor supplementation could not maintain the expression of hair cell-specific markers by cells obtained from the cochlear membrane.

Oral administration of Lactococcus lactis-expressed recombinant porcine epidermal growth factor stimulates the development and promotes the health of small intestines in early-weaned piglets.

We previously generated Lactococcus lactis-expressed recombinant porcine epidermal growth factor (LL-pEGF), and demonstrated improved growth performance in early-weaned piglets. This study investigates the effect of LL-pEGF on the development and expression of genes that maintain the structural integrity and function of the small intestine in early-weaned piglets. The mitogenic effect of porcine epidermal growth factor (pEGF) was tested in vitro with the 5-Bromodeoxyuridine (BrdU) incorporation assay in fibroblast cells. In the in vivo study, 40 weaned piglets were randomly allocated to control, antibiotic control, Lc. lactis-expressing empty vector (LL-EV) and LL-pEGF treatment groups. Cells treated with LL-pEGF had higher BrdU-positive stained cells than those in the control and the LL-EV treatments (P < 0·05). Scanning electron microscope and histological examination demonstrated that the small intestinal villi treated with LL-pEGF were higher (P < 0·05) than in the other treatments. LL-pEGF increased the messenger RNA (mRNA) expression levels of the intestinal structural integrity proteins trefoil factor 3, claudin 1 (CLDN1), occludin and zonula occludens 1 (ZO-1), the digestive enzymes sucrose, aminopeptidase A, and aminopeptidase N, and the nutrient transporters sodium/glucose cotransporter 1 (SGLT1), glucose transporter 2, and peptide transporter 1 (PEPT1) as compared with the control (P < 0·05) in the small intestine. Meanwhile, the mRNA levels of CLDN1 in the jejunum and ZO-1 in the ileum were higher in the LL-EV group than in the control group (P < 0·05). LL-EV and the antibiotic control increased SGLT1 mRNA in the jejunum and PEPT1 mRNA in the ileum compared with the control (P < 0·05). Recombinant pEGF promotes cell mitosis. Oral administration of Lc. lactis-expressing pEGF stimulated intestinal development by upregulating the gene expression of the intestinal structural integrity proteins, the digestive enzymes and the nutrient transporters. The combination of epidermal growth factor and genetically modified micro-organisms may be used as dietary supplements to reduce intestinal stress in animals and even humans.

Efficient Generation of NKX6-1+ Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines

SummaryHuman pluripotent stem cells (hPSCs) represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF) and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10), and inhibitors of bone morphogenetic protein (BMP) and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG) recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

Neurotoxic effects of local anesthetics on the mouse neuroblastoma NB2a cell line

Local anesthetics are used clinically for peripheral nerve blocks, epidural anesthesia, spinal anesthesia and pain management; large concentrations, continuous application and long exposure time can cause neurotoxicity. The mechanism of neurotoxicity caused by local anesthetics is unclear. Neurite outgrowth and apoptosis can be used to evaluate neurotoxic effects. Mouse neuroblastoma cells were induced to differentiate and generate neurites in the presence of local anesthetics. The culture medium was removed and replaced with serum-free medium plus 20 μl combinations of epidermal growth factor and fibroblast growth factor containing tetracaine, prilocaine, lidocaine or procaine at concentrations of 1, 10, 25, or 100 μl prior to neurite measurement. Cell viability, iNOS, eNOS and apoptosis were evaluated. Local anesthetics produced toxic effects by neurite inhibition at low concentrations and by apoptosis at high concentrations. There was an inverse relation between local anesthetic concentrations and cell viability. Comparison of different local anesthetics showed toxicity, as assessed by cell viability and apoptotic potency, in the following order: tetracaine > prilocaine > lidocaine > procaine. Procaine was the least neurotoxic local anesthetic and because it is short-acting, may be preferred for pain prevention during short procedures.

EGF–FGF2 stimulates the proliferation and improves the neuronal commitment of mouse epidermal neural crest stem cells (EPI-NCSCs)

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF2) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF2, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF–FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF–FGF2 in neuronal differentiation protocols.

