Novel porcine model of acute severe cardiogenic shock developed by upper-body hypoxia.

Despite the urgent need for experimental research in the field of acute heart failure and, particularly cardiogenic shock, currently there are only limited options in large animal models enabling research using devices applied to human subjects. The majority of available models are either associated with an unacceptably high rate of acute mortality or are incapable of developing sufficient severity of acute heart failure. The objective of our research was to develop a novel large animal model of acute severe cardiogenic shock. Advanced left ventricular dysfunction was induced by global myocardial hypoxia by perfusing the upper body (including coronary arteries) with deoxygenated venous blood. The model was tested in 12 pigs: cardiogenic shock with signs of tissue hypoxia developed in all animals with no acute mortality. Cardiac output decreased from a mean (+/- SD) of 6.61+/-1.14 l/min to 2.75+/-0.63 l/min, stroke volume from 79.7+/-9.8 ml to 25.3+/-7.8 ml and left ventricular ejection fraction from 61.2+/-4.3 % to 17.7+/-4.8 % (P

[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.

[Dominant-negative inactivation of p53: the effect of the proportion between trans-dominant inhibitor and its target]. / Dominantno-negativnaia inaktivatsiia p53: vliianie kolichestvennykh sootnoshenii transdominantnogo ingibitora i ego misheni.

Dominant-negative mutations of the p53 tumor suppressor gene and oligomerization of the mutant and wild-type p53 are considered responsible for functional inactivation of the p53 tetramer. Although dominant-negative inactivation of p53 is well reproducible in experimental systems, its contribution to processes occurring in tumor cells heterozygous at p53 is still unclear. To study the effect of dominant-negative inhibitor GSE22 on the p53 activity, cultures coexpressing GSE22 and tetracycline-suppressible p53 were derived from p53-negative cell lines. Transcriptional activity and expression of p53 proved to depend on the proportion between p53 and GSE22. The dominant-negative effect was observed only when GSE22 was in a multifold excess to p53. GSE22 was shown to be suitable for complete reversible inactivation of p53.

[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.