Redifferentiation and induction of tumor suppressors miR-122 and miR-375 by the PAX8/PPARγ fusion protein inhibits anaplastic thyroid cancer: a novel therapeutic strategy.

Anaplastic thyroid cancer (ATC) is an aggressive, fatal disease unresponsive to traditional therapies, generating a need to develop effective therapies. The PAX8/PPARγ fusion protein (PPFP) has been shown to favorably modulate tumor growth in follicular thyroid cancer, prompting our evaluation of its efficacy to inhibit ATC cell and tumor growth in vitro and in vivo. PPFP was constitutively expressed in five ATC cell lines: BHT-101, FRO, C-643, KTC-2 and KTC-3, and inhibited cell growth in four of five cell lines and xenograft tumor growth in four of four cell lines. PPFP-mediated growth inhibition involved multiple mechanisms, including upregulation of miR-122 and miR-375, associated with decreased angiogenesis and AKT pathway inactivation, respectively. Also, PPFP expression resulted in marked increase of thyroid-specific marker transcripts, including PAX8, thyroid peroxidase (TPO), sodium iodide symporter (NIS) and thyroglobulin, to varying degrees by activating their respective promoters, suggesting that PPFP induced cellular redifferentiation. Functional studies demonstrate that increased NIS messenger RNA is not associated with increased 125I uptake. However, ectopic expression of wild-type NIS-induced perchlorate-sensitive iodine uptake, suggesting that endogenous NIS in ATC cell lines is defective. As current treatment for ATC is only palliative, overexpression of PPFP may offer a novel therapeutic strategy for the treatment of ATC.

Preclinical efficacy of the oncolytic measles virus expressing the sodium iodide symporter in iodine non-avid anaplastic thyroid cancer: a novel therapeutic agent allowing noninvasive imaging and radioiodine therapy.

Anaplastic thyroid cancer is an extremely aggressive disease resistant to radioiodine treatment because of loss of sodium iodide symporter (NIS) expression. To enhance prognosis of this fatal cancer, we validated the preclinical efficacy of measles virus (MV)-NIS, the vaccine strain of the oncolytic MV (MV-Edm), modified to include the NIS gene. Western blotting analysis confirmed that a panel of eight anaplastic thyroid cancer (ATC)-derived cell lines do not express NIS protein, but do express CD46, the MV receptor. In vitro cell death assays and in vivo xenograft studies demonstrate the oncolytic efficacy of MV-NIS in BHT-101 and KTC-3, ATC-derived cell lines. Radioactive iodine uptake along with single-photon emission computed tomography (SPECT)-computed tomography imaging of KTC-3 xenografts after (99)Tc(m) administration confirmed NIS expression in vitro and in vivo, respectively, after virus treatment. Adjuvant administration of RAI, to MV-NIS-treated KTC-3 tumors showed a trend for increased tumor cell killing. As current treatment for ATC is only palliative, and MV-NIS is currently Food and Drug Administration approved for human clinical trials in myeloma, our data indicate that targeting ATC with MV-NIS could prove to be a novel therapeutic strategy for effective treatment of iodine-resistant ATC and will expedite its testing in clinical trials for this aggressive disease.

Cellular and physiological mechanisms of new-onset diabetes mellitus after solid organ transplantation.

New-onset diabetes after transplantation is recognized as one of the metabolic consequences which may increase the risk of morbidity and mortality after solid organ transplantation. The pathophysiology of new-onset diabetes after transplantation has not been clearly defined and may resemble that of Type 2 diabetes, characterized by predominantly insulin resistance or defective insulin secretion, or both. This review aims to summarize the current state of knowledge regarding the prevalence, consequences, pathogenesis, and management of new-onset diabetes after transplantation, with a major focus on the possible mechanisms involved in the pathogenesis of the disorder. The aetiology of new-onset diabetes after transplantation is multifactorial, with diabetogenic immunosuppressive drugs playing a major role. Multiple cellular and physiologic mechanisms are involved in the process. Selection of an appropriate maintenance immunosuppressive regimen should involve balancing the risk of patient and graft survival vs. the potential for new-onset diabetes after transplantation.

Single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models.

