IL1B represses gastrin promoter by inducing S536 phosphorylation of NFkB p65.

<p>(A) Effect of NFkB p65 on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with 0.2µg of NFkB p65 (pWTp65NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10ng/ml of IL1B for two hr and then harvested for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (B and C) Effect of NFkB p65 mutants on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with (B) either NFkB p65 (pWTp65NFkB) or the lysine NFkB p65 mutants (pK310R p65 NFkB and pK221R p65 NFkB) and (C) with either NFkB p65 (pWTp65NFkB) or with the serine NFkB p65 mutants (pS536A p65 NFkB, pS529A p65 NFkB and pS276A p65 NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10 ng/ml of IL1B for two hr and then harvested for luciferase assay. 0.2µg of wild type or mutant NFkB p65 were used for transfection. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (D) Analysis of IL1B induced phosphorylation of NFkB p65 at ser536 residue. AGS cells were treated with varying concentrations of recombinant IL1B protein for two hr and then lysed and immunoblotted with anti p-p65NFkB (S536) and anti α-tubulin antibodies. A representative blot is shown. The star in panel C indicates that the repression of transcription of pGAS-Luc with pWTp65NFkB is significantly alleviated when p65S536ANFkB was used both in presence and absence of IL1B.NS indicates that the differences between the repressive activity of pWTp65NFkB and that of the mutant clones on gastrin promoter are not significant.</p

IL1B-induced TAK1 activates NFkB to inhibit gastrin expression.

<p>(A) Dose dependent activation of MyD88 by IL1B in AGS cells. Total RNA was extracted from AGS cells treated with increasing concentration of recombinant IL1B (0, 5, 10ng/ml) for 10 min. Real time PCR analysis for MyD88 was performed in those RNA samples. β-actin was taken as the endogenous control. The graph represents the mean of relative quantification measured from three different experiments +/- SD. (B) Analysis of IL1B induced phosphorylation of MAP3K TAK1. AGS cells were treated with 10ng/ml recombinant IL1B protein for 0, 15 and 30 min and then lysed for western blot analysis with p-TAK1, TAK1 and β–actin antibodies. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (C) Dose dependent activation of NFkB p50 and p65 by TAK1. AGS cells were co-transfected in a dose dependent manner with pCMVTAK1 along with its activator pCMVTAB1 and western blot analysis was done for NFkB p50 and p65 respectively. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (D and E) Effect of TAK1/TAB1on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) and either with (D) varying concentrations of pCMVTAK1 along with 0.2 µg of pCMVTAB1 or (E) varying concentrations of pCMVTAB1 along with 0.5 µg of pCMVTAK1.The IL1B (10ng/ml) treatment as control has been included in panel E. Cells were harvested after 48 hr of transfection for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (F) Knock down of TAK1 in IL1B-treated AGS cells releases gastrin repression. AGS cells were first transfected with either TAK1 siRNA (80 nM) or control siRNA(80 nM). After twenty four hr these cells were transfected with 0.5 µg of pGAS-Luc.Forty six hr post pGAS-Luc transfection, cells were treated with IL1B (10ng/ml) for two hr and subsequently harvested. Control experiments with only pGAS-Luc transfected and IL1B treated pGAS-Luc transfected AGS cells are also shown. (G) A cartoon showing that IL1B induces NFkB via MyD88/TAK1 to regulate gastrin expression. Stars in panel C indicate statistical significance of the observations.</p

Modulation of p300 and HDAC1 alters endogenous gastin expression in AGS cells.

<p>(A) Knock down of p300 down regulates basal expression of endogenous gastrin. AGS cells were incubated with either scrambled control siRNA or with siRNA against p300 for 48 hr. Cell lysates were prepared and protein estimation was done. Equal amount of total protein was used for ELISA of Gastrin. (B) HDAC1 functionally attenuates gastrin expression. AGS cells were treated with either IL1B or IL1B along with TSA or left untreated. Gastrin ELISA was performed with equal amount of protein lysates prepared from these cells. (C) Gastrin expression is modulated by a stimulus specific alteration between NFkB-p300 and NFkB-HDAC1 recruitment (Upper panel). NFkB basally activates gastrin expression with p300 as a co-activator (Lower panel). In presence of IL1B there is an increased recruitment of HDAC1 and divestment of p300 that result in inhibition of gastrin transcription.</p

NFkB hetero-dimerize on the gastrin promoter and associate with either p300 or HDAC1 in a stimulus specific manner.

<p>(A) Direct binding of NFkB heterodimers to gastrin promoter. EMSA was performed with nuclear extracts prepared from IL1B treated (10ng/ml for 2hr.) AGS cells. Supershift assay was performed by incubating the nuclear lysates first with either NFkB p50 or p65 antibody for 30 min and then with radiolabelled gastrin EMSA probe. The chase was performed using corresponding unlabelled oligo probes. NE stands for Nuclear Extract. (B) Gastrin promoter occupancy by NFkB heterodimers and other co-factors. ChIP qPCR analysis was done with anti p50, p65, HDAC1 and p300 antibodies in IL1B (10ng/ml) treated and untreated AGS cells. (C) NFkB associates with HDAC1 and NCoR on the gastrin promoter to bring about histone methylation. ChIP qPCR analysis was done with anti NFkB p65, anti-H3K9Me3 and anti-NCoR in IL1B (10ng/ml) treated and untreated AGS cells.</p

Differential transmission of the molecular signature of RBSP3, LIMD1 and CDC25A in basal/ parabasal versus spinous of normal epithelium during head and neck tumorigenesis: A mechanistic study

<div><p>Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.</p></div

Deletion mapping of 240bp human gastrin promoter for IL1B responsive elements.

