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

Tumor suppressor p53, hypoxia-inducible factor 1 (HIF-1) and heat-shock factor 1 (HSF-1) are involved as the key transcription factors in cellular response to stress, induced by genetic material damage, hypoxia and heat shock respectively. The protein factors listed above also play an integral part in tumor development and progression. Thus, modulation of their activity may be important for treatment of cancer. In our work we obtained the reporter constructs for quantitative assessment of p53, HIF-1 and HSF-1 transcriptional activity on the basis of retro- and lentiviruses, allowing to obtain reporter cell lines almost out of any cell type. Induction of beta-galactosidase reporter gene expression, reflecting the activity of p53 and HIF-1 factors, depends on dose of treatment and also correlates with the induction of the endogenous target genes expression. The observed effect of activating treatments completely disappeared when the expression of p53 and HIF-1 genes was inhibited with specific siRNAs. The obtained reporter constructs may find the application in the screening of chemical and genetic (such as siRNA- and cDNA-libraries) modulators of transcriptional activity along with the investigation of components of signal transduction pathways modulating the transcriptional activity of those factors.

[Construction of a reporter system for fine quantitative assessment of activity of p53 protein in cultured cells]. / Razrabotka reporternoi sistemy dlia tonkoi kolichestvenoi otsenki aktivnosti belka p53 v kul'turakh kletok.

Since transcriptional activation of genes downregulating cell proliferation mediates the tumor suppressor activity of p53, induction of p53 targets was assumed to adequately reflect the state of p53-dependent pathways. To estimate the p53 activity in cultured cells, self-inactivating retrovirus constructs pSIP-ConA-GFP and pSIP-ConA-LacZ were obtained to express the GFP or beta-galactosidase reporter gene under the control of a promoter containing p53-responsive elements. The advantages of these constructs were efficient delivery, comparable expression in different cells of a culture, and, consequently, the possibility of quantitating the p53 activity induced by various agents. With pSIP-ConA-LacZ, p53 activation in response to 12 chemotherapeutic agents was analyzed in human carcinoma cell line HCT116 and its derivatives HCT116/mdm2 and HCT116ARF, which expressed genes affecting the p53 activity. The analysis was also carried out with human cell lines HEF, WI-38, U2OS, and HT1080 originating from connective tissue. The construct proved suitable for detecting fine differences in p53 activation induced by various stress factors. The constructs were proposed for generating reporter cell lines from various cultures in order to identify the genetic or chemical factors that modulate the p53 activity and may be employed in new antitumor drugs.