Streptozotocin (STZ)-induced diabetes mellitus (DM) offers a very cost-effective and expeditious technique that can be used in most strains of rodents, opening the field of DM research to an array of genotypic and phenotypic options that would otherwise be inaccessible. Despite widespread use of STZ in small animal models, the data available concerning drug preparation, dosing and administration, time to onset and severity of DM, and any resulting moribundity and mortality are often limited and inconsistent. Because of this, investigators inexperienced with STZ-induced diabetes may find it difficult to precisely design new studies with this potentially toxic chemical and account for the severity of DM it is capable of inducing. Until a better option becomes available, attempts need to be made to address shortcomings with current STZ-induced DM models. In this paper we review the literature and provide data from our pancreatic islet transplantation experiments using single high-dose STZ-induced DM in NCr athymic nude mice with hopes of providing clarification for study design, suggesting refinements to the process, and developing a more humane process of chemical diabetes induction.

Antitumor Activity of VB-111, a Novel Antiangiogenic Virotherapeutic, in Thyroid Cancer Xenograft Mouse Models.

VB-111 is an engineered antiangiogenic adenovirus that expresses Fas-c in angiogenic blood vessels and has previously been shown to have significant antitumor activity in vitro and in vivo in Lewis lung carcinoma, melanoma, and glioblastoma models. To evaluate the efficacy of VB-111 in thyroid cancer, we conducted in vivo xenograft nude mouse studies using multiple thyroid cancer-derived cell lines models. VB-111 treatment resulted in 26.6% (P = 0.0596), 34.4% (P = 0.0046), and 37.6% (P = 0.0249) inhibition of tumor growth in follicular, papillary and anaplastic thyroid cancer models, respectively. No toxicity was observed in any model. All tumor types showed a consistent and significant reduction of CD-31 staining (P < 0.05), reflecting a reduction of angiogenic activity in the tumors, consistent with the intended targeting of the virus. A phase 2 clinical trial of VB-111 in patients with advanced differentiated thyroid cancer is ongoing.

Expression of the PAX8/PPARγ Fusion Protein Is Associated with Decreased Neovascularization In Vivo: Impact on Tumorigenesis and Disease Prognosis.

The PAX8/PPARγ fusion protein (PPFP) occurs in 36% of human follicular thyroid carcinoma (FTC) and is associated with favorable prognosis. To elucidate the function of PPFP in FTC, we analyzed the consequences of PPFP expression in immortalized thyrocytes in vitro and in vivo via xenograft tumorigenesis. While PPFP-expressing cells exhibited oncogenic hallmarks, including increased growth and decreased apoptosis, in vitro, xenograft tumors were initiated but not sustained in vivo. PPFP xenograft tumors exhibited reduced CD31 staining and VEGF expression, suggesting that PPFP modulates neovascularization. Microarray analysis demonstrated increased expression of tissue inhibitor of metalloproteinase (TIMP-3), an inhibitor of angiogenesis, in PPFP cells and tumors, a finding confirmed by quantitative PCR and immunohistochemistry. Immunohistochemical staining of archival human thyroid tumors demonstrates a significant decrease in CD31 staining in all adenomas and carcinomas containing the PAX8/PPARγ rearrangement. Decreased angiogenesis in PPFP-containing tumors is directly correlated with our observations in the xenograft model and provides evidence for the first time that PPFP may impact FTC tumorigenesis by modulating angiogenesis in vivo.

ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice.

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1-2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials.

Most multifocal papillary thyroid carcinomas acquire genetic and morphotype diversity through subclonal evolution following the intra-glandular spread of the initial neoplastic clone.

Papillary thyroid carcinoma (PTC) is frequently multifocal (mPTC), with synchronous tumour foci often showing varied morphology. The genetic mechanisms underlying the development of multiple and histologically diverse tumour foci remain uncertain. Different tumour foci might develop either through intrathyroidal dissemination of a single malignant clone, with morphotype differentiation occurring as a result of subclonal progression, or they may stem from independent transformational events involving multiple progenitor clones. To determine the clonal derivation of multiple tumour foci and to map their clonal relationships and genetic progression in mPTC, we evaluated genome-wide allelic imbalances (AI) and BRAF V600E mutation status in 55 synchronous tumour foci from 18 mPTC patients. For apparently monoclonal tumours, we calculated the probabilities of monoclonal derivation and used phylogenetic analysis to model clonal evolution. Genome-wide allelotyping and BRAF mutation analysis showed genetic alterations consistent with monoclonal origin in 83% of cases, mostly with evidence of subclonal evolution. BRAF V600E mutations were early events during clonal evolution of most, but not all cases. MPTC with morphologically diverse tumour foci also arose through monoclonal derivation in 75% of cases, demonstrating that morphotype-determining genetic changes can be acquired during clonal diversification, subsequent to the spread of the original malignant progenitor clone. In 17% of patients, discordant AI or BRAF V600E profiles implied that mPTCs can occasionally develop from distinct transformation events. This study suggests that mPTC originates usually from neoplastic transformation and subsequent intrathyroidal spread of a single malignant progenitor clone. Clonal progression and morphotype differentiation occur through progressive acquisition of genetic alterations subsequent to the initial intra-glandular spread. In monoclonal BRAF V600E-positive mPTCs, BRAF V600E is not always present in all tumour foci, indicating that other tumour-genetic factors in the primary progenitor clone can also trigger PTC neoplastic transformation.

