Induction of apoptosis signal-regulating Kinase 1 by E2F-1 may not be essential for E2F-1-mediated apoptosis in melanoma cells.
OBJECTIVE
In the present study, we investigate the role of apoptosis signal-regulating kinase 1 (ASK1) mitogen-activated protein (MAP) kinase signal pathways in E2F-1-mediated apoptosis.


METHODS
A gene expression profile in response to E2F-1 overexpression was performed by cDNA microarray analysis and confirmed by real-time reverse-transcription polymerase chain reaction. Kinase activities were assayed by Western blot analysis or kinase assay. Apoptosis was assessed by morphologic inspection and flow-cytometric analysis. Cytotoxicity was monitored by MTT assay.


RESULTS
E2F-1 upregulated the expression of ASK1 8-fold compared to the Ad-LacZ-infected control in SK-MEL-2 melanoma cells, which was confirmed by reverse-transcription polymerase chain reaction. Sequence analysis showed that there are 2 putative E2F-1 DNA binding sites in the ASK1 promoter region. Truncated E2F-1 protein, which lacks the transactivation domain, failed to upregulate ASK1, suggesting that ASK1 was regulated at the transcriptional level by E2F-1. E2F-1 overexpression resulted in the transient activation of c-Jun N-terminal kinase (JNK); however, dominant negative mutant ASK1 had no effect on E2F-1 cytotoxicity and JNK activation. p38 was not activated by E2F-1, and inhibition of p38 had no effect on E2F-1-mediated cell death. The ASK1 kinase assay showed that ASK1 activity was not upregulated in response to E2F1 overexpression. The inhibition of ASK1 upstream kinase-AKT can enhance E2F-1-mediated cell death. Moreover, an adenovirus expressing truncated E2F-1 keeps the ability of inducing apoptosis in melanoma cells.


CONCLUSIONS
ASK1 expression is upregulated by E2F-1 at the transcription level, but the upregulation of ASK1 expression by E2F-1 was not coordinated with an increased ASK1 activity. The ASK1-JNK/p38 pathway does not appear to play a crucial role in E2F-1-induced apoptosis.
Dong
Yan Bin
YB
Department of Surgery, University of Louisville School of Medicine, Louisville, Ky., USA.
Phelps
Allison M
AM
Yang
Hai Liang
HL
Jamshidi-Parsian
Azemat
A
Chen
Canming
C
Hao
Hongying
H
Gomez-Gutierrez
Jorge G
JG
Zhou
Heshan Sam
HS
McMasters
Kelly M
KM
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism*; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human