Stress response of a p53 homologue in the radioresistant Sf9 insect cells.
OBJECTIVE
To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance.


METHODS
Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcription-polymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in gamma-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required.


RESULTS
A 47-49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following gamma-irradiation and etoposide.


CONCLUSIONS
A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following gamma-irradiation indicate functional differences from human cells.
Suman
Shubhankar
S
Natural Radiation Response Mechanisms Group, Department of Zoology, University of Delhi, India.
Khaitan
Divya
D
Pati
Uttam
U
Seth
Rakesh Kumar
RK
Chandna
Sudhir
S
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D005720:Gamma Rays; D006801:Humans; D019476:Insect Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011836:Radiation Tolerance; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D005047:Etoposide; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases