Shoumin Zhu1, Mohammed Soutto1, Zheng Chen1, DunFa Peng1, Judith Romero-Gallo2, Uma S Krishna2, Abbes Belkhiri1, M Kay Washington3, Richard Peek2, Wael El-Rifai1,4,5. 1. Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 2. Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 3. Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 4. Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 5. Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, Tennessee, USA.
Abstract
OBJECTIVE: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pylori infection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
OBJECTIVE:DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pyloriinfection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pyloriinfection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pyloriinfection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pyloriinfection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pyloriinfection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pyloriinfection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pyloriinfection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
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