[Clinical characteristics of "classical" and "non-classical" paraneoplastic neurological syndrome].

Objective: To explore the clinical features of classical and non-classical paraneoplastic neurological syndrome (PNS). Methods: From 2015 to 2020, 48 cases of definite PNS admitted to the First Affiliated Hospital of University of Science and Technology of China were retrospectively collected, and classification, clinical characteristics, onconeural antibodies and primary tumors were analyzed. The included cases were divided into classical and non-classical groups according to Graus criteria, and the differences of clinical characteristics, onconeural antibodies, combined tumors, time of diagnosis and mortality were compared between the two groups. Results: Among the 48 confirmed patients, 21 (43.8%) were positive for well-characterized onconeural antibodies. There were 28 cases (58.3%) and 20 cases (41.7%) in classic and non-classical PNS groups, respectively. No significant differences of age, sex, clinical involvement site, characteristic positive antibody type, tumor diagnosis rate and follow-up mortality were found between the two groups (all P>0.05). The time of diagnosis in the non-classical PNS group was 3.0 (2.0, 6.5) months, which was significantly longer than that in the classical PNS group 1.0(0.6, 3.0) months (P<0.05). Meanwhile, the combination rate of non-characteristic antibodies in the classical PNS group (10 cases, 35.7%) was significantly higher than that in the non-classical PNS group (1 case, 5.0%) (P=0.016). During the follow-up, 39 patients (81.3%) with tumor were confirmed, and 29 patients (60.4%) were diagnosed with PNS before the tumor was found. Conclusions: The"non-classical"PNSs are common in clinical settings. Diagnosis may be delayed due to the nonclassical symptoms of the patients. When patients have clinical symptoms related to PNS, onconeural antibodies should be detected and the relevant tumors should also be screened. Patients have positive antibodies but with no tumors should be closely followed up for more than 5 years.

Oncogenic activation of JAK3-STAT signaling confers clinical sensitivity to PRN371, a novel selective and potent JAK3 inhibitor, in natural killer/T-cell lymphoma.

Aberrant activation of the JAK3-STAT signaling pathway is a characteristic feature of many hematological malignancies. In particular, hyperactivity of this cascade has been observed in natural killer/T-cell lymphoma (NKTL) cases. Although the first-in-class JAK3 inhibitor tofacitinib blocks JAK3 activity in NKTL both in vitro and in vivo, its clinical utilization in cancer therapy has been limited by the pan-JAK inhibition activity. To improve the therapeutic efficacy of JAK3 inhibition in NKTL, we have developed a highly selective and durable JAK3 inhibitor PRN371 that potently inhibits JAK3 activity over the other JAK family members JAK1, JAK2, and TYK2. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of the JAK3-STAT signaling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3 compared to tofacitinib in vitro, leading to significant tumor growth inhibition in a NKTL xenograft model harboring JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL.

The miR-181d-regulated metalloproteinase Adamts1 enzymatically impairs adipogenesis via ECM remodeling.

The extracellular matrix (ECM) maintenance is crucial to the structural integrity of adipocytes and whole adipose tissue formation. However, the potential impact of the ECM on adipocyte lineage commitment is unclear. Herein, we demonstrate that forced expression of matrix-associated metalloproteinase Adamts1 (a disintegrin and metalloproteinase with thrombospondin motifs 1), which we show is targeted by microRNA-181d (miR-181d) during BMP4-induced adipocytic lineage commitment, markedly impairs adipocyte commitment. Conversely, siRNA-induced inhibition of Adamts1 promotes adipocyte commitment. Adamst1 metalloprotease activity is required for this inhibition and is determined to function via remodeling ECM components followed by activating FAK-ERK signaling pathway during the commitment process. Furthermore, ablation of Adamts1 in adipose tissue increases adipose tissue mass, reduces insulin sensitivity, and disrupts lipid homeostasis. This finding is consistent with Adamts1 decreased expression in the adipose tissue of obese mice and an inverse correlation of Adamts1 expression with body mass index in humans. Collectively, our results indicate that Adamts1 acts as an ECM 'modifier', with miR-181d-induced downregulation, that regulates adipocyte lineage commitment and obesity.

