Planarians Customize Their Stem Cell Responses Following Genotoxic Stress as a Function of Exposure Time and Regenerative State.

Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short- (3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 µM) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed.

Stem cell proliferation patterns as an alternative for in vivo prediction and discrimination of carcinogenic compounds.

One of the major challenges in the development of alternative carcinogenicity assays is the prediction of non-genotoxic carcinogens. The variety of non-genotoxic cancer pathways complicates the search for reliable parameters expressing their carcinogenicity. As non-genotoxic and genotoxic carcinogens have different cancer risks, the objective of this study was to develop a concept for an in vivo test, based on flatworm stem cell dynamics, to detect and classify carcinogenic compounds. Our methodology entails an exposure to carcinogenic compounds during the animal's regeneration process, which revealed differences in proliferative responses between non-genotoxic and genotoxic carcinogens during the initial stages of the regeneration process. A proof of concept was obtained after an extensive study of proliferation dynamics of a genotoxic and a non-genotoxic compound. A pilot validation with a limited set of compounds showed that the proposed concept not only enabled a simple prediction of genotoxic and non-genotoxic carcinogens, but also had the power to discriminate between both. We further optimized this discrimination by combining stem cell proliferation responses with a phenotypic screening and by using specific knockdowns. In the future, more compounds will be tested to further validate and prove this concept.

Redox-Related Mechanisms to Rebalance Cancer-Deregulated Cell Growth.

A delicate balance exists between the process of carcinogenesis and tissue regeneration. A number of malignant tumours are considered the outcome of an impaired or incomplete regeneration process, resulting in persistently dividing cells. Regeneration-competent tissues and animals are able to prevent and counteract growth abnormalities and seem to have a low vulnerability to chemical carcinogenesis. Cancer cell survival depends, among other things, on various redox-related mechanisms, which are targets of currently developed therapies. Disadvantages of these therapies are a lack of specificity and drug resistance. As the majority of these redox-related mechanisms also play an important role in successful and coordinated cell functioning and reproduction, the regeneration process offers a unique parallel context for modern cancer research. This review focuses on the interconnections between regeneration and carcinogenesis and how an understanding of regenerative forces and redox-controlled mechanisms could contribute to the identification of new therapeutic targets to block the growth and survival of cancer cells.

Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

Toxicity profiles and solvent-toxicant interference in the planarian Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure.

To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound-induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent-toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short- (1 day) and long-term (2 weeks) exposures to low DMSO concentrations (up to 1000 µl l(-1) ) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long-term exposure set-up, a concentration of 500 µl l(-1) DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 µl l(-1) DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 µl l(-1) DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 µl l(-1) in this organism. In the second part of our study, we propose a statistical approach to account for solvent-toxicant interactions and discuss full-scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea.

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea.

The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neoblasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induction of stem cell activity may underlie this 'restoring' effect, although a carcinogenic outcome after longer exposure times cannot be excluded.