bafilomycin-a1 and Starvation

Connexin (Cx) proteins form large conductance channels which function as regulators of communication between neighboring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signaling, survival and death processes. Connexin 43 (Cx43), a major connexin isoform in brain and heart, is rapidly turned over. Recent studies implicated that autophagy, a lysosomal degradation pathway induced upon nutrient starvation, mediates connexins, including Cx43, degradation. Here, we examined the impact of nutrient starvation on endogenous Cx43-protein levels and endogenous Cx43-driven intercellular communication in primary bovine corneal endothelial cells (BCECs). Hank's Balanced Salt Solution (HBSS) was used as a starvation condition that induces autophagic flux without impacting the survival of the BCECs. Nutrient starvation of BCECs caused a rapid decline in Cx43-protein levels, both as gap junctions and as hemichannels. The time course of the decline in Cx43-protein levels coincided with the time course of the decline in intercellular communication, assessed as intercellular Ca(2+)-wave propagation in BCECs exposed to a single-cell mechanical stimulus. The decline in Cx43-protein levels, both as gap junctions and as hemichannels, could be prevented by the addition of bafilomycin A1, a lysosomal inhibitor, during the complete nutrient starvation period. Consistent with this, bafilomycin A1 significantly alleviated the decrease in intercellular Ca(2+)-wave propagation. This study further underpins the importance of autophagy as an important degradation pathway for Cx43 proteins during periods of nutrient deprivation, thereby impacting the ability of cells to perform intercellular communication.

Topics: Animals; Apoptosis; Calcium; Cattle; Cell Communication; Connexin 43; Connexins; Epithelial Cells; Epithelium, Corneal; Gap Junctions; Lysosomes; Macrolides; Signal Transduction; Starvation

Yeast Atg8 and mammalian microtubule-associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. Sequence analysis reveals that Atg8 proteins are highly conserved in lepidopteran species. The abundance of endogeous Atg8 and the ratios of Atg8 conjugation to phosphatidylethanolamine (Atg8-PE)/Atg8 are different among several lepidopteran cell lines and different tissues of Helicoverpa armigera larvae. Both the density of fluorescent pre-autophagosomal structures with GFP-Ha Atg8 and the abundance of Atg6 are positively correlated with levels of Atg8-PE in different cell lines. The mutant GFP-Atg8(G116A) has lost the function in punctual formation, suggesting that G116 is important for autophagy. Exogenous factors have significant influences on the conversion of Atg8 in lepidopteran cells. Bacillus thuringiensis enhances the degradation of Atg8 in Spodoptera litura Sl-HP cells. Atg8-PE degrades gradually with extension of amino acid starvation, and bafilomycin A1 can block the decrease through the inhibition of autophagosome fusion with lysosome. Interestingly, high pH is more effective than amino acid starvation in Bombyx mori Bme cells to induce the conversion of BmAtg8 to BmAgt8-PE. Change of the quality of fetal bovine serum in the culture medium results in alteration of the ratio of Atg8-PE/Atg8 in some lepidopteran cell lines.

Topics: Amino Acid Sequence; Animals; Autophagy; Bacillus thuringiensis; Cell Line; Hydrogen-Ion Concentration; Insect Proteins; Macrolides; Molecular Sequence Data; Moths; Phosphatidylethanolamines; Phylogeny; Starvation

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

Topics: Animals; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 12; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chloroquine; Chromones; Cisplatin; Drug Screening Assays, Antitumor; Female; Gene Knockdown Techniques; Humans; Macrolides; Mice; Morpholines; Proteins; Sirolimus; Starvation

In hydra, the regulation of the balance between cell death and cell survival is essential to maintain homeostasis across the animal and promote animal survival during starvation. Moreover, this balance also appears to play a key role during regeneration of the apical head region. The recent finding that autophagy is a crucial component of this balance strengthens the value of the Hydra model system to analyze the implications of autophagy in starvation, stress response and regeneration. We describe here how we adapted to Hydra some established tools to monitor steady-state autophagy. The ATG8/LC3 marker used in biochemical and immunohistochemical analyses showed a significant increase in autophagosome formation in digestive cells after 11 days of starvation. Moreover, the maceration procedure that keeps intact the morphology of the various cell types allows the quantification of the autophagosomes and autolysosomes in any cell type, thanks to the detection of the MitoFluor or LysoTracker dyes combined with the anti-LC3, anti-LBPA, and/or anti-RSK (ribosomal S6 kinase) immunostaining. The classical activator (rapamycin) and inhibitors (wortmannin, bafilomycin A(1)) of autophagy also appear to be valuable tools to modulate autophagy in hydra, as daily-fed and starved hydra display slightly different responses. Finally, we show that the genetic circuitry underlying autophagy can be qualitatively and quantitatively tested through RNA interference in hydra repeatedly exposed to double-stranded RNAs.

Topics: Androstadienes; Animals; Autophagy; Biological Assay; Biomarkers; Fluorescent Dyes; Hydra; Macrolides; Models, Biological; Regeneration; RNA Interference; Sirolimus; Staining and Labeling; Starvation; Wortmannin