Induction of p14ARF by E2F1 contributes to 8-chloro-adenosine-induced apoptosis in human lung cancer H1299 cells.
BACKGROUND
8-Chloro-adenosine (8-Cl-Ado) inhibits tumor cell proliferation by inducing cell-cycle arrest and apoptosis. We speculate that upregulation of p14ARF by E2F1 might contribute to 8-Cl-Ado-induced late apoptosis.


METHODS
Hoechst staining, cell proliferation and TUNEL assays, real-time quantitative PCR, Western blotting, chromatin immunoprecipitation and RNA interference were employed in investigating the role of induction of p14ARF by E2F1 in 8-Cl-Ado-induced apoptosis in human lung cancer H1299 cells.


RESULTS
Exposure of H1299 to 8-Cl-Ado led to apoptosis after long exposure (48 h), revealed by the appearance of nucleus fragmentation and apoptotic bodies and the activation of procaspase-3 pathway. Western blotting and RT-PCR showed that the upregulation of p14ARF was in parallel with E2F1 expression during exposure. Furthermore, induction of p14ARF was attributed to increased E2F1 expression, evidenced by E2F1 transfection and chromatin immunoprecipitation/real-time quantitative PCR. Knockdown of p14ARF expression in H1299 decreased TUNEL-positive cell numbers and relatively increased survival cell numbers during 8-Cl-Ado exposure, indicating insensitivity of p14ARF-knocked down cells to 8-Cl-Ado.


CONCLUSIONS
Induction of p14ARF by E2F1 contributes to 8-Cl-Ado-induced late apoptosis.
Li
Wen-Juan
WJ
Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, PR China.
Gu
Yan-Yan
YY
Zhang
Hai-Jun
HJ
Zhou
Jing
J
Jia
Hong-Ti
HT
D015762:2-Chloroadenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects; D034622:RNA Interference; D025803:Tumor Suppressor Protein p14ARF / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D025803:Tumor Suppressor Protein p14ARF; D015762:2-Chloroadenosine; C075440:8-chloroadenosine