ABSTRACT
We have developed a hypoxia-inducible gene therapy approach for the expression of the mature form of human endonuclease G to facilitate cell death in hypoxic regions of the tumor. The chimeric therapeutic gene is placed under the control of a hypoxia response element based promoter and contains a translocation motif linked in frame to an oxygen-dependent degradation domain and the endonuclease G gene. Transient expression of the chimeric therapeutic gene in breast and prostate cancer cell lines resulted in efficient cell death under hypoxia-mimetic conditions. Stable MDA-MB-435 cells expressing the chimeric therapeutic gene under 1% O2 showed an increase in stable HIF-1alpha protein levels and synthesis of the endonuclease G protein in a time-dependent manner. In normoxic conditions, these stable transgenic cells exhibited no change in growth rate, invasion and motility when compared to parental cells. Moreover, xenografts generated using the transgenic cells exhibited highly significant suppression of tumor growth in a preclinical cancer model compared to the parental cell line. Thus, the hypoxia-modulated endonuclease G expression has the potential to be used as a gene-based-therapy system to kill malignant cells within hypoxic regions of tumors.
Subject(s)
Endodeoxyribonucleases/metabolism , Genetic Therapy/methods , Neoplasms/therapy , Xenograft Model Antitumor Assays , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cobalt/pharmacology , Endodeoxyribonucleases/genetics , Female , Humans , Hypoxia/physiopathology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, SCID , Microscopy, Fluorescence , Neoplasms/genetics , Neoplasms/pathology , Promoter Regions, Genetic/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , Tumor BurdenABSTRACT
Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.
