Retroviral reporter systems for the assessment of activity of stress-induced signal transduction pathways controlled by p53, HIF-1 and HSF-1 transcription factors

The tumor suppressor p53, hypoxia-inducible factor 1 (HIF-1), and heat-shock factor 1 (HSF-1) are the key transcription factors involved in the stress response to damage of the genetic material, hypoxia, or heat shock, respectively. Since these factors play a considerable role in tumor development and progression, modulation of their activities may be used in cancer therapy. To quantitate the transcriptional activities of p53, HIF-1, and HSF-1, reporter constructs were obtained on the basis of retroviral and lentiviral vectors, allowing generation of reporter cultures from almost all cell types. Induction of the reporter ?-galactosidase gene, which reflected the activity of p53 or HIF-1, proved to depend on the concentration of an activating agent and to correlate with induction of the endogenous target genes of the transcription factors. Inhibition of p53 or HIF-1? expression with specific small interfering RNAs (siRNAs) completely abolished the activating effect of stress conditions. The reporter constructs were proposed for screening chemical compounds or genetic elements (siRNA or cDNA libraries) that modulate the activity of p53, HIF-1, or HSF-1 and for studying the components of the relevant signaling pathways

Transcriptional Inhibition of the Human Papilloma Virus Reactivates Tumor Suppressor p53 in Cervical Carcinoma Cells

Inactivation of tumor suppressor p53 accompanies the majority of human malignancies. Restoration of p53 function causes death of tumor cells and is potentially suitable for gene therapy of cancer. In cervical carcinoma, human papilloma virus (HPV) E6 facilitates proteasomal degradation of p53. Hence, a possible approach to p53 reactivation is the use of small molecules suppressing the function of viral proteins. HeLa cervical carcinoma cells (HPV-18) with a reporter construct containing the ?-galactosidase gene under the control of a p53-responsive promoter were used as a test system to screen a library of small molecules for restoration of the transcriptional activity of p53. The effect of the two most active compounds was studied with cell lines differing in the state of p53-dependent signaling pathways. The compounds each specifically activated p53 in cells expressing HPV-18 and, to a lesser extent, HPV-16 and exerted no effect on control p53-negative cells or cells with the intact p53-dependent pathways. Activation of p53 in cervical carcinoma cells was accompanied by induction of p53-dependent CDKN1 (p21), inhibition of cell proliferation, and induction of apoptosis. In addition, the two compounds dramatically decreased transcription of the HPV genome, which was assumed to cause p53 reactivation. The compounds were low-toxic for normal cells and can be considered as prototypes of new anticancer drugs