Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation.
RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.
Zhou
J
J
Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Zhang
X-Q
XQ
Ashoori
F
F
McConkey
D J
DJ
Knowles
M A
MA
Dong
L
L
Benedict
W F
WF
D000256:Adenoviridae; D017209:Apoptosis / Q000235:genetics; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D020287:In Situ Nick-End Labeling; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D016160:Retinoblastoma Protein / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy
C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D020033:Protein Isoforms; D016160:Retinoblastoma Protein; D020169:Caspases