A chimeric fusion of the hASH1 and EZH2 promoters mediates high and specific reporter and suicide gene expression and cytotoxicity in small cell lung cancer cells

Abstract

Transcriptionally targeted gene therapy is a promising experimental modality for treatment of systemic malignancies such as small cell lung cancer (SCLC). We have identified the human achaete-scute homolog 1 (hASH1) and enhancer of zeste homolog 2 (EZH2) genes as highly upregulated in SCLC compared to a panel of representative normal tissues. Here, we evaluate the use of regulatory regions from the hASH1-and EZH2-promoter regions alone and in combination for suicide gene therapy of SCLC. Two hASH1-promoter regions comprising 0.3 and 0.7 kb immediately upstream of (and including) the transcription start site were tested. Both constructs induced reporter gene activity (up to sevenfold SV40-promoter activity) in all tested classic (hASH1 positive) SCLC and in two hASH1-negative SCLC cell lines, whereas gene activity was low or absent (o4% of SV40 activity) in one hASH1-negative SCLC and in all control cell lines tested. To evaluate its therapeutic potential, the 0.7 kb hASH1 proximal-promoter region was evaluated for cytotoxicity in a suicide gene assay. The construct induced SCLC cytotoxicity at levels equivalent to those observed with the SV40 promoter, while control cells remained unaffected by the treatment. Analogously, a 1.1 kb EZH2-promoter region was evaluated by reporter and suicide gene assays. The EZH2 promoter potently induced reporter gene activity in SCLC (up to 25-fold of SV40 activity) while moderate reporter activity (up to 12% of SV40 activity), was detected in the control cells. However, in the suicide gene assay both control and SCLC cells demonstrated sensitivity indicating lack of promoter specificity. Finally, we fused the 0.7 kb hASH1 promoter to the EZH2 promoter generating a chimeric hASH1EZH2 regulatory construct. The chimeric promoter demonstrated increased activity in SCLC cells compared to the hASH1 promoter alone while retaining specificity in control cells. The hASH1EZH2 promoter thus constitutes a promising transcriptional regulator for SCLC gene therapy.

High-grade neuroendocrine carcinomas of the lung highly express enhancer of zeste homolog 2, but carcinoids do not

“…EZH2 has been shown to be overexpressed in a number of malignant neoplasms, including endometrial cancer, prostate cancer, breast cancer, hepatocellular carcinoma, oral mucosal squamous cell carcinoma, urothelial carcinoma, cutaneous melanoma, SCLC, gastric carcinoma, and renal cell carcinoma [6][7][8][9][10][11][12][13][14][15]. In addition, EZH2 expression has been shown to be linked with aggressive behavior in Hodgkin-type lymphoma, and increased EZH2 levels are frequently associated with poor prognosis in multiple human malignancies, including B-cell non-Hodgkin-type lymphomas, breast, prostate, and bladder cancer [7][8][16][17][18][19][20][21].…”

“…The current literature on EZH2 expression in neuroendocrine tumors of the lung is limited, with no literature discussing EZH2 expression in pulmonary carcinoids and LCNEC [12]. To test the hypothesis that EZH2 expression is associated with higher grade and thus potentially more biologically aggressive tumors, immunohistochemical studies were performed to examine EZH2 expression in TC, AC, LCNEC, and SCLC.…”

PRIMA-1Met/APR-246 Induces Apoptosis and Tumor Growth Delay in Small Cell Lung Cancer Expressing Mutant p53

“…The human non-immortalized lung fibroblast cell line, CCD32Lu, and the breast carcinoma cell line, MDA-MB-231, were obtained from American Type Culture Collection (ATCC) and the non-small cell lung carcinoma cell line, H1299, was provided by Dr. R.J. Christiano (Houston, TX). Propagation of CCD32Lu, MDA-MB-231, and H1299 cell lines is described elsewhere (20,21). The cell lines were grown in monolayer, suspension or semiadherent/suspension cultures (Table 1).…”

In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection

“…Initially, the cytotoxicity of endocytosis inhibitors: chloroquine (inhibiting acidification of endosomes and lysosome fusion), 27 chlorpromazine ( inhibiting clathrin-mediated endocytosis), 28 filipin ( inhibiting caveaolae-mediated endocytosis), 29,30 and cytochalasin B (inhibiting macropinocytosis) 31 was determined in NCI-H69 and H1299 cells in the range reported previously by others. Concentrations causing approximately 30% decrease in MTT cell viability 32 were selected (data not shown, see legend of Figure 6). Twenty microlitres (0.8 µmoL) of liposome preparations with varying amounts of PEG-lipid were diluted and mixed by rapid pipetting up and down with DNA solutions containing pEGFP-N1 and pCMV-LUC (22.5 µg each) yielding a total volume of 100 µL DNA/lipoplex solution.…”