Global expression analysis of ECL cells in Mastomys natalensis gastric mucosa identifies alterations in the AP-1 pathway induced by gastrin-mediated transformation

2004 ◽  
Vol 20 (1) ◽  
pp. 131-142 ◽  
Author(s):  
M. Kidd ◽  
T. Hinoue ◽  
G. Eick ◽  
K. D. Lye ◽  
S. M. Mane ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Mucosa ◽  
Western Blot ◽  
Cell Transformation ◽  
Mastomys Natalensis ◽  
Rt Pcr ◽  
Cell Hyperplasia ◽  
Ecl Cells ◽  
Ecl Cell ◽  
Gene Expression Alterations

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8–16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (<20) in hyperplastic and tumor mucosa. The histamine H1 receptor was consistently increased in proliferating mucosa. This gene change was confirmed by Q-RT-PCR. Other genes showing alterations included neural-(chromogranin A and somatostatin), cell-cycle-, and AP-1-associated genes. Immunostaining confirmed alterations in neural markers. Cluster analysis of ECL cell-enriched samples demonstrated that c- fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.

scholarly journals Study on histogenesis of enterochromaffin-like carcinoid in autoimmune atrophic gastritis associated with pernicious anemia

2007 ◽  
Vol 64 (8) ◽  
pp. 543-548 ◽  
Author(s):  
Lana Macukanovic-Golubovic ◽  
Vuka Katic ◽  
Gorana Rancic ◽  
Mladen Milenovic ◽  
Goran Marjanovic ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Pernicious Anemia ◽  
Histopathological Examination ◽  
Serum Levels ◽  
Control Group ◽  
Specific Marker ◽  
Cell Hyperplasia ◽  
Ecl Cells ◽  
G Cell ◽  
Ecl Cell

Background/Aim. Autoimmune atrophic fundic gastritis induces the pernicious anemia (PA), as well as the changes in both epithelium and endocrine cells of gastric mucosa. The most important complications are: achlorhydria, hypergastrinemia, gastric cancer and enterochromaffin-like ( ECL) carcinoid. The aim of this study was to examine ECL carcinoid histogenesis in A-gastritis associated with PA. Methods. During the period from 2000?2006, 65 patients with PA and 30 patients of the control group were examined. Histopathological examination was done in endoscopical biopsies of gastric mucosa fixed in 10% formaldehyde. Paraffin sections were stained with classic hematoxylin-eosin (HE); histochemical AB-PAS (pH 2.5), cytochemical argyrophilic Servier-Munger?s and immunocytochemical PAP methods for G cell identification and chromogranin A antibodies - specific marker for neuroendocrine ECL cells. Both G and ECL cells were counted per 20 fields, of surface 0.0245312 mm2 by a field. Basal gastrin serum levels were also examined by using radioimmunoassay (RIA) method. The obtained results were statisticaly calculated by using Student?s t test. Results. Marked antral G cell hyperplasia associated with corporal ECL hyperplasia was found. ECL cell hyperplasia was of simplex, linear, adenomatoid type to the pattern of intramucous ECL cell carcinoid. An average number of G cells was statistically significant in the patients with PA as compared to the control group (p < 0.05) as well as an average number of ECL cells. Conclusion. We concluded that antral G cell hyperplasia accompanied by gastrinemia induces ECL hyperplasia and ECL corporal carcinoid in A-gastritis and that their histogenesis develops trough simple, linear and adenomatoide hyperplasia. .

Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3078-3078
Author(s):  
Joellen H. H. Lin ◽  
Michael B. Boffa ◽  
Marlys L. Koschinsky
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Mrna Expression ◽  
Fibrin Clot ◽  
Mrna Abundance ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Monocytes And Macrophages ◽  
Fibrinolysis Inhibitor ◽  
Flanking Region

Abstract Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.

Proteomic Signature Predicting Achievement of Very Good Partial Response In Patients with Multiple Myeloma Based On Complementary Label-Free and iTRAQ Quantitative Proteome Analysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1902-1902
Author(s):  
Dominik Dytfeld ◽  
Malathi Kandarpa ◽  
John R Strahler ◽  
Dattatreya Mellacheruvu ◽  
Suchitra Subramani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Western Blot ◽  
Differential Expression ◽  
Proteomic Analysis ◽  
Quantitative Proteomics ◽  
Proteomic Profile ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Rt Pcr

Abstract Abstract 1902 Introduction: Multiple myeloma (MM) remains mostly incurable. Novel therapies have improved response rates, which are now reaching 100%. More importantly, number of recent studies showed that the depth of response, e.g. achievement of at least 90% reduction of the disease (≥VGPR) is associated with longer disease control. Therefore, improving VGPR rates and establishing predictors of VGPR to a given regimen may be an important clinical goal. High throughput quantitative proteomics may offer greater insight into the actual biology of the malignant cell than genome analysis and therefore, may be more useful in the development of personalized therapy. The objective of this study is to establish a proteomic signature predicting achievement of at least VGPR to initial treatment with bortezomib (Velcade®), pegylated liposomal doxorubicin, and dexamethasone (VDD). We previously reported preliminary proteomic profile of malignant plasma cells (PCs) obtained from a set of naïve MM pts enrolled in the VDD trial (Dytfeld et al., ASH 2009). Here we present the results of differential proteomic analysis of MM PCs of all available samples from the frontline VDD study (≥VGPR vs. <VGPR) using two independent and complementary quantitative proteomic platforms. We also compared the proteomic profile with gene expression data. Preliminary validation of the biomarkers of response prediction is presented. Methods: PCs were acquired from pre-treatment bone marrow specimens after obtaining informed consent from patients (pts), and were thereafter enriched with a RosetteSep® negative selection kit. Quantitative proteomic analysis of PCs from 17 naïve pts with MM from the VDD study was performed using iTRAQ approach in 8-plex variant. To increase confidence of analysis, label-free quantitative proteomics (LF) based on spectra counting was conducted on PCs from 12 pts. In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as a reference. The data were analyzed using ProteinPilot™. For LF analysis, proteins were fractionated before trypsin digestion on Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). A database search was carried out using X!Tandem followed by Trans-proteomic Pipeline. At least 1.5-fold difference in expression in both platforms was used as a cut-off value. To correlate proteomics with gene expression of dysregulated proteins of interest, mRNA levels were analyzed by quantitative real time PCR (RT-PCR). Validation of proteomic findings on proteins of interest was performed using Western Blot. Results: We identified a total of 894 proteins in 3 iTRAQ experiments with high confidence (FDR<1%) and 1058 proteins by LF approach. Based on iTRAQ analysis, 20 proteins were found up-regulated in samples from pts with ≥VGPR (8 out of 17 pts) while 14 were down- regulated. Using LF approach, 284 proteins were elevated in the ≥VGPR group (6 out of 12 pts) while 315 proteins were down-regulated. Both iTRAQ and LF methods showed 15 differentially expressed proteins in common and 14 of them showed identical up or down trends. Interestingly, among differentially expressed proteins, there were proteins involved in proteasome activation (PSME1 and TXNL1), protection against oxidative stress (TXN and TXNDC5), glucose and cholesterol metabolism (TP1, APOA1 and ACAT1) and apoptosis (MX1). RT-PCR performed on a subset of genes confirmed the trend in differential expression between pts with ≥VGPR and <VGPR for TXNDC5 and PSME1. No change in mRNA expression levels was observed in TXN, APOA1, TPI1 and MX1while the trend in expression was reversed for ACAT1. Western blot analysis performed to date validated differential expression of PSME1. Conclusions: We present patient-derived proteomic characteristics of MM cells using two independent proteomic platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline VDD study shows differential expression of 34 proteins in pts who achieved ≥VGPR vs. pts with <VGPR. Correlation with gene expression and further validation and functional analysis are in progress. This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Jakubowiak: Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

Differentiation of the Gastric Mucosa I. Role of histamine in control of function and integrity of oxyntic mucosa: understanding gastric physiology through disruption of targeted genes

2006 ◽  
Vol 291 (4) ◽  
pp. G539-G544 ◽  
Author(s):  
Duan Chen ◽  
Takeshi Aihara ◽  
Chun-Mei Zhao ◽  
Rolf Håkanson ◽  
Susumu Okabe
Keyword(s):  
Gastric Mucosa ◽  
Histidine Decarboxylase ◽  
Parietal Cells ◽  
Oxyntic Mucosa ◽  
Mucosal Integrity ◽  
Ecl Cells ◽  
Gastric Physiology ◽  
Ecl Cell ◽  
Insight Into

Many physiological functions of the stomach depend on an intact mucosal integrity; function reflects structure and vice versa. Histamine in the stomach is synthesized by histidine decarboxylase (HDC), stored in enterochromaffin-like (ECL) cells, and released in response to gastrin, acting on CCK2 receptors on the ECL cells. Mobilized ECL cell histamine stimulates histamine H2 receptors on the parietal cells, resulting in acid secretion. The parietal cells express H2, M3, and CCK2 receptors and somatostatin sst2 receptors. This review discusses the consequences of disrupting genes that are important for ECL cell histamine release and synthesis (HDC, gastrin, and CCK2 receptor genes) and genes that are important for “cross-talk” between H2 receptors and other receptors on the parietal cell (CCK2, M3, and sst2 receptors). Such analysis may provide insight into the functional significance of gastric histamine.

Overexpression of the Anti-Apoptotic Genes mcl-1, bcl-w, bcl-xL and a1 Is Correlated with Chronic Myelogenous Leukemia Progression and Resistance to Gleevec.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2880-2880
Author(s):  
F. A. Castro ◽  
J. F. Jacysyn ◽  
A. G. Ulbrich ◽  
P. R. Tobo ◽  
L. R. Lopes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Real Time ◽  
Chronic Phase ◽  
Rt Pcr ◽  
Blastic Crisis ◽  
Gapdh Gene ◽  
Blastic Phase ◽  
Cytogenetic Remission ◽  
Apoptotic Genes

Abstract Chronic myeloid leukemia (CML) is a stem cell disease where t(9;22) translocation is considered the primary molecular event leading to the appearance of the bcr-abl fusion gene and consequent cellular transformation. Bcr-Abl tyrosine-kinase inhibitors have been developed and are fairly successful in the treatment of CML. Despite their outstanding clinical activity in CML, they are not a definitive cure: the efficacy of imatinib mesylate (Gleevec®), for instance, in CML-blastic phase is reduced and reports of resistance and intolerance to it have been published. Since Bcr-abl initiates cellular modifications leading to an extreme resistance to apoptosis, we decided to investigate possible secondary targets for CML therapy. We evaluate the expression of known anti and pro-apoptotic genes in terms of CML progression and response to Gleevec. We studied 10 health controls and 71 CML patients in different phases (20 chronic phase, 20 accelerated phase, 10 blastic phase, 15 cytogenetic remission post-Gleevec® and 6 Gleevec® refractory patients). CML group was constituted by 26 men and 35 women, median age of 51.7 years (range 23–73 years), 5 men and 5 women, median age of 49.3 (range 25–72 years), were healthy controls. Peripheral blood mononuclear cells were isolated and expression of bax, bcl-w, mcl-1, bcl-2, a1 and bcl-xL was analyzed by real-time RT-PCR. Protein expression of Bcl-2 and Bcl-xL were analyzed by western-blot. The results of real-time RT-PCR and western-blot are expressed by relative expression, e.g. ratio of investigated genes or protein to the reference GAPDH gene and protein, respectively. We observed an increase of bcl-w (p<0.001), mcl-1 (p< 0.001), a1 (p<0.01) and bcl-xL (p<0.001) gene expression and a remarkable reduction of bcl-2 (p<0.001) in CML-BP patients (table 1). Patients in remission post-Gleevec® presented an anti-apoptotic gene expression profile similar to controls (p>0.05) and refractory patients profile seem to be analogous to blastic crisis (p>0.05). bax levels did not show significant changes in CML patients in different phases (p>0.05). Bcl-2 and Bcl-xL protein data support real-time RT-PCR findings. Taken together these results suggest that mcl-1, bcl-w, bcl-xL and a1 contribute to disease progression and resistance to treatment in CML patients. Further investigations on the state of the apototic machinery in CML patients should provide new approaches for drug design and consequently new efficient treatment for AP, BC and refractory CML patients. Table 1. Ratio of amplicons of the investigated genes to housekeeping (GAPDH). Gene C CP-CML AP-CML BP-CML CCR-CML R-CML C: control; CP: chronic phase; AP: accelerated phase; BP: blastic crisis; CCR: complete cytogenetic remission; R: refractory patients. Results expressed by mean /SD. mcl-1 3.6/0.9 4.4/1.0 8.5/5.0 12.6/2.7 2.4/0.7 13.7/3.6 a1 329.9/153.3 564.1/349.1 1,7000/564.4 972.4/564.0 434.2/98.1 968.8/2.4 bcl-w 4.2/1.1 12.8/3.2 3.4/0.6 17.2/8.6 3.5/0.8 19.8/3.2 bcl-xL 2.9/1.3 5.4/1.1 10.6/3.5 39.4/6.2 3.0/2.1 42.0/3.5 bcl-2 21.1/5.2 13.8/7.0 6.1/3.1 3.1/1.3 21.7/6.4 3.6/2.2 bax 3.2/1.3 4.2/0.8 3.7/1.3 5.2/2.5 4.5/2.7 4.2/2.7

Fasting-induced changes in ECL cell gene expression

2007 ◽  
Vol 31 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Nils W. G. Lambrecht ◽  
Iskandar Yakubov ◽  
George Sachs
Keyword(s):  
Gene Expression ◽  
Protein Content ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Types ◽  
Mucosal Cell ◽  
Ecl Cells ◽  
Ecl Cell ◽  
Histamine Uptake ◽  
Cell Gene Expression ◽  
Fasted Rats

Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histamine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate l-histidine but significantly decreased expression of the cellular l-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase α1-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

A polyamine pathway-mediated mitogenic mechanism in enterochromaffin-like cells of Mastomys