Neurogenesis of neural crest-derived periodontal ligament stem cells by EGF and bFGF.

Neuroregenerative medicine is an ever-growing field in which regeneration of lost cells/tissues due to a neurodegenerative disease is the ultimate goal. With the scarcity of available replacement alternatives, stem cells provide an attractive source for regenerating neural tissue. While many stem cell sources exist, including: mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells, the limited cellular potency, technical difficulties, and ethical considerations associated with these make finding alternate sources a desirable goal. Periodontal ligament stem cells (PDLSCs) derived from the neural crest were induced into neural-like cells using a combination of epidermal growth factor, and basic fibroblast growth factor. Morphological changes were evident in our treated group, seen under both light microscopy and scanning electron microscopy. A statistically significant increase in the expression of neuron-specific β-tubulin III and the neural stem/progenitor cell marker nestin, along with positive immunohistochemical staining for glial fibrillary acidic protein, demonstrated the success of our treatment in inducing both neuronal and glial phenotypes. Positive staining for synaptophysin demonstrated neural connections and electrophysiological recordings indicated that when subjected to whole-cell patch clamping, our treated cells displayed inward currents conducted through voltage-gated sodium (Na(+) ) channels. Taken together, our results indicate the success of our treatment in inducing PDLSCs to neural-like cells. The ease of sourcing and expansion, their embryologic neural crest origin, and the lack of ethical implications in their use make PDLSCs an attractive source for use in neuroregenerative medicine.

The combination of epidermal growth factor and glycogen synthase kinase 3 inhibitor support long-term self-renewal of Sca-1 positive hepatic progenitor cells from normal adult mice

Isolation and long-term maintenance of hepatic progenitor cells (HPCs) from healthy, non-injured adult livers remains challenging due to the lack of specific surface markers for selection and a limited understanding of the mechanisms for maintaining self-renewal. Previously, we identified a Sca-1 positive, bipotent HPC population in the peri-portal region of adult liver, and found MAPK/ERK and Wnt/β-Catenin pathways to be synergistically involved in their proliferation. In this study, we report the long-term culture of Sca-1 positive HPCs with epidermal growth factor (EGF) and CHIR99021, a small molecule inhibitor of glycogen synthase kinase 3 (GSK-3). Sca-1+ HPCs remain non-tumorigenic when passaged 35 times in vitro over 1 year. Flow cytometric analysis indicates that HPCs are positive for Sca-1 and putative liver progenitor cell markers, including CD13, CD24 and Prominin-1, but negative for hematopoietic/endothelial cell markers CD31, CD34, CD45, CD90 and CD117. Immunocyto-chemistry and RT-PCR indicate Sca-1+ HPCs express albumin (ALB), α-fetoprotein (AFP), cytokeratin19 (CK19), Sox9 and a panel of special hepatic progenitor transcriptional factors. Moreover, Sca-1+ HPCs are able to differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions in vitro and can take part in liver repopulation in an acetaminophen (APAP) induced liver injury mouse model. This study provides a paradigm to capture and maintain HPCs from naive liver tissue and offers a valuable cell model for investigating the molecular mechanisms underlying the cell lineage relationship in normal liver.

Raising Serum Gastrin to Improve Glycemic Control in (Type 2) Diabetes: Another Limb of the Enteroinsular Axis?

Gastrin has long been considered a candidate incretin hormone, i.e. an enteral factor responsible for the more than 2-fold increase in insulin secretion when glucose is ingested orally than when administered iv to match peripheral glucose levels. In healthy humans, acute administration of gastrin to achieve supraphysiological levels leads to a modest release of insulin under basal glucose conditions and a more pronounced potentiation of insulin release when coadministered with glucose (1), evidence supporting an incretin role for gastrin. Nevertheless, resection of the gastric antrum as part of Whipple’s operation that reduced serum gastrin levels to very low or undetectable levels was not associated with any diminution of the incretin effect (2), suggesting that whatever role gastrin played was dispensable, at least in nondiabetic individuals. With the identification of gastric inhibitory polypeptide, now known as glucose-dependent insulinotropic polypeptide, and subsequently glucagon-like peptide-1 as more potent incretin hormones whose secretion may be impaired in type 2 diabetes, attention largely shifted away from gastrin playing an important role in the enteroinsular axis. Like gastric inhibitory polypeptide and glucagon-like peptide-1, gastrin has also been shown to induce -cell proliferation and neogenesis in various model systems, and also appears to increase the insulin content of individual -cells. Zollinger and Ellison’s original description of peptic ulcer disease attributable to purported gastrinoma included one case where “the pancreatic tissue removed with the tumor showed normal islands with many large -cells...” (3). Examination of another case with resected pancreas adjacent to an antral gastrinoma “showed a marked proliferation of ducts and islet tissue” with “development of islets from cells (nesidioblasts) within the duct epithelium (4). More recently, Meier et al. (5) showed that in cases of intrapancreatic gastrinoma, there was an increase in fractional -cell area and -cell replication in pancreatic tissue adjacent to but not distant from gastrin-producing tumors, suggesting that either an extremely elevated level of gastrin acting in a paracrine fashion or some other growth factor released by the tumor is required for such trophic -cell effects in humans (and perhaps only in those predisposed to endocrine neoplasia). In isolated human islets, a combination of epidermal growth factor and gastrin but not gastrin alone was required to increase the numbers of -cells in culture, although both gastrin and epidermal growth factor independently increased the insulin content of islets (6). Thus, evidence exists to support an effect of gastrin on increasing insulin production in human -cells. By blocking gastric acid production, proton pump inhibitors (PPIs) remove negative feedback on gastrin production by enterochromaffin cells. In a rodent model of type 2 diabetes treatment with the PPI lansoprazole increased serum gastrin that was associated with improved glycemia and increased pancreatic insulin content (7). Now comes a randomized placebo-controlled trial of the PPI pantoprazole in type 2 diabetes that reduced the glycosylated hemoglobin by approximately 1 percentage point (8). The improvement in glycemic control was associated with a decrease in fasting glucose and increase in fasting insulin such that homeostasis model assessment surrogate measures suggested an improvement in -cell function and no change in insulin sensitivity. Although

The effects of BMP6 overexpression on adipose stem cell chondrogenesis: Interactions with dexamethasone and exogenous growth factors

Adipose-derived stem cells (ASCs) are multipotent progenitors that can be chondrogenically induced by growth factors such as bone morphogenetic protein 6 (BMP-6). We hypothesized that nonviral transfection of a BMP-6 construct (pcDNA3-BMP6) would induce chondrogenic differentiation of ASCs encapsulated in alginate beads and that differentiation would be enhanced by the presence of the synthetic glucocorticoid dexamethasone (DEX) or the combination of epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor beta-1 (TGF-beta1), collectively termed expansion factors (EFs). Chondrogenesis was assessed using quantitative real-time polymerase chain reaction for types I, II, and X collagen, aggrecan, and BMP6. Immunohistochemistry was performed with antibodies for types I, II, and X collagen and chondroitin-4-sulfate. BMP6 overexpression alone induced a moderate chondrogenic response. The inclusion of EFs promoted robust type II collagen expression but also increased type I and X collagen deposition, consistent with a hypertrophic chondrocyte phenotype. Early gene expression data indicated that DEX was synergistic with BMP-6 for chondrogenesis, but immunohistochemistry at 28 days showed that DEX reduced glycosaminoglycan accumulation. These results suggest that chondrogenic differentiation of ASCs depends on complex interactions among various growth factors and media supplements, as well as the concentration and duration of growth factor exposure.

Pancreatic ductal transdifferentiation for β-cell neogenesis

Background: Transdifferentiation of exocrine pancreatic cells into insulin-secreting cells may be an alternative or a support to islet transplantation-based therapy of Type 1 diabetes. The application relates to a method of generating in vitro insulin-producing β-cells from dedifferentiated exocrine pancreatic cells using a new formulation of growth factors based on the combination of epidermal growth factor and leukemia-inhibitory factor. Objective/methods: This evaluation summarizes the current literature and the potential therapeutic application of the present patent for β-cell neogenesis. Results/conclusion: As suggested by this patent, the combination of growth factors acting on different intracellular pathways may reverse autoimmunity and increase the results of islet transplantation.

