Overexpression of the R2 subunit of ribonucleotide reductase in human nasopharyngeal cancer cells reduces radiosensitivity.
OBJECTIVE
Ribonucleotide reductase is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleotide triphosphates, which are utilized in both DNA synthesis and DNA repair. We reported previously that RR enzyme activity and R2 (catalytic subunit of RR) protein levels were increased after exposure to ionizing radiation (IR) in growth-arrested human tumor cells, suggesting that R2 protein expression regulates RR activity to allow for IR damage repair. Using isogenic human nasopharyngeal carcinoma cells in this study, we examine the relationship of overexpression of either the R1 regulatory subunit or the R2 catalytic subunit of RR to the cellular response of IR damage.


METHODS
We used three isogenic human nasopharyngeal cancer cell lines previously derived by Zhou et al, including KB, the parental tumor cell line; KB/M1, an R1 protein-overexpressing clone stably transfected with human R1 complementary DNA; and KB/M2, a R2 protein-overexpressing clone stably transfected with human R2 complementary DNA. We initially characterized these isogenic human tumor cell lines in exponential growth for R2 protein expression, RR enzyme activity, and R2 protein changes during the cell cycle by flow cytometry. Subsequently, the IR response in these cell lines was determined by clonogenic survival, cell cycle changes occurring after IR, and an analysis of IR DNA damage determined by pulsed field gel electrophoresis. The effect of combining IR and hydroxyurea, a RR (R2) inhibitor, was also studied in KB and KB/M2 cells.


RESULTS
KB/M2 cells were found to have 4.5-fold higher R2 protein expression and a threefold higher RR enzyme activity in exponential growth than KB and KB/M1. Although R2 protein levels increased at the G1/S transition in all cell lines, KB/M2 cells also demonstrated consistently higher R2 protein levels throughout the cell cycle. Using a linear-quadratic analysis of IR clonogenic survival data, KB/M2 cells were more radioresistant than KB and KB/M1 cells, including both decreased alpha and decreased beta values, a finding that correlates with increased reparable IR damage. KB/M2 cells also show a reduced G2 cell cycle arrest and fewer DNA double strand breaks 18 hours after IR (6 Gy). Exposure of KB/M2 cells to hydroxyurea (300 microM) after exposure to IR restored in vitro radiosensitivity in a manner similar to that found in KB and KB/M1 cells.


CONCLUSIONS
An increase in R2 protein levels and RR activity in KB/M2 cells results in IR resistance, which appears mediated by enhanced IR damage repair during G2. R1 protein overexpression in these isogenic human tumor cells (KB/M1) did not affect RR activity or IR response.
Kuo
Mei-Ling
ML
Department of Radiation Oncology, University Hospitals of Cleveland/Ireland Cancer Center and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-6068, USA.
Hwang
Hwa-Shin
HS
Sosnay
Patrick R
PR
Kunugi
Keith A
KA
Kinsella
Timothy J
TJ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002277:Carcinoma / Q000188:drug therapy / Q000201:enzymology / Q000532:radiotherapy; D002453:Cell Cycle; D045744:Cell Line, Tumor; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000528:radiation effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D066298:In Vitro Techniques; D007624:KB Cells / Q000187:drug effects / Q000528:radiation effects; D009303:Nasopharyngeal Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000532:radiotherapy; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D012262:Ribonucleoside Diphosphate Reductase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000528:radiation effects
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D011838:Radiation-Sensitizing Agents; C019857:CDP reductase; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea