Characterization of the GADD45 response to ionizing radiation in WI-L2-NS cells, a p53 mutant cell line.
We have previously reported that WI-L2-NS, a human lymphoblastoid cell line, has very high basal levels of GADD45 mRNA and protein in spite of a p53 mutation at amino acid 237. Regardless of the amount of Gadd45 in this cell line, no growth suppression activity was detected. We report here that in WI-L2-NS, the mutated p53 protein adopts predominantly a wild type (wt) conformation and binds to the p53 binding site in the GADD45 third intron. In this cell line, the already high levels of mutated p53 protein can be induced further by ionizing radiation (IR) but the response of the p53 downstream effector genes is altered. Induction of GADD45 and CIP1/WAF1 is reduced compared to p53 wt cell lines but is still substantially higher than the average fold induction obtained from 39 p53 mutant cell lines. Induction of the MDM2 gene was not detected in WI-L2-NS following IR. The induction pattern of the three p53 effector genes by the alkylating agent methylmethane sulfonate (MMS) was also attenuated in WI-L2-NS cells. In TK6 cells, a WI-L2-NS sister cell line having a p53 wt genotype, the induction of the p53 downstream effectors is normal, i.e. induced, both at the protein and the mRNA levels. These results indicate that the DNA binding activity of the mutated p53 protein in WI-L2-NS might be responsible, at least in part, for the high basal levels of GADD45 but can not mediate the full induction of the p53 downstream effector genes. The reason(s) for the inability of Gadd45 to suppress growth in this cell line remains however unknown.
Carrier
F
F
Laboratory of Molecular Pharmacology, DTP, DCT, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. carrier@Box-c.nih.gov
Bae
I
I
Smith
M L
ML
Ayers
D M
DM
Fornace
A J
AJ
D000477:Alkylating Agents / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007438:Introns / Q000235:genetics; D008214:Lymphocytes; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009154:Mutation; D009687:Nuclear Proteins; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000096:biosynthesis; D011839:Radiation, Ionizing; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097642:GADD45 Proteins
D000477:Alkylating Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004247:DNA; D047908:Intracellular Signaling Peptides and Proteins; D008741:Methyl Methanesulfonate; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human; C497206:MDM2 protein, human