The human ribonucleotide reductase subunit hRRM2 complements p53R2 in response to UV-induced DNA repair in cells with mutant p53.
Ribonucleotide reductase (RR) is responsible for the de novo conversion of the ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. RR consists of two subunits, hRRM1 and hRRM2. p53R2 is a new RR family member. Because the majority of human tumors possess mutant p53, it is important to know the molecular mechanism by which mutant p53 regulates RR and to what extent. In this study, we investigated the expression and function of p53R2 and hRRM2 after UV treatment in human prostate cancer PC3 cells, which possess mutant p53 with a truncated COOH-terminal, and in human oropharyngeal cancer KB cells, which possess wild-type p53. p53R2 (analyzed by Western blot and standardized relative to Coomassie Blue-stained band) was down-regulated in PC3 cells and up-regulated in KB cells after UV exposure. In contrast, hRRM2 was up-regulated by UV in both PC3 cells and KB cells. hRRM2 and p53R2 mRNA levels were assessed by Northern blot, and the results paralleled that of the Western blot. Coimmunoprecipitation assays using agarose-conjugated goat antihuman RRM1 antibody confirmed that the p53R2 binding to hRRM1 decreased in PC3 cells but increased in KB cells after UV treatment. hRRM2 binding to hRRM1 increased in both cell lines under the same conditions. These results suggest that PC3 cells are deficient in both transcription of p53R2 and binding to hRRM1 in response to UV irradiation. Confocal microscopy further confirmed that these findings were not due to translocation of hRRM2 and p53R2 from the cytoplasm to the nucleus. RR activity was measured following UV treatment and shown to increase in PC3 cells. It was unchanged in proportional of KB cells. The RR activity is consistent with the expression of hRRM2 seen in the Western blots. Thus, we hypothesize that hRRM2 complements p53R2 to form RR holoenzyme and maintain RR activity in PC3 cells after UV treatment. To further confirm this hypothesis, we examined the effect of RRM2 inhibitors on cells exposed to UV. In PC3 cells, hydroxyurea inhibited hRRM2 and resulted in increased sensitivity to UV irradiation. We also examined the effect of UV treatment on the colony-forming ability of cells transfected with hRRM2 as well as p53R2 sense or antisense expression vectors. Expression of antisense hRRM2 in PC3 cells led to decreased hRRM2 expression and resulted in greater sensitivity to UV than observed in wild-type PC3 cells. Taken together, we conclude that UV-induced activation of p53R2 transcription and binding of p53R2 to hRRM1 to form RR holoenzyme are impaired in the p53-mutant cell line PC3.
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010, USA.
Bingsen
B
Zhou
Xiyong
X
Liu
Xueli
X
Mo
Lijun
L
Xue
Dana
D
Darwish
Weihua
W
Qiu
Jennifer
J
Shih
Edward B
EB
Hwu
Frank
F
Luh
Yun
Y
Yen
D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D004249:DNA Damage; D004260:DNA Repair; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology / Q000528:radiation effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007624:KB Cells; D008297:Male; D009154:Mutation; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012262:Ribonucleoside Diphosphate Reductase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C405947:RRM2B protein, human; D012264:Ribonucleotide Reductases; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea