t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism

Abbes Belkhiri, Altaf A. Dar, Alexander Zaika, Mark Kelley, Wael El-Rifai

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of Mr 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 μmol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to ≥2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser 473 Akt and p-Ser9 GSK3β. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.

Original languageEnglish (US)
Pages (from-to)395-403
Number of pages9
JournalCancer Research
Volume68
Issue number2
DOIs
StatePublished - Jan 15 2008
Externally publishedYes

Fingerprint

Cell Survival
Up-Regulation
Apoptosis
Ceramides
Neoplasms
Activating Transcription Factor 1
Dopamine and cAMP-Regulated Phosphoprotein 32
Activating Transcription Factors
CREB-Binding Protein
Proteins
Caspase 9
Response Elements
Electrophoretic Mobility Shift Assay
Cytochromes c
Tetracycline
Luciferases
Drug Resistance
Caspase 3
Cyclic AMP
Membrane Potentials

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism. / Belkhiri, Abbes; Dar, Altaf A.; Zaika, Alexander; Kelley, Mark; El-Rifai, Wael.

In: Cancer Research, Vol. 68, No. 2, 15.01.2008, p. 395-403.

Research output: Contribution to journalArticle

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abstract = "t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of Mr 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 μmol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to ≥2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser 473 Akt and p-Ser9 GSK3β. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.",
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