Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma.
OBJECTIVE
Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2.


METHODS
Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays.


RESULTS
Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival.


CONCLUSIONS
Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.
Martin
Juliette
J
Institute for Clinical Pharmacology, University of Bern, Bern, Switzerland.
Magnino
Fabrice
F
Schmidt
Karin
K
Piguet
Anne-Christine
AC
Lee
Ju-Seog
JS
Semela
David
D
St-Pierre
Marie V
MV
Ziemiecki
Andrew
A
Cassio
Doris
D
Brenner
Charles
C
Thorgeirsson
Snorri S
SS
Dufour
Jean-François
JF
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D046228:Microarray Analysis; D051336:Mitochondrial Membranes / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D012334:RNA, Neoplasm