Interaction of the epidermal growth factor receptor and the DNA-dependent protein kinase pathway following gefitinib treatment.
The epidermal growth factor receptor (EGFR) is an important target for cancer therapy. We previously showed that the EGFR inhibitor gefitinib modulated repair of DNA damage following exposure to cisplatin and etoposide involving the DNA-dependent protein kinase (DNA-PK) pathway. In this study, we specifically investigated the effect of EGFR inhibition by gefitinib on functional activity of DNA-PK in cancer cell lines and the interaction between EGFR and DNA-PK. The effects of DNA-PK inhibition by wortmannin and small interfering RNA to the catalytic subunit of DNA-PK (DNA-PK(CS)) on cell proliferation and DNA interstrand cross-link repair were investigated in the human MCF-7 breast cancer cell line and compared with the effects of gefitinib. DNA-PK activity was quantitated and expression measured by immunoblotting following gefitinib treatment. Immunoprecipitation experiments were done with and without gefitinib in MCF-7 cells, the AR42J pancreas cell line with high EGFR, and the human MDA-453 breast cancer cell line expressing low EGFR. Nuclear and cytoplasmic extracts were immunoblotted with antibody to DNA-PK(CS) to determine if gefitinib treatment altered cellular expression. Reduction of DNA-PK activity by wortmannin and expression by small interfering RNA to DNA-PK(CS) sensitized cells to cisplatin and inhibited repair of cisplatin-induced interstrand cross-links. Gefitinib treatment reduced DNA-PK activity in MCF-7 and AR42J but not MDA-453 cells. Immunoprecipitation experiments showed interaction between EGFR and DNA-PK(CS) in a dose-dependent and time-dependent manner following gefitinib treatment in MCF-7 and AR42J but not MDA-453 cells. Gefitinib treatment reduced nuclear expression and increased cytosolic expression of DNA-PK(CS) in MCF-7 and AR42J but not MDA-453 cells. Treatment with gefitinib modulates association of EGFR and DNA-PK(CS). This is correlated with decreased function of DNA-PK(CS). Inhibition of DNA-PK(CS) may be an important factor in sensitization to chemotherapy and radiation following treatment with inhibitors of the EGFR pathway.
Friedmann
Benjamin J
BJ
Cancer Research UK Drug-DNA Interactions Research Group, Royal Free and University College Medical School, University College London.
Caplin
Martyn
M
Savic
Boris
B
Shah
Tahir
T
Lord
Christopher J
CJ
Ashworth
Alan
A
Hartley
John A
JA
Hochhauser
Daniel
D
D000730:Androstadienes / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D000971:Antineoplastic Combined Chemotherapy Protocols; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D002945:Cisplatin / Q000627:therapeutic use*; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D005047:Etoposide / Q000627:therapeutic use; D000077156:Gefitinib; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D011799:Quinazolines / Q000627:therapeutic use*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051381:Rats; D000077191:Wortmannin
D000730:Androstadienes; D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D034741:RNA, Small Interfering; D005047:Etoposide; D004247:DNA; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase; D002945:Cisplatin; D000077156:Gefitinib; D000077191:Wortmannin