Low-dose radiation response of the p21(WAF1/CIP1) gene promoter transduced by adeno-associated virus vector

Mitsuru Nenoi,Sachiko Ichimura,Shin-Ichiro Takahash,Kazuhiro Daino,Teruo Akuta

doi:10.1038/emm.2006.65

Abstract

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

Highlights

There has been increasing interest in the use of gene therapy for the treatment of malignant tumors
In order to examine the usability of the p21 gene promoter in the development of a low-dose IRinducible vector for gene therapy, we first analyzed the ionizing radiation (IR)-response of endogenous p21 gene expression in human breast cancer cell line MCF-7, which contains wild-type p53
In vivo DNase Ihypersensitive site analyses showed that p53 recognition sites in the p21, 14-3-3σ, and KARP-1 genes are resistant to nuclease digestion even after gene induction by irradiation (Braastad et al, 2003)

Summary

Introduction

There has been increasing interest in the use of gene therapy for the treatment of malignant tumors. Gene therapy for cancer is based on tumor-specific delivery of genes encoding proteins that are directly toxic to tumor cells, induce anti-tumor immune responses, or sensitize tumor cells to prodrugs. A number of strategies have been developed to localize therapeutic genes to tumor cells, such as those using tissue-specific promoters/enhancers. A MUC1 gene promoter-driven transgene could be preferentially expressed in MUC1-positive breast cancer cells (Kurihara et al, 2000). A carcinoembrionic antigen (CEA) promoter could be utilized in preferential expression of transgenes in CEA-producing colon and lung cancer cells (Konishi et al, 1999). Tumor-specific gene delivery by use of restricted replication-competent viruses has been developed, such as E1B55K-deficient adenovirus selectively targeting p53-mutated tumor cells (Kirn, 2000). Transgenes under the control of an IR-inducible promoter are expressed selectively in the radiation field. The most widely utilized promoter for this purpose is that of the early

Methods

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Conclusion