СЕЛЕКТИВНОЕ ИНГИБИРОВАНИЕ KRAS-СИГНАЛЬНОГО КАСКАДА ПРИ КОМБИНИРОВАННОМ ВОЗДЕЙСТВИИ НИЗКИХ ДОЗ РАПАМИЦИНА И ПАКЛИТАКСЕЛА IN VIVO

Background. Therapy with compounds potentially capable to block KRAS oncogene signaling pathway is perspective direction in modern oncopharmacology. The aim of current study was to investigate effects of the combined treatment with rapamycin (RAP) and paclitaxel (PAC) in transgenic zebrafish (Danio rerio) with constant expression of mutant KRASV12 oncogene conjugated to green fluorescent protein (GFP) in epidermal cells. This strain has a modified phenotype due to epidermal hyperplasia and expression of GFP reporter at skin of embryos and adult fish.Materials and methods. Fish embryos 6 hpf were exposed to 0.1 % DMSO solution (control) and various doses of the drugs or combinations thereof. GFP expression in epidermal cells was morphometrically measured at 72 hpf.Results. Dose-related decrease in phenotypic changes up to complete epidermal normalization under RAP 50–400 nM treatment was observed. Treatment with nontoxic for embryos doses of PAC 50–250 nM increased fluorescence level in a dose-dependent manner, indicating an activation of KRAS signaling. Using of lower doses of RAP (10 and 25 nM) or PAC (10 nM) had no statistically significant effect on expression of transformed phenotype. Whereas combined treatment (RAP 10–25 nM and PAC 10–50 nM) dramatically decreased level of epidermal fluorescence and completely normalized phenotype of transgenic fish.Conclusions. Thus, mutual potentiating effect of RAP and PAC in low doses which leads to selective inhibition of the KRAS signaling pathway was revealed, indicating the prospect of further studies of these drugs combination for targeted cancer therapy.Введение. Применение соединений, потенциально способных блокировать функционирование сигнального каскада онкогена KRAS, является одним из перспективных направлений современной онкофармакологии.Цель исследования – изучить эффекты комбинированного действия рапамицина (RAP) и паклитаксела (РАС) на трансгенной линии зебрафиш (Danio rerio), характеризующейся постоянной экспрессией в клетках эпидермиса онкогена KRASV12, конъюгированного c зеленым флуоресцентным белком (green fluorescent protein, GFP). Эта линия имеет измененный фенотип, обусловленный гиперплазией кератиноцитов и флуоресценцией в них GFP-репортера.Материалы и методы. Эмбрионы рыб в возрасте 6 ч помещали в среду с добавлением 0,1 % раствора диметилсульфоксида (контроль) и различных доз исследуемых препаратов или их комбинаций. Время инкубации составило 72 ч, после чего проводили количественную оценку интенсивности флуоресценции GFP-репортера в клетках эпидермиса с помощью компьютерной морфометрии.Результаты. При воздействии RAP выраженность фенотипических изменений уменьшалась вплоть до полной нормализации фенотипа в дозе 50–400 нмоль. РАС в дозе 50–250 нмоль не оказывал токсического влияния на развитие эмбрионов, однако дозозависимо повышал уровень флуоресценции репортера, что свидетельствует об усилении экспрессии онкогена KRAS. Воздействие низких доз RAP (10–25 нмоль), а также PAC (10 нмоль) по отдельности не оказывало статистически значимого влияния на выраженность трансформированного фенотипа. В то же время использование различных комбинаций низких доз этих препаратов (RAP в дозе 10–25 нмоль в сочетании с PAC в дозе 10–50 нмоль) существенно снижало регистрируемый уровень флуоресценции, полностью нормализуя фенотип трансгенных рыб.Заключение. Выявлено взаимное потенцирующее действие низких доз RAP и PAC, приводящее к избирательному ингибированию сигнального каскада онкогена KRAS, что свидетельствует о перспективности дальнейших исследований комбинации этих препаратов для таргетной терапии опухолей

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines

SELECTIVE INHIBITION OF KRAS SIGNALING BY COMBINATION OF LOW DOSE RAPAMYCIN AND PACLITAXEL IN VIVO

Background. Therapy with compounds potentially capable to block KRAS oncogene signaling pathway is perspective direction in modern oncopharmacology. The aim of current study was to investigate effects of the combined treatment with rapamycin (RAP) and paclitaxel (PAC) in transgenic zebrafish (Danio rerio) with constant expression of mutant KRASV12 oncogene conjugated to green fluorescent protein (GFP) in epidermal cells. This strain has a modified phenotype due to epidermal hyperplasia and expression of GFP reporter at skin of embryos and adult fish.Materials and methods. Fish embryos 6 hpf were exposed to 0.1 % DMSO solution (control) and various doses of the drugs or combinations thereof. GFP expression in epidermal cells was morphometrically measured at 72 hpf.Results. Dose-related decrease in phenotypic changes up to complete epidermal normalization under RAP 50–400 nM treatment was observed. Treatment with nontoxic for embryos doses of PAC 50–250 nM increased fluorescence level in a dose-dependent manner, indicating an activation of KRAS signaling. Using of lower doses of RAP (10 and 25 nM) or PAC (10 nM) had no statistically significant effect on expression of transformed phenotype. Whereas combined treatment (RAP 10–25 nM and PAC 10–50 nM) dramatically decreased level of epidermal fluorescence and completely normalized phenotype of transgenic fish.Conclusions. Thus, mutual potentiating effect of RAP and PAC in low doses which leads to selective inhibition of the KRAS signaling pathway was revealed, indicating the prospect of further studies of these drugs combination for targeted cancer therapy

Characterization of CoCas9 nuclease from <i>Capnocytophaga ochracea</i>

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5’-NRRWC-3’ PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.</p

High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal