Darpp-32: a novel antiapoptotic gene in upper gastrointestinal carcinomas.
We show the molecular mechanisms involved in Darpp-32 overexpression and its biological role in upper gastrointestinal adenocarcinomas (UGC). A tumor tissue array of 377 samples was developed and used to detect DARPP-32 DNA amplification and protein overexpression, which occurred in 32% and 60% of UGCs, respectively. Concomitant overexpression of mRNA for Darpp-32 and its truncated isoform t-Darpp was observed in 68% of tumors (P < 0.001). When Darpp-32 and t-Darpp were overexpressed in AGS and RKO gastrointestinal cells, up to a 4-fold reduction in the apoptosis rate was observed (terminal deoxynucleotidyl transferase-mediated nick-end labeling and Annexin V assays) in response to camptothecin, sodium butyrate, and ceramide. However, the introduction of mutations in phosphorylation sites abrogated this effect. Expression of Darpp-32 and t-Darpp preserved the mitochondrial transmembrane potential and was associated with increased levels of Bcl2 protein. A reversal of Bcl2 protein level was obtained using small interfering RNAs for Darpp-32 and t-Darpp. Luciferase assays using the p53 and p21 reporter plasmids and probing of immunoblots with antibodies specific for p53 transcriptional targets, such as Hdm2 and p21, indicated that neither Darpp-32 nor t-Darpp interfere with p53 function. Altogether, we show more frequent mRNA and protein overexpression of Darpp-32 than DNA amplification, suggesting that, in addition to amplification, transcriptional or posttranscriptional mechanisms may play an important role. The expression of Darpp-32 and t-Darpp is associated with a potent antiapoptotic advantage for cancer cells through a p53-independent mechanism that involves preservation of mitochondrial potential and increased Bcl2 levels.
Belkhiri
Abbes
A
Digestive Health Center of Excellence and Department of Pathology, University of Virginia Health System, Charlottesville, Virginia 22908-0708, USA.
Zaika
Alexander
A
Pidkovka
Nataliya
N
Knuutila
Sakari
S
Moskaluk
Christopher
C
El-Rifai
Wa'el
W
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002166:Camptothecin / Q000494:pharmacology; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018628:Gene Dosage; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D015854:Up-Regulation
D000972:Antineoplastic Agents, Phytogenic; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D002166:Camptothecin