Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway.
ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.
Bao
Jia-Ju
JJ
Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Le
Xiao-Feng
XF
Wang
Rui-Yu
RY
Yuan
Jiuhong
J
Wang
Lin
L
Atkinson
Edward N
EN
LaPushin
Ruth
R
Andreeff
Michael
M
Fang
Bingliang
B
Yu
Yinhua
Y
Bast
Robert C
RC
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D005260:Female; D016147:Genes, Tumor Suppressor; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D061945:Caspase Inhibitors; C117386:DIRAS3 protein, human; D002154:Calpain; D020169:Caspases; D020741:rho GTP-Binding Proteins