Cellular autophagic capacity is highly increased in azaserine-induced premalignant atypical acinar nodule cells

Gábor Réz1, Szilveszter Tóth and Zsolt Pálfia

Department of General Zoology, Loránd Eötvös University, pf 330, H-1445 Budapest, Hungary


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.

Abbreviations: AAC, atypical acinar cell; AACN, atypical acinar cell nodule; AV, autophagic vacuole; AV1, AV2, AV3, AVc, AVt, early, advanced, late, complex and total autophagic vacuole respectively; CHI, cycloheximide; CVF, cytoplasmic volume fraction; VBL, vinblastine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
It became generally accepted in the last decade that cellular autophagy is an indispensable pathway of the catabolic side of cellular steady-state or growth regulation (1,2). In spite of its importance, surprisingly little and sporadic data are available on the intracellular degradation of endogenous proteins and structures of tumor cells. Relatively few papers have dealt with lysosomal proteolysis and/or cellular autophagy in malignant cells (319), where the rate of degradation seemed low (814,1622). As in normal cells (2), cellular autophagy in malignant cells in vitro was shown to be regulated by nutrition (5), but there are practically no data available regarding the activity or regulation of autolysosomal degradation in tumors in vivo. Extension of research efforts to this field seems desirable from both a cell biological and a pathological point of view.

To answer the question what differences exist between the autophagic degradation of normal and altered cells, fully comparable cell types and experimental conditions are needed. A pancreatic, in vivo model system allows us to compare the autophagic reactions of normal, premalignant and malignant cells in different stages of tumor progression. The exocrine pancreatic cell is a useful model system that is extremely sensitive to agents affecting autophagy (23,24). Its further advantage is that it can be subjected to a well-established, simple, efficient, although time consuming, carcinogenesis protocol (25). Using azaserine as initiator and a pancreatotrophic diet as promoter it is possible to produce in one and the same pancreas several tumors, some of which are in different defined stages of tumor progression. A number of data show that 3–4 months after initiation, premalignant lesions called atypical acinar cell nodules (AACN) appear in the pancreas. Later, after 12–19 months of treatment, pancreatic adenomas, adenocarcinomas and, occasionally, metastases can be present in the same animal (2529). Thus, this in vivo pancreatic azaserine carcinogenesis model offers a system in which the following normal, altered and transformed cell phenotypes of common origin may be influenced by in vivo treatment of the animals and their reactions compared: control normal pancreatocytes of untreated animals; control pancreatocytes from animals not treated with the initiator but kept on the trophic diet; control pancreatocytes from the host tissue of tumor-bearing animals; premalignant atypical acinar cells (AACs); pancreatic adenoma cells; pancreatic adenocarcinoma cells; metastatic cells originating from pancreatic adenocarcinoma (i.e. all the significant stages of tumor progression can be studied and compared).

Our present approach to the problem is electron microscopical quantitative morphology. Morphometrical description of volume changes in the autophagic compartment and its subcompartments has already been succesfully used for comparison of the autophagic capacity of different cell types (23,3032). Significant quantitative differences were found in the autophagic activities of liver and pancreatic cells under different physiological (nutritional) conditions (3335). The vinblastine (VBL)-induced increase in cytoplasmic volume fraction (CVF) of autophagic vacuoles correlated well with the metabolic state of ad libitum fed, fasted and fasted and refed animals (35). It was expected, therefore, that quantitative measurements of extension of the autophagic compartment upon VBL treatment will also reveal possible differences between normal and altered phenotypes of pancreatocytes. We also intended to measure the rate of autophagic degradation (% cytoplasmic volume degraded/min) in the aforementioned cells. The method relies on the fact that if an inhibitor of segregation [i.e. of autophagic vacuole (AV) formation] is given, the volume of the autophagic/lysosomal compartment will decrease due to ongoing intralysosomal digestion (2,3642). The rate of this regression is proportional to the rate of autophagic degradation of the cytoplasm. In the present experiment cycloheximide (CHI) was used as a segregational inhibitor (32,3743). At a given time point, the rate of segregation of the cytoplasm quanta into AVs can be calculated as the sum of expansional and regressional rates (42).

The present study focused on the first stage of azaserine-induced tumor progression. Our results show that AACs of primary premalignant acidophilic nodules are characterized by highly increased autophagic capacity when compared with either of the controls.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Animals and treatments
Five-week-old male Wistar rats were purchased from Humán Co. (Gödöllõ, Hungary) and ad libitum fed with a commercial standard laboratory diet (Charles River Hungary Ltd, Budapest, Hungary) for 2 weeks, when their body weight reached 200–250 g. Then they were divided into three experimental groups. The group designated normal controls were further kept on the aforementioned commercial diet throughout the experiment. The food of the other two groups was changed to a semi-synthetic pancreatotrophic raw soybean flour-based diet (25,44,45) containing 25% w/w raw soybean flour and 75% semi-synthetic diet containing casein (20%), DL-methionine (0.3%), wheat starch (11.7%), sucrose (38.3%), cellulose fiber (5%), sunflower oil (20%), choline bitartrate (0.2%), vitamin premix (1%) and salt premix (3.5%). Both premixes were prepared (Bio-Serv Co., Holton Industries, Frenchtown, NJ) according to the recommendations of the American Institute of Nutrition (44). The latter two groups of rats were kept on this diet for a further 6 month period. One of them, designated soya controls, remained untreated, whereas animals in the other one (azaserine group) received a single initiator injection (30 mg/kg body wt i.p.) of aqueous azaserine (Sigma-Aldrich Ltd, Budapest, Hungary) solution (30 mg/ml).

