Shared developmental roles and transcriptional control of autophagy and apoptosis in Caenorhabditis elegans.

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells that, depending on the cellular milieu, can either promote survival or act as an alternative mechanism of programmed cell death (PCD) in terminally differentiated cells. Despite the important developmental and medical implications of autophagy and the main form of PCD, apoptosis, orchestration of their regulation remains poorly understood. Here, we show in the nematode Caenorhabditis elegans, that various genetic and pharmacological interventions causing embryonic lethality trigger a massive cell death response that has both autophagic and apoptotic features. The two degradation processes are also redundantly required for normal development and viability in this organism. Furthermore, the CES-2-like basic region leucine-zipper (bZip) transcription factor ATF-2, an upstream modulator of the core apoptotic cell death pathway, is able to directly regulate the expression of at least two key autophagy-related genes, bec-1/ATG6 and lgg-1/ATG8. Thus, the two cell death mechanisms share a common method of transcriptional regulation. Together, these results imply that under certain physiological and pathological conditions, autophagy and apoptosis are co-regulated to ensure the proper morphogenesis and survival of the developing organism. The identification of apoptosis and autophagy as compensatory cellular pathways in C. elegans might help us to understand how dysregulated PCD in humans can lead to diverse pathologies, including cancer, neurodegeneration and diabetes.

Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates.

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.

Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds.

INTRODUCTION: The model organism Caenorhabditis elegans is widely used for genetic studies as well as a living biomonitor in ecotoxicology. In this study, we investigated whether C. elegans may represent a suitable model for rapid preliminary toxicity studies of pharmaceutical compounds. METHODS: For this purpose, we used the EGFR kinase inhibitors BIBU1361, BIBX1382, and an inactive chemical analogue BIBU1476. As a first parameter to score for toxicity, we determined lethality of the wild-type C. elegans strain N2 (Bristol) in the presence of the compounds. The transgenic C. elegans strain PC72 (lacZ, heat shock protein-16 [hsp-16] construct) was used as a report organism for toxic effects. PC72 expresses beta-Galactosidase which is induced by hsp-16 in direct response when exposed to toxic compounds. The expression of beta-Galactosidase in cells was subsequently visualized by histochemical staining with X-Gal. RESULTS: A rank order of potency with respect to lethality was established: BIBU1361>BIBX1382>>BIB1476. The induction of beta-Galactosidase was concentration-dependent for each compound and demonstrated the same order of potency as observed for lethality. Furthermore, these compounds showed the same order for lethality in rodents, the first requirement of validation. DISCUSSION: These results indicate that wild-type C. elegans and the transgenic strain PC72 are both suitable models to determine the toxicity of pharmaceutical compounds. This approach allows for an easy and fast ranking of compound toxicity, which may lead to a more rational choice for further in vivo tests.