Regulation of gene expression in prostate cancer cells with an artificially constructed promoter responsive to radiation

Abstract

A promoter library was developed that is composed of DNA fragments constructed by randomly elongating the cis-acting elements of transcription factors presumably activated in prostate cancer by radiation, and linking to the TATA-box sequence. One promoter with the strongest reactivity to X-ray in the LNCap cells of the library was chosen and improved by the introduction of random mutations. The resultant promoter was designated clone 880-8, showing the highest dose-dependent activity enhancement with X-ray irradiation (X-irradiation). A recombinant retrovirus expressing the luciferase gene under the control of clone 880-8 was infected into LNCap cells that showed 9.12±0.36-fold enhancement of luciferase activity 12 h after X-irradiation at 10 Gy. When the infected cells were inoculated onto nude mice, enhancement of luciferase expression was 4.27 ± 1.36-fold 12 h after X-irradiation at 10 Gy. When LNCap was infected with another recombinant carrying the fcy::fur gene downstream from clone 880-8, fcy::fur expression was enhanced by X-irradiation. It was also shown to increase the dosedependent cell killing ratio with 5-FC as compared with a counterpart without X-irradiation. These results suggest that the method used in this study is effective to construct a promoter responsive to stimulation. Such promoters can be used for stimulation-controlled gene therapies.

An artificially constructed radiation-responsive promoter is activated by doxorubicin

“…6 Similarly, enhancement of luciferase expression induced by Dox treatment was also suppressed with D-mannitol ( Figure 1d). Therefore, oxidative stress is assumed to be involved in the activation of the promoter.…”

“…Concrete processes to construct the promoter were described elsewhere. 6 In brief, a promoter probe vector, pGL3-DU-TATA, was constructed by cloning a polymerase chain reaction (PCR)-amplified DNA fragment containing the TATA box signal out of the human heme oxygenase I gene promoter into upstream of the luciferase gene of pGL3-Control (Promega, Madison, WI). Equimolar amount of synthesized cis-elements of transcription factors that is responsive to radiation in prostate cancer cells, including NF-kB (5 0 -gggactttcc-3 0 ), AP-1 (5 0 -tgactca-3 0 ), Oct-1 (5 0 -atgcaaat-3 0 ), p53 (5 0 -gaacatgtctaagcatgctg-3 0 ) and Nrf-2 (5 0 -tgctgagtca-3 0 ) in addition to 1/100 molar amounts of DNA fragments containing recognition sequences of HindIII and KpnI restriction enzymes, were randomly ligated.…”

“…6 We thus investigated whether anticancer drugs that could induce oxidative stress in treated cells enhance clone 880-8 activity.…”

“…5 We previously developed artificially constructed promoters responsive to radiation stimulation in prostate cancer cells and reported our attempts to use them for artificial regulation of gene expression in vitro and in vivo. 6 These promoters were constituted with randomly combined cis-elements of transcription factors that are activated in response to radiation in prostate cancer cells and are linked to a DNA fragment containing the TATA box signal. The most actively responsive promoter was designated clone 880-8 that was also activated in a living body by radiation.…”

Utilization of microRNAs with decreased expression levels in response to X-ray irradiation for fine-tuning radiation-controlled gene regulation

“…In the case of malignant LNCaP cells implanted into a mouse, the promoter also significantly enhanced the luciferase gene expression after X-ray irradiation, although to a lesser degree. However, in an in vitro simulation study of suicide gene therapy, when a prodrug was applied at high concentrations, significant cell death was observed even without X-ray irradiation (4). microRNAs (miRNAs) are low-molecular-weight RNAs (19-24 nucleotides in length) that do not code for polypeptides and control gene expression mainly by interfering with the translation process after binding target sequences complimentary to the miRNA seed sequence present in messenger RNAs.…”