Activity of enzalutamide in men with metastatic castration-resistant prostate cancer is affected by prior treatment with abiraterone and/or docetaxel.

BACKGROUND: Enzalutamide and abiraterone are new androgen-axis disrupting treatments for metastatic castration-resistant prostate cancer (mCRPC). We examined the response and outcomes of enzalutamide-treated mCRPC patients in the real-world context of prior treatments of abiraterone and/or docetaxel. METHODS: We conducted a seven-institution retrospective study of mCRPC patients treated with enzalutamide between January 2009 and February 2014. We compared the baseline characteristics, PSA declines, PSA progression-free survival (PSA-PFS), duration on enzalutamide and overall survival (OS) across subgroups defined by prior abiraterone and/or docetaxel. RESULTS: Of 310 patients who received enzalutamide, 36 (12%) received neither prior abiraterone nor prior docetaxel, 79 (25%) received prior abiraterone, 30 (10%) received prior docetaxel and 165 (53%) received both prior abiraterone and prior docetaxel. Within these groups, respectively, ⩾30% PSA decline was achieved among 67, 28, 43 and 24% of patients; PSA-PFS was 5.5 (95% CI 4.2-9.1), 4.0 (3.2-4.8), 4.1 (2.9-5.4) and 2.8 (2.5-3.2) months; median duration of enzalutamide was 9.1 (7.3-not reached), 4.7 (3.7-7.7), 5.4 (3.8-8.4) and 3.9 (3.0-4.6) months. Median OS was reached only for the patients who received both prior abiraterone and docetaxel and was 12.2 months (95% CI 10.7-16.5). 12-month OS was 78% (59-100%), 64% (45-90%), 77% (61-97%) and 51% (41-62%). Of 70 patients who failed to achieve any PSA decline on prior abiraterone, 19 (27%) achieved ⩾30% PSA decline with subsequent enzalutamide. CONCLUSIONS: The activity of enzalutamide is blunted after abiraterone, after docetaxel, and still more after both, suggesting subsets of overlapping and distinct mechanisms of resistance.

miR-34a functions as a tumor suppressor modulating EGFR in glioblastoma multiforme.

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice.

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.

Basic helix-loop-helix transcription factors and enteroendocrine cell differentiation.

For over 30 years it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently little was understood about the events that result in commitment to endocrine differentiation or the eventual segregation of over 10 different hormone-expressing cell types in the gastrointestinal tract. Enteroendocrine cells arise from pluripotent intestinal stem cells. Differentiation of enteroendocrine cells is controlled by the sequential expression of three basic helix-loop-helix transcription factors, Math1, Neurogenin 3 (Neurog3) and NeuroD. Math1 expression is required for specification and segregation of the intestinal secretory lineage (Paneth, goblet,and enteroendocrine cells) from the absorptive enterocyte lineage. Neurog3 expression represents the earliest stage of enteroendocrine differentiation and in its absence enteroendocrine cells fail to develop. Subsequent expression of NeuroD appears to represent a later stage of differentiation for maturing enteroendocrine cells. Enteroendocrine cell fate is inhibited by the Notch signalling pathway, which appears to inhibit both Math1 and Neurog3. Understanding enteroendocrine cell differentiation will become increasingly important for identifying potential future targets for common diseases such as diabetes and obesity.

Capsaicin causes cell-cycle arrest and apoptosis in ER-positive and -negative breast cancer cells by modulating the EGFR/HER-2 pathway.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is an ingredient of chili peppers with inhibitory effects against cancer cells of different origin. We examined the activity of capsaicin on breast cancer cells in vitro and in vivo. The drug potently inhibited growth of ER-positive (MCF-7, T47D, BT-474) and ER-negative (SKBR-3, MDA-MB231) breast cancer cell lines, which was associated with G(0)/G(1) cell-cycle arrest, increased levels of apoptosis and reduced protein expression of human epidermal growth factor receptor (EGFR), HER-2, activated extracellular-regulated kinase (ERK) and cyclin D1. In contrast, cell-cycle regulator p27(KIP1), caspase activity as well as poly-ADP ribose polymerase (PARP) cleavage were increased. Notably, capsaicin blocked breast cancer cell migration in vitro and decreased by 50% the size of MDA-MB231 breast cancer tumors growing orthotopically in immunodeficient mice without noticeable drug side effects. in vivo activation of ERK was clearly decreased, as well as expression of HER-2 and cyclin D1, whereas caspase activity and PARP cleavage products were increased in tumors of drug-treated mice. Besides, capsaicin potently inhibited the development of pre-neoplastic breast lesions by up to 80% without evidence of toxicity. Our data indicate that capsaicin is a novel modulator of the EGFR/HER-2 pathway in both ER-positive and -negative breast cancer cells with a potential role in the treatment and prevention of human breast cancer.

NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor.

The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in beta-cells and either absent or expressed below the limit of lacZ detection in mature alpha-, delta-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic delta-cell line, resulted in potent repression of somatostatin concomitant with induction of the beta-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in beta- but not delta-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.

HMG box transcriptional repressor HBP1 maintains a proliferation barrier in differentiated liver tissue.

We previously isolated HBP1 as a target of the retinoblastoma (RB) and p130 family members and as the first of the HMG box transcriptional repressors. Our subsequent work demonstrated that HBP1 coordinates differentiation in cell culture models. In the present study, we show that HBP1 regulates proliferation in a differentiated tissue of an animal. Using transgenic mice in which HBP1 expression was specifically increased in hepatocytes under control of the transthyretin promoter, we determined the impact of HBP1 on synchronous cell cycle reentry following partial hepatectomy. Modest overexpression of HBP1 yielded a detectable cell cycle phenotype. Following a mitogenic stimulus induced by two-thirds partial hepatectomy, mice expressing the HBP1 transgene showed a 10- to 12-h delay in progression through G(1) to the peak of S phase. There was a concomitant delay in mid-G(1) events, such as the induction of cyclin E. While the delay in G(1) and S phases correlated with the slight overexpression of transgenic HBP1, the level of the endogenous HBP1 protein itself declined in S phase. In contrast, the onset of the immediate-early response following partial hepatectomy was unchanged in HBP1 transgenic mice. This observation indicated that the observed delay in S phase did not result from changes in signaling pathways leading into the G(0)-to-G(1) transition. Finally, transgenic mice expressing a mutant HBP1 lacking the N-terminal RB interacting domain showed a stronger S-phase response following partial hepatectomy. These results provide the first evidence that HBP1 can regulate cell cycle progression in differentiated tissues.

Role of the amino-terminal domain of simian virus 40 early region in inducing tumors in secretin-expressing cells in transgenic mice.

BACKGROUND & AIMS: The early region of simian virus 40 (SV40) encodes 2 transforming proteins, large T (Tag) and small t antigen, that produce neuroendocrine tumors in the intestine and the pancreas when expressed in secretin cells of transgenic mice. METHODS: Two SV40 early-region transgenes containing a deletion that eliminated expression of the small t antigen were expressed in transgenic mice under control of the secretin gene. The 2 lines of mice, one expressing the native large T antigen and the other T antigen with a mutation in its N-terminal J domain, were examined to determine which biological activities of the SV40 early region were required for tumorigenesis. RESULTS: Most animals expressing wild-type large T antigen developed pancreatic insulinomas and lymphomas and died between 3 and 6 months of age. However, small intestinal neoplasms were extremely rare in the absence of small t antigen expression. Transgenic lines expressing the J domain mutant failed to develop tumors. CONCLUSIONS: Transformation of secretin-producing enteroendocrine cells by SV40 requires functional cooperation between intact large T and small t oncoproteins. In contrast, large T antigen alone is sufficient to induce tumors in the endocrine pancreas and thymus.

Review article: transcriptional events controlling the terminal differentiation of intestinal endocrine cells.

Secretin-producing enteroendocrine cells arise from a multipotential endocrine progenitor in the crypts of the small intestine. As these cells migrate up the crypt-villus axis, they produce secretin and stop dividing as they terminally differentiate and die. Transcription of the secretin gene is controlled by a complex enhancer binding to multiple transcription factors. The basic helix-loop-helix protein, BETA2, binds to an E box sequence and associates with the p300 coactivator to activate transcription of the secretin gene. Basic helix-loop-helix proteins appear to play a pivotal role in the control of cellular differentiation. BETA2 induces cell cycle arrest and apoptosis in addition to activating secretin gene expression. Thus BETA2 may function as a master regulatory gene to coordinate terminal differentiation of secretin cells.

Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells.

The basic helix-loop-helix protein BETA2 interacts with p300 to coordinate differentiation of secretin-expressing enteroendocrine cells.

The major epithelial cell types lining the intestine comprise a perpetually self-renewing population of cells that differentiate continuously from a stem cell in the intestinal crypts. Secretin-producing enteroendocrine cells represent a nondividing subpopulation of intestinal epithelial cells, suggesting that expression of the hormone is coordinated with cell cycle arrest during the differentiation of this cell lineage. Here we report that the basic helix-loop-helix protein BETA2 associates functionally with the coactivator, p300 to activate transcription of the secretin gene as well as the gene encoding the cyclin-dependent kinase inhibitor p21. Overexpression of BETA2 in cell lines induces both cell cycle arrest and apoptosis suggesting that BETA2 may regulate proliferation of secretin cells. Consistent with this role, we observed both reentry of normally quiescent cells into the cell cycle and disrupted cell number regulation in the small intestine of BETA2 null mice. Thus, BETA2 may function to coordinate transcriptional activation of the secretin gene, cell cycle arrest, and cell number regulation, providing one of the first examples of a transcription factor that controls terminal differentiation of cells in the intestinal epithelium.

Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice.

Candidate transcription factors involved in pancreatic endocrine development have been isolated using insulin gene regulation as a paradigm. The cell-type restricted basic helix-loop-helix (bHLH) gene, BETA2/NeuroD, expressed in pancreatic endocrine cells, the intestine, and the brain, activates insulin gene transcription and can induce neurons to differentiate. To understand the importance of BETA2 in pancreatic endocrine cell differentiation, mice lacking a functional BETA2 gene were generated by gene targeting experiments. Mice carrying a targeted disruption of the BETA2 gene developed severe diabetes and died perinatally. Homozygous BETA2 null mice had a striking reduction in the number of insulin-producing beta cells and failed to develop mature islets. Islet morphogenesis appeared to be arrested between E14.5 and E17.5, a period characterized by major expansion of the beta cell population. The presence of severe diabetes in these mice suggests that proper islet structure plays an important role in blood glucose homeostasis. In addition, secretin- and cholecystokinin-producing enteroendocrine cells failed to develop in the absence of BETA2. The absence of these two pancreatic secretagogs may explain the abnormal cellular polarity and inability to secrete zymogen granules in pancreatic acinar exocrine cells. The nervous system appeared to develop normally, despite abundant expression of BETA2 in differentiating neurons. Thus, BETA2 is critical for the normal development of several specialized cell types arising from the gut endoderm.

The basic helix-loop-helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene expression.

The gene encoding the hormone secretin is expressed only in enteroendocrine S cells and insulin-producing pancreatic beta cells during development. A 120-bp enhancer directs cell-specific expression of the rat secretin gene in secretin-expressing cells. The enhancer includes an E-box sequence, CAGCTG, which is important for transcriptional activity. To further characterize the role of the E box, a consensus binding site for basic helix-loop-helix (bHLH) proteins, we have examined factors that interact with this element in the secretin gene. The results suggest that transcription is activated by a recently isolated tissue-specific bHLH protein, BETA2, heterodimerized to the ubiquitously expressed bHLH proteins, Pan 1 and Pan 2, the rodent homologues of E47 and E12. The importance of BETA2 for transcriptional activation of secretin is further illustrated by antisense experiments inhibiting BETA2 expression in secretin-producing cell lines, which resulted in the inhibition of most E box-dependent transcription. Expression of BETA2 in a nonendocrine cell line conferred the ability to express secretin-reporter genes that are transcribed at minimal levels in the absence of BETA2. Secretin-producing enteroendocrine cells in the murine small intestine showed specific immunostaining with BETA2 antibodies, corroborating observations in cell lines. Thus BETA2 is to our knowledge the first transcription factor identified that specifically activates cell type-specific expression of an intestinal hormone gene.

Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice.

The hormone peptide YY is produced by endocrine cells in the pancreas, ileum and colon. We have previously shown that peptide YY is coexpressed in all four islet cell types in the murine pancreas when they first appear, suggesting a common peptide YY-producing progenitor. In the colon, peptide YY has been frequently identified in glucagon-expressing L-type endocrine cells. Characterization of colonic endocrine tumors in transgenic mice expressing simian virus 40 large T antigen under the control of the peptide YY gene 5' flanking region revealed tumor cells producing not only peptide YY and glucagon, but also neurotensin, cholecystokinin, substance P, serotonin, secretin, and gastrin. This suggested that multiple enteroendocrine lineages were related to peptide YY-producing cells. Subsequent examination of the ontogeny of colonic endocrine differentiation in nontransgenic mice revealed that peptide YY was the first hormone to appear during development, at embryonic day 15.5. Between embryonic days 16.5 and 18.5, cells expressing glucagon, cholecystokinin, substance P, serotonin, secretin, neurotensin, gastrin and somatostatin first appeared and peptide YY was coexpressed in each cell type at this time. Peptide YY coexpression continued in a significant fraction of most enteroendocrine cell types throughout fetal and postnatal development and into adulthood, with the exception of serotonin-producing cells. This latter population of cells expanded dramatically after birth with rare coexpression of peptide YY. These studies indicate that expression of peptide YY is an early event in colonic endocrine differentiation and support the existence of a common progenitor for all endocrine cells in the colon.

Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells.

Transcriptional regulation of secretin gene expression.

Expression of the gene encoding the hormone secretin is restricted to a specific enteroendocrine cell type and to beta-cells in developing pancreatic islets. To characterize regulatory elements in the secretin gene responsible for its expression in secretin-producing cells, we used a series of reporter genes for transient expression assays in transfection studies carried out in secretin-producing islet cell lines. Analysis of the transcriptional activity of deletion mutants identified a positive cis regulatory domain between 174 and 53 base pairs upstream from the transcriptional initiation site which was required for secretin gene expression in secretin-producing HIT insulinoma cells. Within this enhancer were sequences resembling two binding sites for the transcription factor Sp1, as well as a consensus sequence for binding to helix-loop-helix proteins. Analysis of these three elements by site-directed mutagenesis suggests that each is important for full transcriptional activity. The role of proximal enhancer sequences in directing secretin gene expression to appropriate tissues is further supported by studies in transgenic mice revealing that 1.6 kilobases of the secretin gene 5' flanking sequence were sufficient to direct the expression of either human growth hormone or simian virus 40 large T-antigen reporter genes to all major secretin-producing tissues.

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1.

The murine neuroendocrine cell line, STC-1, was found to contain 296.8 +/- 1.8 fmol of cholecystokinin-like immunoreactivity (CCK-LI) per mg cell protein. Immunocytochemical stain of STC-1 cells maintained in monolayer culture indicated that CCK-LI activity was present in 93% of the cells. Analysis by reverse-phase high-performance liquid chromatography indicated that STC-1 cells contained CCK-8 and an unidentified form as the predominant storage form. form. However, only CCK-8 was released into the culture medium upon stimulation by various secretagogues. The release of CCK-LI from STC-1 cells was stimulated by dibutyryl cAMP, forskolin, KCl, A23187, 4 beta-phorbol 12-myristate 13-acetate and luminal stimulants, e.g., sodium oleate, L-tryptophan, camostat and plaunotol. The release of CCK-LI from STC-1 cells was also stimulated by a neuropeptide, bombesin. The stimulatory effects of most of these agents were dose dependent. The stimulatory effects of dibutyryl cAMP, forskolin, and plaunotol were potentiated by 3-isobutyl-1-methyl xanthine, while that of camostat was not. The results obtained in this study indicate that the release of CCK from STC-1 cells shares the same characteristics of CCK release as from the CCK-secreting cells of the intestinal mucosa observed both in the dog and the rat in vitro and in vivo. Thus, the cellular mechanism of CCK release which appears to be cAMP- and Ca(2+)-dependent may be modulated by cellular protein kinase C activity. The STC-1 cell appears to be a suitable model for studying the mechanism of CCK release.

Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor.

The islets of Langerhans contain four distinct endocrine cell types producing the hormones glucagon, insulin, somatostatin and pancreatic polypeptide. These cell lineages are thought to arise from a common, multipotential progenitor cell whose identity has not been well established. The pancreatic and intestinal hormone, peptide YY, has been previously identified in glucagon-producing cells in islets; however, transgenic mice expressing Simian Virus 40 large T antigen under the control of the peptide YY gene expressed the oncoprotein in beta, delta and pancreatic polypeptide cells, and occasionally developed insulinomas, suggesting relationships between peptide YY-producing cells and several islet cell lineages. The four established pancreatic islet cell types were examined for coexpression of peptide YY in islets of normal and transgenic mice throughout development. Peptide YY immunoreactivity was identified in the earliest endocrine cells in the fetal pancreas and was coexpressed in each islet cell type during development. Peptide YY showed a high degree of co-localization with glucagon- and insulin-producing cells in early pancreatic development, but by adulthood, peptide YY was expressed in less than half of the alpha cells and was no longer expressed in beta cells. Peptide YY was also coexpressed with somatostatin and pancreatic polypeptide when these cell types first appeared, but most delta and pancreatic polypeptide cells continued to express peptide YY throughout development. The use of conditions that distinguish peptide YY from the related peptides, pancreatic polypeptide and neuropeptide Y, as well as the ability of the peptide YY gene to direct expression of a reporter gene in islets of transgenic mice, establishes expression of peptide YY in the earliest pancreatic endocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene.

It is well established that the gene encoding the hormone secretin is expressed in a specific enteroendocrine cell, the S cell. We now show that the secretin gene is transiently expressed in insulin-producing B cells of the developing pancreatic islets in addition to the intestine. Furthermore, secretin is produced by most established islet cell lines. In order to identify and characterize the regulatory elements within the secretin gene that control tissue-specific expression, we have introduced secretin reporter gene constructions into the secretin-producing HIT and STC-1 cell lines as well as the nonexpressing INR1-G9 glucagonoma line. Analysis of deletion mutants revealed that sequences between 174 and 53 bp upstream from the transcriptional start site are required for maximal expression in secretin-producing cells. This positive element functioned independently of position and orientation. Further deletions into the enhancer resulted in a stepwise loss of transcriptional activity, suggesting the presence of several discrete control elements. The sequence CAGCTG within the secretin enhancer closely resembles that of the core of the B-cell-specific enhancer in the insulin gene. Point mutations introduced into this putative element led to greater than 85% reduction in transcriptional activity. Gel mobility shift assays suggested that a factor in B cells closely related or identical to proteins that bind to the insulin enhancer interacts with the CAGCTG motif in the secretin gene.

The secretin gene: evolutionary history, alternative splicing, and developmental regulation.

The gene encoding the hormone secretin has been isolated and structurally characterized. The transcriptional unit is divided into four exons spanning 813 nucleotides. Comparison of the rat secretin gene to the other members of the glucagon-secretin gene family reveals that similarities are restricted to the exons encoding the biologically active peptides. Analysis of RNA from porcine intestine indicates that at least two transcripts are generated from the porcine secretin gene as a result of differential splicing. The longer and more abundant transcript appears to be identical to a previously isolated cDNA, which encodes a precursor that includes a 72-amino acid C-terminal extension peptide. The shorter transcript does not contain the third exon and, as a result, encodes only 44 residues beyond the C terminus of secretin. The amino acid sequence deduced from the shorter transcript is identical to a precursor form of secretin recently isolated from porcine duodenum [Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. Sci. USA 87, 6781-6785]. Developmental studies reveal that both secretin mRNA and peptide levels in the intestine are highest just before birth, prior to the onset of gastric acid secretion and feeding. This observation implies that secretin biosynthesis in developing animals is controlled independently of the principal factors known to regulate secretin release in adult animals.