Remnant lipoproteins induce endothelial plasminogen activator inhibitor-1.

Remnant lipoproteins (RLPs) accumulate in type III hyperlipoproteinemia, a condition associated with significant cardiovascular morbidity. The effect of RLPs on fibrinolysis is unknown. Our aim was to study the effect of RLPs on endothelial expression of plasminogen activator inhibitor-1 (PAI-1). After 24-h culture of human aortic endothelial cells with RLPs at concentrations of 0 (control), 0.038, or 0.076 mg triglyceride/mL, postculture PAI-1 antigen concentrations were: 870 +/- 80, 1963 +/- 183 (P = 0.005), and 3551 +/- 177 ng/mL (P < 0.001), respectively. Furthermore, after 24-h incubation of endothelial cells with RLPs (0 or 0.076 mg triglyceride/mL), PAI-1 activity increased from 0.667 +/- 0.144 to 1.268 +/- 0.198 U/mL, respectively (P = 0.008) and endothelial PAI-1 mRNA increased to 2.7 +/- 0.66 that of control (P = 0.048). In conclusion, RLPs from patients with type III hyperlipoproteinemia induce endothelial cell PAI-1 expression, which may contribute to a prothrombotic state.

DNA binding of TEA/ATTS domain factors is regulated by protein kinase C phosphorylation in human choriocarcinoma cells.

Transcription enhancer factor 1 (TEF-1) controls the expression of a diverse set of genes. Previous studies implicated protein kinase C (PKC)-mediated signal transduction in modulating TEF function. We demonstrate that in human choriocarcinoma BeWo cells, the PKC activator 12-O-tetradecanoyl phorbol 13-acetate and PKC inhibitor bisindolylmaleimide reciprocally down- and up-regulate, respectively, TEF-mediated GGAATG core enhancer activity. In vitro TEF-1 phosphorylation with several PKC isozymes and phosphoamino acid analysis confirmed that TEF-1 is a potential PKC substrate. TEF-1.DNA complexes formed by BeWo nuclear extracts are supershifted by phosphoserine- and phosphothreonine- but not phosphotyrosine-specific antibodies, indicating that TEF-1 is phosphorylated in vivo at serine and threonine residues. The TEF-1 phosphorylation domain was localized to the third alpha-helix of the DNA binding domain and adjacent hinge region by phosphopeptide analysis. TEF-1 phosphorylation significantly reduced its DNA binding activity both in vitro and in vivo, providing a possible mechanism for the inhibitory action of PKC. Finally, BeWo cells contained abundant levels of gamma and delta PKC isoforms, and their overexpression resulted in even greater inhibition of GGAATG core enhancer activity after 12-O-tetradecanoyl phorbol 13-acetate treatment. These data strongly suggest that PKC-mediated phosphorylation is a key factor controlling TEF function.

S20G mutant amylin exhibits increased in vitro amyloidogenicity and increased intracellular cytotoxicity compared to wild-type amylin.

Human amylin, a major constituent of pancreatic amyloid deposits, may be a pathogenetic factor for noninsulin-dependent diabetes mellitus (NIDDM). We demonstrated that the human amylin S20G gene mutation (S20G) was associated with a history of early onset, more severe type of NIDDM, linking the amylin gene to this disease. Also, we demonstrated that expression of human wild-type (WT) amylin in COS-1 cells leads to intracellular amyloidogenesis and induction of apoptosis, suggesting a possible mechanism for disease induction. Therefore we compared the abilities of S20G and WT amylin to induce apoptosis in transfected COS-1 cells and form amyloid in vitro. We transfected the rat (RAT), mutated human (MUT), WT, and S20G amylin genes into COS-1 cells and measured apoptosis using fluorescent-activated cell sorting analysis at 48, 72, and 96 hours. At 96 hours apoptosis increased significantly (P < 0.01) in cells transfected with WT and S20G over RAT or MUT (WT, 19%; S20G, 25%; RAT, 13%; and MUT, 12%) and the difference between WT and S20G was significant (P < 0.05). Synthetic WT and S20G monomeric peptides were used to generate amyloid fibrils in vitro as measured by the thioflavin T binding assay. The S20G amylin formed approximately twofold more amyloid at a rate approximately threefold higher than WT. Electron micrography indicated that the in vitro amyloid generated by WT and S20G amylins were morphologically indistinguishable. The results suggest that increased cytotoxicity by S20G is because of increased amyloidogenicity, which may be a causative factor in the early development of NIDDM, possibly through loss of ss cell mass.

