Gadd45alpha does not modulate the carboplatin or 5-fluorouracil-induced apoptosis in human papillomavirus-positive cells.
Gadd45alpha is shown to be induced by a wide spectrum of DNA-damaging agents and implicated in negative regulation of cell growth by causing G2-M arrest or induction of apoptosis. In the present study, we explored the involvement of p53 in the promoter activation of Gadd45alpha as well as the role of Gadd45alpha in carboplatin (Carb) or 5-fluorouracil (5-FU)-induced apoptosis in human papillomavirus virus (HPV)-positive HEp-2 and HeLa cells. We report that Carb or 5-FU upregulate Gadd45alpha and p53 in both these cells. Transient transfection of chloramphenicol acetyl transferase (CAT)-reporter construct driven by Gadd45alpha promoter clearly indicated that Gadd45alpha upregulation was mediated through activation of its promoter. Inhibition of p53 function by dominant-negative-p53 expression partially suppressed the activation of Gadd45alpha promoter. Further, the induction of apoptosis was assessed by detection of poly (ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. Inhibition of upregulated Gadd45alpha expression by antisense expression vector did not modulate the Carb or 5-FU-induced apoptosis. Overall, we conclude that Gadd45alpha promoter activation partially depends on p53 function in HPV-positive cells. Moreover, Gadd45alpha protein does not modulate Carb or 5-FU-induced apoptosis in these cells.
Singh
Sandeep
S
National Centre for Cell Science, Ganeshkhind, Pune 411 007, India.
Upadhyay
Ankur Kumar
AK
Ajay
Amrendra Kumar
AK
Bhat
Manoj Kumar
MK
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D016190:Carboplatin / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005472:Fluorouracil / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae; D030361:Papillomavirus Infections / Q000188:drug therapy*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000097642:GADD45 Proteins
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D016190:Carboplatin; D018797:Cell Cycle Proteins; D005472:Fluorouracil; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; C439070:GADD45A protein, human; D000097642:GADD45 Proteins