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Although cellular autophagy is recognized as a major pathway of macromolecular catabolism, little data are available regarding its activity or regulation in tumor cells. We approach this problem by morphometrical investigation into the possible changes in autophagic activity during progression of rat pancreatic adenocarcinoma induced by azaserine and promoted by a raw soya flour-containing pancreatotrophic diet. In the present study, the autophagic capacity of the carcinogen-induced premalignant atypical acinar nodule cells was characterized and compared with controls (normal tissue of rats kept on standard laboratory or pancreatotrophic diet and host tissue of the premalignant nodules of the azaserine-treated rats). Given for 90 min, vinblastine, an enhancer of autophagic segregation (i.e. formation of autophagic vacuoles), caused a one to two orders of magnitude larger expansion of the autophagic compartment in atypical nodule cells than in the controls. Then a 20 min blockade of segregation by cycloheximide led to regression of the autophagic compartment, which was barely measurable or moderate in the controls but exceeded 50% in the premalignant cells. At the same time, the cytoplasmic volume fraction of early autophagic vacuoles regressed to a near zero value in each cell type. Expansion and regression rates of these nascent vacuoles showed that both segregation and degradation were 6-20 times faster in the nodule than in normal tissue cells. These results show that the autophagic capacity of the premalignant cells in our system is greatly increased, possibly making these cells unusually sensitive to up-regulation of their self-digesting activity in response to different extracellular signals or drugs.

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.