Cooperative binding of TEF-1 to repeated GGAATG-related consensus elements with restricted spatial separation and orientation.

The human transcriptional enhancer factor (TEF) family includes TEF-1, TEF-3, TEF-4, and TEF-5. The TEFs share a highly conserved 68-amino acid TEA/ATTS DNA-binding domain, which binds to SV40 GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and muscle-specific M-CAT (GGTATG) enhansons. We determined the optimal DNA-binding consensus sequence for TEF-1. Using a purified GST-TEF-1 fusion protein and a random pool of synthetic oligonucleotides, 31 independent clones were obtained after six rounds of binding site selection. DNA sequences analysis revealed that 16 clones contained direct repeats with a 3-bp spacer (DR3), and 15 clones contained a single binding site. The predominate consensus half-site was GGAATG (67%), and the other elements were of the form G(A)GA(T/C)ATG. The TEF-1 bound to the DR3 as a dimer in a cooperative manner. Cooperative binding was dependent on the spacing and orientation of the half-sites and was inhibited by deoxycholate treatment, providing evidence that protein-protein interactions were involved. The data suggest that TEF dimerization is important for its ability to modulate gene transcription.

eNOS gene transfer to vascular smooth muscle cells inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis.

OBJECTIVE: Smooth muscle cell (SMC) proliferation is a critical component of vascular diseases such as atherosclerosis and restenosis. Nitric oxide (NO) donors and gene transfer of endothelial nitric oxide synthase (eNOS) have been shown to inhibit SMC proliferation. NO may cause this effect by delaying cell cycle progression and/or induction of apoptosis. The aim of the current study was to examine the mechanism of eNOS-mediated inhibition of SMC proliferation. In addition, the effect of eNOS expression in vascular SMCs on the expression of the cyclin dependent kinase inhibitors, p27 and p21 was examined. METHODS: SMCs were transduced with an adenoviral vector encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta Gal) at a multiplicity of infection of 100. Non-transduced cells served as additional controls. Transgene expression was sought by NADPH diaphorase staining, immunohistochemistry and Western Blotting. Functionality of the recombinant protein was assessed by measurement of cGMP. Cell cycle analysis was performed by flow cytometry and p27 and p21 expression were studied by western blot analysis. Apoptosis was sought by Annexin V staining and DNA laddering. RESULTS: eNOS expression was detected in transduced SMCs. cGMP levels were increased in eNOS-transduced compared to control cells. Expression of eNOS in SMCs resulted in a delay in cell cycle progression and upregulation of p27 and p21. There was no increase in apoptosis detected in eNOS transduced cells after 24 or 72 h. CONCLUSION: eNOS gene transfer to vascular SMCs inhibits cell proliferation via upregulation of p27 and p21 resulting in a delay in cell cycle progression.

FHIT and TSG101 in thyroid tumours: aberrant transcripts reflect rare abnormal RNA processing events of uncertain pathogenetic or clinical significance.