Multi-targeted inhibition of the epidermal growth factor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways: A phase I study of cetuximab (C), erlotinib (E), and bevacizumab (B) in patients with solid tumors

3005 Background: Complex interrelationships exist between the EGFR and VEGFR pathways. EGFR activation elicits cell proliferation, and downstream effects increase expression of VEGF. In renal cell carcinoma, mutations increase hypoxia inducible factor-1alpha, stimulating VEGF and transforming growth factor expression. Moreover, there is additive tumor inhibition from combined EGFR targeting with C, and a tyrosine kinase inhibitor. To maximally inhibit EGFR, and then inhibit downstream VEGFR activity, this phase I study was initiated to determine the maximum tolerated dose (MTD) of E with a fixed dose of C, and then the MTD of B with combined E and C in patients with advanced malignancies. Methods: Patients with advanced malignancies likely to express EGFR were entered in part 1 to daily oral E (starting at 100mg, planned initially to increase to 150 mg), with fixed dose C (400 mg/m2 loading and 250 mg/m2 IV weekly). Once the MTD was determined for E in combination C, part 2 incorporated the addition of escalating doses of B (5 mg/kg IV every 2 weeks, to increase to 10 mg/kg) to the combination of E and C. Results: 27 patients were entered and received 84 courses over 3 dose levels. In part 1 grade 3 rash occurred in 2 patients at E at 100 mg daily, and the MTD of E for this combination was 50 mg daily with standard dose C (12 patients treated). Other adverse events included rash, diarrhea, hypomagnesemia, and nausea. Part 2: B at 5 mg/kg IV q14 days can be added to the MTD of E with C, with additional non-dose limiting toxicities of headache, proteinuria, and hypertension. Durable stable disease has been observed in 4 patients with metastatic disease for 7 (sqaumous cell); 10+, 12, and 12+ (renal cell) months. Conclusions: The MTD for E combined with standard C is 50 mg daily. B at 5mg/kg can be combined safely with this combination and dose escalation is ongoing. [Table: see text]

New sources of pancreatic β-cells

Two major initiatives are under way to correct the beta-cell deficit of diabetes: one would generate beta-cells ex vivo that are suitable for transplantation, and the second would stimulate regeneration of beta-cells in the pancreas. Studies of ex vivo expansion suggest that beta-cells have a potential for dedifferentiation, expansion, and redifferentiation. Work with mouse and human embryonic stem (ES) cells has not yet produced cells with the phenotype of true beta-cells, but there has been recent progress in directing ES cells to endoderm. Putative islet stem/progenitor cells have been identified in mouse pancreas, and formation of new beta-cells from duct, acinar and liver cells is an active area of investigation. Peptides, including glucagon-like peptide-1/exendin-4 and the combination of epidermal growth factor and gastrin, can stimulate regeneration of beta-cells in vivo. Recent progress in the search for new sources of beta-cells has opened promising new opportunities and spawned clinical trials.

Combination Therapy with Epidermal Growth Factor and Gastrin Induces Neogenesis of Human Islet β-Cells from Pancreatic Duct Cells and an Increase in Functional β-Cell Mass

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.

Induction of mammotroph development by a combination of epidermal growth factor, insulin, and estradiol-17beta in rat pituitary tumor GH3 cells.

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.

Growth Hormone-dependent Differentiation of 3T3-F442A Preadipocytes Requires Janus Kinase/Signal Transducer and Activator of Transcription but Not Mitogen-activated Protein Kinase or p70 S6 Kinase Signaling

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42(MAPK)/p44(MAPK) mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70(s6k) was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70(s6k), had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42(MAPK)/p44(MAPK) and p70(s6k) is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl4 and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [3H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [3H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [3H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [3H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.