1998 ◽  
Vol 275 (2) ◽  
pp. G370-G376 ◽  
Author(s):  
M. Kidd ◽  
L. H. Tang ◽  
S. W. Schmid ◽  
K. Miu ◽  
I. M. Modlin
Keyword(s):  
Dna Synthesis ◽  
Proliferative Response ◽  
Transforming Growth Factor ◽  
Cell Transformation ◽  
Polyamine Biosynthesis ◽  
Transforming Growth Factor Α ◽  
Ecl Cells ◽  
Ecl Cell ◽  
Factor Α ◽  
Rate Limiting

We have previously demonstrated that in Mastomys species proliferation of gastric enterochromaffin-like (ECL) cells is predominantly regulated by gastrin and by transforming growth factor-α (TGF-α) in the naive and neoplastic state, respectively. In this study we examined whether these intracellular mitogenic responses are mediated by polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis. An ECL cell preparation of high purity was used to measure the effect of the polyamine derivatives putrescine, spermidine, and spermine on DNA synthesis by bromodeoxyuridine uptake. Both putrescine and spermidine augmented gastrin-stimulated, but not basal, DNA synthesis in naive cells. This proliferative response correlated with an increase in ODC activity that was partially inhibited (20%) by difluoromethylornithine (DFMO), an inhibitor of ODC (IC50, 30 pM). In contrast, all polyamines increased both basal and TGF-α-stimulated DNA synthesis as well as ODC activity in tumor ECL cells. DFMO completely inhibited the proliferative response of TGF-α (IC50, 3 pM). Thus polyamine biosynthesis is involved in proliferation of ECL cells and in particular the mitogenesis of tumor cells, suggesting a role for this pathway in the regulation of ECL cell transformation.

scholarly journals Assessment of Helicobacter pylori Gene Expression within Mouse and Human Gastric Mucosae by Real-Time Reverse Transcriptase PCR

2001 ◽  
Vol 69 (8) ◽  
pp. 4759-4766 ◽  
Author(s):  
Bachra Rokbi ◽  
Delphine Seguin ◽  
Bruno Guy ◽  
Véronique Mazarin ◽  
Emmanuel Vidor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
16S Rrna ◽  
Mouse Model ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
H Pylori

ABSTRACT Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA >katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, andkatA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification ofH. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.

Osteonectin/SPARC Is Epigenetically Silenced in AML with MLL Gene Rearrangements and Selectively Inhibits the Growth of MLL Rearranged Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1106-1106
Author(s):  
Jorge F. DiMartino ◽  
Norman J. Lacayo ◽  
Marie Varadi ◽  
Yaddanapudi Ravindranath ◽  
Ron Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Cancer ◽  
Western Blot ◽  
Cell Lines ◽  
Chromatin Remodeling ◽  
Promoter Methylation ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Mll Gene ◽  
Sparc Expression

Abstract Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most significant genes was osteonectin, also known as secreted protein, acidic, rich in cysteine (SPARC). This gene encodes a matricellular glycoprotein with diverse functions in cell-matrix interactions. SPARC has been identified as a target of epigenetic silencing in pancreatic cancer and addition of exogenous SPARC to cancer cell lines induces growth arrest and apoptosis. To determine if leukemia cell lines could be used as a model to study the basis for SPARC silencing and its role in cell growth and survival we measured SPARC expression in AML cell lines with rearranged and germline MLL genes. By real-time quantitative reverse transcriptase PCR (Q-RT-PCR), the cell lines THP-1 (MLL-AF-9) and ML-2 (MLL-AF6) expressed SPARC mRNA levels 40 to 1000 fold lower than Kasumi-1 (t(8;21)) and KG1a. By Western blot, SPARC was easily detectable in Kasumi-1 and KG1a but undetectable in the MLL rearranged lines. Bisulfite sequencing revealed extensive methylation of CpG dinucleotides in the promoter region and first exon of SPARC in THP-1 and ML-2 but a complete lack of methylation in KG1a. Treatment with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine (DAC) restored SPARC expression to nearly normal levels in THP-1 cells by Q-RT-PCR and Western blot, suggesting that promoter methylation is critical to the silencing of this gene. To determine if SPARC was contributing to the growth inhibitory effect of DAC, cells were cultured in varying concentrations of exogenous purified SPARC. Concentrations of SPARC that reduced the growth of ML-2 and THP-1 by 30 to 40% had no effect on the growth of KG1a cells. We conclude that SPARC, a putative tumor suppressor that is epigenetically silenced in pancreatic cancer, is also silenced by promoter methylation in AML with MLL rearrangements. These studies suggest that SPARC expression may constitute a reliable pharmacodynamic endpoint for clinical studies of chromatin remodeling agents in patients with MLL rearranged AML. Whether SPARC is a direct target of MLL and how MLL rearrangements are related to SPARC silencing are the subject of future studies.

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4213-4213
Author(s):  
Priya Khoral ◽  
Robert J Guo ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Blot Analysis ◽  
Drug Combination ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

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