OBJECTIVE: The chromosomal regions containing the two putative tumour suppressors, fragile histidine triad gene (FHIT) and tumour suppressor gene 101 (TSG101), are deleted frequently in thyroid tumours. We therefore analysed FHIT and TSG101 transcripts in a group of advanced thyroid tumours to establish their role in thyroid tumorigenesis. DESIGN: Retrospective analysis of FHIT and TSG101 mRNA transcripts and genomic DNA from cryo-preserved thyroid tumours. TP53, previously shown at the genomic level not to be mutated in this cohort of tumours, served as a control. PATIENTS: We analysed nine follicular thyroid carcinomas (FTC), six papillary thyroid carcinomas and six follicular adenomas (FA) and histologically normal thyroid tissue from four of the FA patients. MEASUREMENTS: Single stage and nested reverse transcription polymerase chain reaction (RT-PCR) products of FHIT, TSG101, and TP53 were analysed by agarose or polyacrylamide gel electrophoresis and sequenced. Genomic DNA was also analysed by polymerase chain reaction and sequencing (FHIT) or by Southern blotting (TSG101). Clinical data were correlated with the results of the mutation analysis. RESULTS: Truncated FHIT transcripts were observed frequently alongside full length transcripts with nested RT-PCR, most often in FTC, while single stage RT-PCR revealed only normal length transcripts in all tumours. Similar results were obtained for TP53, while abnormal TSG101 transcripts were detectable by single stage RT-PCR. Sequence analysis of the truncated FHIT and TSG101 transcripts revealed mainly exon skipping and alternate RNA processing events. Only a single point mutation (of TSG101) was found. Southern blotting for the TSG101 gene, and PCR amplification and sequencing of the FHIT gene showed no evidence of genomic abnormalities in either case, and there was no evidence of splice site mutations in the FHIT gene, suggesting that the truncated transcripts result from altered RNA processing. There was no relationship between tumour stage, grade or survival and the presence of FHIT or TSG101 abnormalities. CONCLUSIONS: Truncated FHIT and TSG101 transcripts in thyroid tumours reflect alternate mRNA splicing events, rather than genomic deletions. Such abnormal RNA processing seems to be common and widespread in thyroid neoplasms, as similar results were obtained by analysis of transcripts of TP53, which we had previously shown not to be mutated in these specimens. Although a pathogenetic role for these aberrant transcripts remains possible, no correlation was found with stage, histological grade or outcome in this small group of advanced thyroid malignancies. Relaxation of mRNA splice control appears to be a feature of follicular cell-derived thyroid neoplasms.

Novel human TEF-1 isoforms exhibit altered DNA binding and functional properties.

The transcriptional enhancer factor-1 (TEF-1) is a member of the TEA/ATTS domain family. TEF-1 binds to GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and M-CAT (GGTATG) response elements and is involved in the transactivation of a variety of genes, including the SV40 large T antigen, mammalian muscle-specific genes, and human chorionic somatomammotropin genes. Also, TEF-1 acts as a transcriptional repressor in placental cells, possibly through interaction with the TATA binding protein (TBP), preventing TBP binding to the TATA box. Here we describe the cloning, tissue-specific expression pattern, and functional characterization of two novel TEF-1 isoforms, TEF-1beta and TEF-1gamma. These isoforms most likely arise from alternative splicing of mRNA transcribed from a single gene and involve substitutions and/or insertions in a region immediately following the DNA binding domain. TEF-1beta appears to be widely distributed like the prototypic TEF-1, designated TEF-1alpha, whereas TEF-1gamma exhibits a narrower tissue-specific expression pattern that includes pancreas, kidney, and skeletal and heart muscle. The relatively limited sequence alterations among these isoforms cause significant changes in their DNA binding and transcriptional activities. TEF-1beta and TEF-1gamma bind to GT-IIC sequences with higher affinity and repress hCS promoter more efficiently than TEF-1alpha. These results suggest that each TEF-1 isoform may play unique regulatory roles in various tissues.

Leptin responses to overfeeding: relationship with body fat and nonexercise activity thermogenesis.

Administration of leptin to rodents results in weight loss through decreased food intake and increased energy expenditure that occurs in part through increased spontaneous activity. In humans, low levels of spontaneous physical activity and below normal plasma leptin concentrations predict subsequent excess weight gain. We recently found that failure to increase nonexercise activity thermogenesis (NEAT) with overfeeding results in greater fat gain in humans, and subsequently evaluated whether changes in leptin are related to NEAT activation. We measured plasma leptin concentrations and adipose tissue leptin messenger ribonucleic acid together with the components of energy expenditure in 16 nonobese humans before and after overfeeding to assess the relationship between leptin responses to overfeeding and the changes in NEAT. Adipocyte leptin expression was up-regulated with overfeeding, and leptin concentrations increased. Leptin concentrations correlated with body fat before and after overfeeding. Changes in leptin with overfeeding were strongly related to changes in body fat, but not to changes in NEAT. Changes in NEAT correlated inversely with fat gain. It is, therefore, unlikely that leptin mediates activation of NEAT with overfeeding in nonobese humans; rather, leptin directly reflects body fat mass and fat mass gain.

Leptin and leptin receptor expression in normal and neoplastic human pituitary: evidence of a regulatory role for leptin on pituitary cell proliferation.