<p>(A) Schematic diagram of serial deletion of putative gastrin promoter cloned upstream of luciferase reporter gene. The boxes and circle represent different transcription factor binding sites within the 240 bp gastrin promoter. The line diagrams depict the different deletion clones used for the study. (B) The fold inhibition in luciferase activity of different deletion constructs of the gastrin promoter in AGS cells in presence and absence of recombinant IL1B (10 ng/ml). AGS cells were transfected with respective gastrin promoter deletion constructs. Forty-six hours after transfection, cells were either treated or left untreated with 10 ng/ml of IL1B for two hours and then harvested. Luciferase activity was measured and the average fold differences between the treated and untreated sets were plotted from three different experiments.</p

Dose dependent effect of IL1B upon Smad7 expression.

<p>(A) Quantitative analysis of IL1B induced <i>Smad7</i> mRNA expression. RNA was extracted from AGS cells treated with variable concentration of recombinant IL1B (0, 2.5, 5, 10 ng/ml) for two hours. Real time PCR analysis for <i>Smad</i> 7 was performed from cDNA prepared from those samples. Beta-actin was taken as the endogenous control. The graph represents the mean of relative quantification measured from three different experiments +_{_} SD. (B) Analysis of IL1B induced Smad 7 protein expression. AGS cells were treated with increasing concentration of recombinant IL1B protein for two hours and then lysed and immunoblotted with Smad7 antibody. The bands were scanned by Image J software and normalized band intensity of Smad7 from three different experiments +_{_} SD was plotted as a histogram. A representative blot is shown.</p

Effect of IL1B promoter polymorphism on Smad 7 expression.

<p>(A) <i>IL1B</i> promoter polymorphisms differentially regulate Smad 7 protein expression in AGS cells. AGS cells were transfected with the IL1B promoter-driven luciferase construct having either the C or T allele at the −31 position (the constructs are represented as the line diagram) and cells were harvested after 48 hours for immuno blot with Smad7 antibody. The band intensity was scanned and normalized with Beta- actin. The normalized band intensity of three different experiments was plotted. (B) <i>IL1B</i> promoter polymorphisms differentially downregulate gastrin via Smad 7, independent of NFkB pathaway. AGS cells were co-transfected with the <i>IL1B</i> promoter-driven luciferase construct having either the C or T allele at the −31 position along with pGas-Luc and after 24 hours of transfection cells were either treated with NBD or left untreated. Beta-Gal plasmid was used as transfection control. Cells were harvested after 48 hours of transfection for luciferase assay. Protein in each case was normalized by Bradford assay. The normalized mean RLU/µg protein +_ SD of three different experiments was plotted. The same lysates were also subjected to immunoblot analysis with Smad 7 and NFkB antibody. Beta-actin was used as input control.</p

Overall concordance obtained between different experiments undertaken in invasion tumours.

<p>Abbreviations: EXPR: Expression (IHC); METH: Methylation (promoter); DEL: Deletion; OVERALL: overall alterations (Deletion+ Methylation);DYS: Dysplasia; HNSCC: Head and neck squamous cell carcinoma; H: High; M: Moderate; L: Low; ND: Not done; NOR: Normal; TUM: Tumor; B: Basal; P: Parabasal; S: Spinous. P value represents level of significance.</p

Promoter methylation analysis.

<p>Promoter methylation of the genes in normal epithelium (basal/ parabasal and spinous layers), dysplastic zone of epithelium and HNSCC. U: Unmethylated; M: Methylated; N: Normal; T: Tumor; B: Basal layer; P: Parabasal layer; S: Spinous layers; L: pUC19/ HpaII molecular weight ladder. a. Representative images depicting step- wise separation of basal/parabasal and spinous layers in normal epithelium.i. Hematoxylin and eosin stained parallel normal section. ii. Unstained parallel section. iii. Laser line (arrow star) demarcating basal/ parabasal and spinous layers. iv. Remaining basal/ parabasal layers after separation of the spinous layers. b. Representative hematoxylin and eosin stained sections for separation of dysplastic and non- dysplastic zone in mild/ moderate and severe dysplasia. i. Mild dysplasia; ii. Moderate dysplasia; iii. Severe dysplasia. Red line represents the line of application of laser beam for separating dysplastic and non- dysplastic zones. c. Representation agarose gel image showing methylation by Methylation Specific Restriction Analysis (MSRA). U: Undigested; H: HpaII digested. d. Representation agarose gel image showing methylation by Methylation Specific PCR (MSP). U: Unmethylated; M: Methylated. e. Histograms depicting percentage of methylation obtained for i. RBSP3; ii. LIMD1. f. Histograms depicting percentage of methylation in etiological groups in i. RBSP3; ii. LIMD1. p value (Fisher’s exact) represents the level of significance during univariate comparison. # Represents sample number; B: Basal; P: Parabasal: S: Spinous: T: Tumor; N: Normal. Group 1: HPV-TOB-; Group 2: HPV+TOB-; Group 3: HPV-TOB+; Group 3: HPV+TOB+.</p