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¹ Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania and ² Department of Research and Development, Ambion Inc., Austin, Texas

Requests for reprints: Iraimoudi S. Ayene, Department of Radiation Oncology, School of Medicine, University of Pennsylvania, 195 John Morgan Building, Philadelphia, PA 19104-6072. Phone: 215-898-9507; Fax: 215-898-0090. E-mail: ayenei{at}mail.med.upenn.edu

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiation-responsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments.

Key Words: Molecular targets of radiation response • Silencing and reactivation of gene expression

Grant support: National Cancer Institute research grant CA 92108.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

³ J.E. Biaglow et al., personal communication.

Received 5/21/04; revised 1/20/05; accepted 2/21/05.