Leptin is a circulating hormone secreted by adipose and a few other tissues. The leptin receptor consists of a single transmembrane-spanning polypeptide that is present as a long physiologically important form as well as in several short isoforms. Recent studies have suggested that the anterior pituitary may have a role in the regulatory effects of leptin in animal models. To test this possibility in human pituitaries, we examined the expression of leptin and OB-R in normal and neoplastic pituitaries, and the possible functions of leptin in the pituitary were also analyzed. Leptin was present in 20-25% of anterior pituitary cells and was expressed in most normal anterior pituitary cells, including ACTH (70% of ACTH cells), GH (21%), FSH (33%), LH (29%), TSH (32%), and folliculo-stellate cells (64%), but was colocalized with very few PRL cells (3%), as detected by double labeling immunohistochemistry with two different antileptin antibodies. In addition, leptin expression was detected by RT-PCR in some pituitary tumors, including ACTH (three of four), GH (one of four), null cells (two of four), and gonadotroph (one of four) tumors as well as in normal pituitary. Immunohistochemical staining showed greater immunoreactivity for leptin in normal pituitaries compared to adenomas. Treatment of an immortalized cultured anterior pituitary cell line, HP75, with leptin stimulated pancreastatin secretion in vitro. Leptin also inhibited cell growth in the human HP75 and in the rat pituitary GH3 cell lines. Both long (OB-Rb) and common (OB-Ra) forms of the leptin receptor messenger ribonucleic acid and leptin receptor protein were expressed in normal and neoplastic anterior pituitary cells. These findings show for the first time that leptin is expressed by most human anterior pituitary cell types and that there is decreased leptin protein immunoreactivity in pituitary adenomas compared to that in normal pituitary tissues. We also show that OB-Rb is widely expressed by normal and neoplastic anterior pituitary cells, implicating an autocrine/paracrine loop in the production and regulation of leptin in the pituitary.

Human placental TEF-5 transactivates the human chorionic somatomammotropin gene enhancer.

Human chorionic somatomammotropin (hCS) gene expression in the placenta is controlled by an enhancer (CSEn) containing SV40-related GT-IIC and SphI/SphII enhansons. These enhancers are controlled by members of the transcription enhancer factor-1 (TEF-1) family. Recently TEF-5, whose mRNA is abundant in placenta, was shown to bind cooperatively to a unique, tandemly repeated element in CSEn2, suggesting that TEF-5 regulates CSEn activity. However, expression of TEF-5 using a cDNA lacking the 5'-untranslated region and containing a modified translation initiation site was not accompanied by CSEn activation. Using nested, degenerate PCR primers corresponding to conserved TEF domains, several novel TEF-1-related cDNAs have been cloned from a human placental cDNA library. The open reading frame of one 3033-bp clone was identical to TEF-5 and contained 300- and 1423-bp 5'- and 3'-untranslated regions, respectively. The in vitro generated approximately 53-kDa TEF-5 polypeptide binds specifically to GT-IIC and SphI/SphII oligonucleotides. Overexpression of TEF-5 in BeWo cells using the intact 3033-bp cDNA transactivates the hCS and SV40 enhancers and artificial enhancers comprised of tandemly repeated GT-IIC enhansons, but not OCT enhansons. The data demonstrate that TEF-5 is a transactivator that is likely involved in the transactivation of CSEn enhancer function. Further, the data suggest that elements within the untranslated regions, initiation site, or both control TEF-5 expression in ways that influence its transactivation function.

Intracellular amyloidogenesis by human islet amyloid polypeptide induces apoptosis in COS-1 cells.

Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet beta cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating beta cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609-616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways.

Role of nonexercise activity thermogenesis in resistance to fat gain in humans.

Humans show considerable interindividual variation in susceptibility to weight gain in response to overeating. The physiological basis of this variation was investigated by measuring changes in energy storage and expenditure in 16 nonobese volunteers who were fed 1000 kilocalories per day in excess of weight-maintenance requirements for 8 weeks. Two-thirds of the increases in total daily energy expenditure was due to increased nonexercise activity thermogenesis (NEAT), which is associated with fidgeting, maintenance of posture, and other physical activities of daily life. Changes in NEAT accounted for the 10-fold differences in fat storage that occurred and directly predicted resistance to fat gain with overfeeding (correlation coefficient = 0.77, probability < 0.001). These results suggest that as humans overeat, activation of NEAT dissipates excess energy to preserve leanness and that failure to activate NEAT may result in ready fat gain.