ERK1/2/p53 and NF-κB dependent-PUMA activation involves in doxorubicin-induced cardiomyocyte apoptosis.

OBJECTIVE: Numerous studies have demonstrated that Doxorubicin (DOX) induces cardiomyocyte apoptosis, which is associated with DOX-induced acute and chronic cardiotoxicity. DOX activated ERP1/2 and NF-KB signals has been linked to DOX-induced apoptosis and cardiotoxicity. However, the underlying mechanisms responsible for DOX-induced apoptosis have not been completely elucidated. In this study, we determine whether both ERK1/2/p53-dependent and NF-κB dependent-PUMA activation was related to DOX-induced apoptosis in H9c2 cells. MATERIALS AND METHODS: H9c2 cells were treated with DOX (1 µM) for 2-48 hours. To explore the effect of ERK1/2, NF-KB, P53 and PUMA on DOX-induced apoptosis in H9c2 cells, H9c2 cells were transfected with PUMA siRNA or p65 siRNA, or treated with PFT-α (a chemical inhibitor of p53), or PD98059 (ERK inhibitor) before DOX treatment. MTT, Flow cytometry, TUNEL, Western blot and EMSA assay was used to detect cell survival, apoptosis, protein expression and NF-KB activity. RESULTS: DOX induced apoptosis and inhibited growth of H9c2 cells in a time-dependent manner. DOX activated ERK1/2, NF-KB, p53 and PUMA. Knockdown of PUMA completely blocked DOX-induced cell apoptosis and survival inhibition. Knockdown of NF-KB or ERK1/2 alone could partly block DOX-induced PUMA upregulation and cell apoptosis. However, knockdown of NF-KB and ERK1/2 together completely blocked DOX-induced cell apoptosis and PUMA upregulation. In addition, knockdown of ERK1/2 blocked p53-dependent PUMA upregulation. CONCLUSIONS: DOX induced apoptosis and inhibited growth of H9c2 cells by activation of ERK1/2/p53 and NF-κB dependent-PUMA signaling pathway.

Effect of SB203580 on pathologic change of pancreatic tissue and expression of TNF-α and IL-1ß in rats with severe acute pancreatitis.

OBJECTIVE: This study aimed to investigate the effect of SB203580 which is the inhibitor of p38 mitogen-activated protein kinase on pathologic change of pancreatic tissue and expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1b) in rats with severe acute pancreatitis (SAP), Hematoxylin-eosin (HE) staining and immunohistochemistry were carried out in the present study. METHODS: Forty-five male Wistar rats were divided into three groups: the SAP group (N=15), SB203580-treated group (SB group) (N=15), and the control group (N=15). Severe acute pancreatitis was induced by injection of sodium taurocholate into the pancreatic duct. For SB203580-treated group, SB203580 were administered via intraperitoneal injection (10 mg/kg). Serum amylase activity was measured 6, 12 and 24 hours respectively after the operation. The pancreas tissue were stained with HE for histopathological evaluation and the expression of TNF-α and IL-1b in the pancreatic tissue were determined through inferior vena cava. RESULTS: The results show that the level of amylase in SAP group was higher than that in the other groups. Further, the pancreas tissues of SB group rats were observed more mildly edematous, hemorrhagic and with monocytes infiltration. Based on immunohistochemical staining, the expression of TNF-α and IL-1ß in SAP rats were significantly increased than those of the control group. However, those of SB203580-treated group were more significantly reduced than those of SAP group (p < 0.05). CONCLUSIONS: Those data suggest that SB203580, down regulating the expression of pro-inflammatory mediators such as TNF-α and IL-1ß, then through p38 MAPK signaling pathway inhibition, plays an important role in the treatment of SAP.

Effects of dietary copper on growth, digestive, and brush border enzyme activities and antioxidant defense of hepatopancreas and intestine for young grass carp (Ctenopharyngodon idella).

To investigate the effects of dietary copper (Cu) on fish growth, digestive and absorptive enzyme activities, and antioxidant status in the hepatopancreas and intestine, young grass carp (Ctenopharyngodon idella) (282±2.8 g) were fed six diets containing 0.74 (basal diet), 2.26, 3.75, 5.25, 6.70, and 8.33 mg Cu /kg diet for 8 weeks. Results showed that percentage weight gain (PWG) and feed intake were increased with dietary Cu levels up to 3.75 mg/kg diet. In addition, the positive effects of dietary Cu at a level 3.75 or 5.25 mg/kg diet on trypsin, chymotrypsin, and lipase activities in the hepatopancreas and of Na(+), K(+)-ATPase, alkaline phosphatase, creatine kinase, and γ-glutamyl transpeptidase activities in three intestine segments produced significantly (P<0.05) better feed efficiency (FE). However, amylase activity in the hepatopancreas was decreased by dietary Cu levels up to 3.75 mg/kg diet (P<0.05). In addition, dietary Cu at 3.75 or 5.25 mg/kg diet decreased malondialdehyde and protein carbonyl content partly by significantly (P<0.05) increasing the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and glutathione content in the hepatopancreas and intestine. Collectively, dietary Cu improved growth and digestive and absorptive capacity and decreased lipid peroxidation and protein oxidation partly by enhancing antioxidant defense in the hepatopancreas and intestine. The dietary Cu requirement for PWG, plasma ceruloplasmin activity, and FE of young grass carp (282-688 g) were 4.78, 4.95, and 4.70 mg/kg diet, respectively.

Histone demethylase Kdm4b functions as a co-factor of C/EBPß to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes.

CCAAT/enhancer-binding protein (C/EBP) ß is required for both mitotic clonal expansion (MCE) and terminal adipocyte differentiation of 3T3-L1 preadipocytes. Although the role of C/EBPß in terminal adipocyte differentiation is well defined, its mechanism of action during MCE is not. In this report, histone demethylase Kdm4b, as well as cell cycle genes Cdc45l (cell division cycle 45 homolog), Mcm3 (mini-chromosome maintenance complex component 3), Gins1 (GINS complex subunit 1) and Cdc25c (cell division cycle 25 homolog c), were identified as potential C/EBPß target genes during MCE by utilizing promoter-wide chromatin immunoprecipitation (ChIP)-on-chip analysis combined with gene expression microarrays. The expression of Kdm4b is induced during MCE and its induction is dependent on C/EBPß. ChIP, Electrophoretic Mobility Shift Assay (EMSA) and luciferase assay confirmed that the promoter of Kdm4b is bound and activated by C/EBPß. Knockdown of Kdm4b impaired MCE. Furthermore, Kdm4b interacted with C/EBPß and was recruited to the promoters of C/EBPß-regulated cell cycle genes, including Cdc45l, Mcm3, Gins1, and Cdc25c, demethylated H3K9me3 and activated their transcription. These findings suggest a novel feed forward mechanism involving a DNA binding transcription factor (C/EBPß) and a chromatin regulator (Kdm4b) in the regulation of MCE by controlling cell cycle gene expression.

Polymorphism of insulin-like growth factor 1 gene and its association with litter size in Small Tail Han sheep.

The insulin-like growth factor 1 (IGF1) gene was studied as a candidate gene for high prolificacy in sheep. Polymorphisms of 5' regulatory region and all four exons of IGF1 gene were detected in Small Tail Han (n = 277), Hu (n = 58), Texel (n = 48) and Dorset (n = 46) sheep by PCR-RFLP and PCR-SSCP analysis. A microsatellite polymorphic site and a restriction fragment length polymorphism were shown in the 5' regulatory region of IGF1 gene. The ewes with genotype 123/123 bp had 0.81 (P < 0.05) or 1.03 (P < 0.01) lambs more than those with genotype 125/125 bp or 125/127 bp, the ewes with genotype 123/125 bp had 0.46 (P < 0.05) or 0.68 (P < 0.01) lambs more than those with genotype 125/125 bp or 125/127 bp. In addition, there were two mutations (C1511G and A1513G) in 5' regulatory region of IGF1 gene. The ewes with genotype BB or AB had 0.96 (P < 0.05) or 0.38 (P < 0.05) lambs more than those with genotype AA, but there were no significant differences between BB and AB genotypes (P > 0.05) in Small Tail Han sheep. These results preliminarily indicated that these polymorphisms of IGF1 gene could be used in molecular marker-assisted selection for sheep breeding programs.

VSV-MP gene therapy strategy inhibits tumor growth in nude mice model of human lung adenocarcinoma.

Vesicular stomatitis virus (VSV) matrix protein (MP) can induce in vitro apoptosis of tumor cells in the absence of other viral components. Here, the antitumor activity of VSV-MP against lung adenocarcinoma was investigated in vivo. A pVAX-plasmid DNA encoding VSV-MP and control empty vectors (pVAX) were constructed and wrapped-up with liposome. A549 and Spc-A1 human lung adenocarcinoma cells were transfected with liposomal-VSV-MP (Lip-MP) or Lip-pVAX and then examined for cell viability or apoptosis using Hoechst/propidium iodide staining by flow cytometry, and further demonstrated by caspase/poly ADP-ribose polymerase (PARP) cleavage analysis. For the in vivo study, A549 and Spc-A1 lung carcinoma models in nude mice were established and randomly assigned into three groups to receive eight 2-weekly intravenous administrations of medium alone as control, Lip-pVAX or Lip-MP, respectively. Subsequently, Lip-MP significantly reduced tumor growth and prolonged the survival of tumor-bearing mice compared with Lip-pVAX and control agents (P<0.05), with much higher apoptosis index of both in vivo and in vitro tumor cells, respectively (P<0.05). In addition, in vivo antitumoral effect was associated with natural killer-(NK) cell congregation without evidence of toxicity. These observations suggest that systemically delivering Lip-MP has a specific dual antitumor activity in human lung adenocarcinoma by inducing apoptosis and possibly stimulating NK-cell responses, it may provide a clue for developing new therapeutic approaches against human lung adenocarcinoma.

Polymorphisms of caprine POU1F1 gene and their association with litter size in Jining Grey goats.

Seven pairs of primers were designed to amplify 5' promoter region, six exons and partial introns and to detect the polymorphisms of POU1F1 gene in five goat breeds with different prolificacy. The results showed that six mutations were identified in caprine POU1F1 gene including C256T in exon 3, C53T and T123G in intron 3, and G682T (A228S), T723G and C837T in exon 6. The former four mutations were novel SNPs in goat POU1F1 gene. The 53 and 123 loci were in complete linkage disequilibrium in five caprine breeds. Regarding the 256 locus, the Jining Grey goat does with genotype CT had 0.66 kids more than those with genotype CC (P < 0.05), while does with genotype GT had 0.63 (P < 0.05) kids more than those with genotype GG at the 682 locus. The present study preliminarily showed an association between allele T at the 256 and 682 loci of POU1F1 gene and high litter size in Jining Grey goats. Totally 16 haplotypes and 50 genotypes were identified at the above six loci in POU1F1 gene of five goat breeds. Three common haplotypes (hap2, hap3 and hap4) were identified in five goat breeds joined. Four specific haplotypes (hap7, hap9, hap11 and hap13) were detected in Jining Grey goats. The predominant haplotype was hap1 (35.29% and 48.25%) in both Jining Grey and Guizhou White goats, while hap4 (50%) in Boer goats, and hap2 (42.86% and 38.75%) in both Wendeng Dairy and Liaoning Cashmere goats. The most frequent genotypes at six loci in the above five goat breeds were hap1/hap2 (14.38%) and hap1/hap4 (14.38%), hap1/hap2 (38.60%), hap4/hap4 (40.91%), hap2/hap4 (26.53%), hap2/hap5 (20.00%), respectively. The Jining Grey goat does with nine genotypes analyzed of POU1F1 gene showed no obvious difference in litter size.

Polymorphism of 5' regulatory region of ovine FSHR gene and its association with litter size in Small Tail Han sheep.

Single nucleotide polymorphisms of 5' regulatory region of follicle-stimulating hormone receptor (FSHR) gene were detected in two high prolificacy sheep breeds (Small Tail Han and Hu sheep) and two low prolificacy sheep breeds (Corriedale and Chinese Merino sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results indicated that there were three genotypes (AA, AB and BB) detected by primer 1 in Hu sheep while only one genotype (AA) in other three sheep breeds, and frequencies of AA, AB and BB genotypes in Hu sheep were 0.700, 0.225 and 0.075, respectively. There were three genotypes (EE, EF and EG) detected by primer 3 in Small Tail Han sheep while only EE genotype occurred in other three sheep breeds, and frequencies of EE, EF and EG genotypes in Small Tail Han sheep were 0.775, 0.200 and 0.025, respectively. No polymorphism was detected in four sheep breeds by primer 2 and primer 4. The sequencing results showed that there were two nucleotide mutations (g. -681T>C and g. -629C>T) in genotype BB compared with AA for primer 1. As for primer 3, two mutations (g. -197G>A and g. -98T>C) in genotype EF compared with EE and two mutations (g. -200G>A and g. -197G>A) in genotype EG compared with EE. The heterozygous ewes with EG or EF had 0.89 (P < 0.05) or 0.42 (P < 0.05) lambs more than homozygous ewes (EE genotype) in Small Tail Han sheep, respectively, while there was no significant difference on litter size between EG and EF ewes.

GDF9 as a candidate gene for prolificacy of Small Tail Han sheep.

Growth differentiation factor 9 (GDF9) which controls the fecundity of Belclare, Cambridge, Santa Ines, Moghani, Ghezel and Thoka ewes was studied as a candidate gene for the prolificacy of Small Tail Han sheep. According to the sequence of ovine GDF9 gene, six pairs of primers were designed to detect single nucleotide polymorphisms of two exons of GDF9 gene in both high fecundity breed (Small Tail Han sheep) and low fecundity breed (Dorset sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Only the products amplified by primers 2-1 and 2-2 displayed polymorphisms. For primer 2-1, three genotypes (AA, AB and BB) were detected in both sheep breeds. Sequencing revealed one silent mutation (G477A) in exon 2 of GDF9 gene in the BB genotype in comparison with the AA, which was known as G3 mutation of GDF9 gene in Belclare and Cambridge ewes. The relationship of least squares means for litter size was AA > AB > BB in Small Tail Han sheep (P > 0.05). For primer 2-2, two genotypes (CC and CD) were detected in both sheep breeds. Sequencing revealed one novel single nucleotide mutation (G729T) in exon 2 of GDF9 gene in the CD genotype in comparison with the CC, which resulted in an amino acid change (Gln243His). The ewes with mutation heterozygous genotype CD had 0.77 (P < 0.01) lambs more than those with wild type CC in Small Tail Han sheep. These results preliminarily indicated that allele D of GDF9 gene was a potential genetic marker for improving litter size in Small Tail Han sheep.

Role of C/EBP homologous protein (CHOP-10) in the programmed activation of CCAAT/enhancer-binding protein-beta during adipogenesis.

Hormone induction of growth-arrested preadipocytes triggers mitotic clonal expansion followed by expression of CCAAT/enhancer-binding protein (C/EBP)alpha and differentiation into adipocytes. The order of these events is critical because C/EBPalpha is antimitotic and its expression prematurely would block the mitotic clonal expansion required for differentiation. C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, is expressed early in the differentiation program, but lacks DNA-binding activity and fails to localize to centromeres until preadipocytes traverse the G(1)-S checkpoint of mitotic clonal expansion. Evidence is presented that dominant-negative CHOP-10 expressed by growth-arrested preadipocytes transiently sequesters C/EBPbeta by heterodimerization. As preadipocytes reach S phase, CHOP-10 is down-regulated, apparently releasing C/EBPbeta from inhibitory constraint and allowing transactivation of the C/EBPalpha gene. In support of these findings, up-regulation of CHOP-10 with the protease inhibitor N-acetyl-Leu-Leu-norleucinal prevents activation of C/EBPbeta, expression of C/EBPalpha, and adipogenesis.

Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation.

Members of the C/EBP family of transcription factors play essential roles in the adipocyte differentiation program. Treatment of growth-arrested 3T3-L1 preadipocytes with appropriate hormonal agents causes the cells to synchronously reenter the cell cycle and to undergo mitotic clonal expansion. Expression of C/EBPbeta and delta occur early in clonal expansion, later followed by C/EBPalpha (which is anti-mitotic) as the cells exit the cell cycle begin to express adipocyte genes. C/EBPalpha serves as transcriptional activator of many adipocyte genes whose expression produce the adipocyte phenotype. Recent work in this laboratory has focussed on the roles of C/EBPbeta and delta in the differentiation program, in particular the mechanisms by which they activate transcription of the C/EBPalpha gene. Several regulatory elements, both repressive and activating, in proximal promoter of the gene have been identified. The cognate transacting factors that interact with these elements have been characterized and their functions elucidated. These factors have been incorporated into a model for a cascade that leads to transcriptional activation of the C/EBPalpha gene and the terminal steps in the differentiation program.

Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation.

Hormonal induction of 3T3-L1 preadipocytes triggers a cascade of events that initiate differentiation into adipocytes. CCAAT/enhancer-binding proteins beta and delta (C/EBPbeta/delta) are expressed early in the differentiation program, but are not immediately active. After a long lag, C/EBPbeta/delta become competent to bind to the C/EBP regulatory element in the C/EBPalpha gene promoter, C/EBPalpha being a transcriptional activator of numerous adipocyte genes. As C/EBPbeta/delta acquire binding activity, they become localized to centromeres as preadipocytes synchronously enter S phase at the onset of mitotic clonal expansion. Localization to centromeres occurs through C/EBP consensus-binding sites in centromeric satellite DNA. C/EBPalpha, which is antimitotic, becomes centromere-associated much later in the differentiation program as mitotic clonal expansion ceases and the cells become terminally differentiated.

Repressive effect of Sp1 on the C/EBPalpha gene promoter: role in adipocyte differentiation.

Expression of C/EBPalpha is required for differentiation of 3T3-L1 preadipocytes into adipocytes. Previous investigations indicated that transcription of the C/EBPalpha gene is sequentially activated during differentiation, initially by C/EBPbeta and C/EBPdelta and later by C/EBPalpha (autoactivation). These events are mediated by a C/EBP regulatory element in the promoter of the C/EBPalpha gene. This article presents evidence that members of the Sp family, notably Sp1, act repressively on the C/EBPalpha promoter prior to the induction of differentiation. Sp1 was shown to bind to a GC box at the 5' end of the C/EBP regulatory element in the C/EBPalpha promoter and, in so doing, to competitively prevent binding to and transactivation of the promoter by the C/EBPs. One of the differentiation inducers methylisobutylxanthine (a cAMP phosphodiesterase inhibitor) or Forskolin, both of which increase the cellular cAMP level, causes down-regulation of Sp1. This decrease in Sp1 level early in the differentiation program appears to facilitate access of C/EBPbeta and/or C/EBPdelta to the C/EBP regulatory element and, thereby, derepression of the C/EBPalpha gene.

Derepression of the C/EBPalpha gene during adipogenesis: identification of AP-2alpha as a repressor.

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBPalpha) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPalpha gene and a putative transacting factor (C/EBPalpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximately 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2alpha. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2alpha. Both AP-2alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2alpha/CUP, an AP-2alpha1 expression vector, cotransfected with a C/EBPalpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPalpha gene is repressed by AP-2alpha/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein alpha gene.

During adipocyte differentiation, the expression of C/EBPalpha is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program.

Transcriptional repression by human adenovirus E1A N terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis.

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.