Six months after initiation each group was subdivided into three subgroups, i.e. untreated, VBL-treated and VBL + CHI-treated animals. The latter two received a single i.p. injection of 5 mg/kg body wt aqueous (5 mg/ml) vinblastine sulfate (Gedeon Richter Pharmaceutical Factory Co., Budapest, Hungary). The VBL-treated subgroup were killed 90 min after injection, when the VBL + CHI-treated animals received an aqueous CHI (Sigma-Aldrich Ltd) injection (0.5 mg/kg body wt in 0.5 mg/ml solution) and were maintained for an additional 20 min. The rats were killed by decapitation under ether anesthesia and their pancreata were processed for electron microscopy.

Body weight of the rats was monitored regularly. No rats died or ceased gaining weight in this experiment so that they reached ~850 g body wt by month 6 after initiation.

Morphological and morphometric methods
Tissue pieces were fixed for 2 h in 0.1 M sodium cacodylate–HCl-buffered (pH 7.2) 1% glutaraldehyde and post-fixed in phosphate-buffered 1% osmic acid solution for 1 h. This was followed by block contrasting in 2% aquaeous uranyl acetate and embedding in Durcupan (Fluka Chemie AG, Buchs, Switzerland).

Morphometric measurements were carried out by the point counting method as described by Weibel (46). Four rats at each experimental point were used and four samples were taken from each rat for sectioning. In the case of the azaserine group, semi-thin sections of the tissue samples were made and stained either with toluidine blue–Azur II or methylene blue–basic fuchsin mixture. Premalignant AACN and the surrounding host tissue cells were distinguished under a light microscope by their differential morphology and basophilic staining in both paraplast sections of Bouin-fixed (Figure 1AGo) and semi-thin sections from the plastic-embedded (Figure 1BGo) tissue samples. Four host tissue and four AACN samples were taken for thin sectioning from each azaserine-treated rat. Five electron micrographs were taken per sample at a primary magnification of 4000x in a Jeol JEM-100 CX II electron microscope operated at 60 kV. The cytoplasmic volume fractions of the compartments and subcompartments of interest were expressed as percentage of the cytoplasmic volume. Regarding the uneven distribution of the primary point counts, the Mann–Whitney U-test, rather than Student's t-test, was used for statistical evaluation of the data.



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Fig. 1. Light microscopic appearance of the azaserine-induced atypical acinar cell nodules, whose borders are marked with arrows. (A) Conventional paraplast section stained with toluidine blue. x900. (B) Semi-thin araldite section stained with methylene blue–basic fuchsin. x1800.

 
The autophagic compartment (total autophagic vacuoles, AVt) was divided into three subcompartments according to functional morphological criteria.

Early autophagic vacuoles (AV1) (Figure 2AGo) contain seemingly undegraded material, whereas advanced AVs (AV2) (Figure 2A and BGo) exhibit degrading substances of recognizable origin. Late AVs (AV3) (Figure 2BGo) are usually large sized secondary lysosomes enclosing highly degraded substances whose origin can no longer be determined. AV1, AV2 and AV3 may appear in the sections as individual vacuoles or as foci in the AV1, AV2 or AV3 stages of degradation within multicenter complex AVs (AVc). AVc were most probably formed by fusion of differently aged individual AVs. In the present study CVF of individual AV1, AV2, AV3 and AVc were measured separately. Thus: AVt = AV1 + AV2 + AV3 + AVc.



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Fig. 2. Electron microscopic appearance of differently aged autophagic vacuoles indicated by the numbers of AV1, AV2 and AV3, respectively. (A) x37 000. (B) x25 000.

 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Our morphometric data are summarized in Table IGo. As was expected, VBL treatment caused a significant enlargement of AVt in each of the four different pancreatic acinar cell phenotypes investigated. The size of the AVt compartment in the VBL-treated normal, soya and host control cells was found to be larger by one order of magnitude, whereas in the AACs it was larger by two orders of magnitude than in the respective untreated cells. In agreement with previous measurements in murine pancreatic acinar cells (23,42), most of this expansion arose from accumulation of AV1 + 2 in the rat pancreas also. Accumulation of AV3 and AVc did not play a significant role in expansion of the AVt compartment 90 min after VBL treatment. Time course studies in our laboratory (23,24) showed that these subcompartments start to expand some hours later in mouse pancreatic exocrine cells. The VBL-induced AV2 subcompartment was larger than AV1 in the normal control, but the opposite was true for the three tissues under the influence of the soya diet. This observation is in line with our earlier observation (35) that the ratio of different AV types at the same time point after the same VBL treatment can be different according to the physiological state of the mouse pancreatic tissue. Apart from this variation, no true differences between the three control cell types could be observed in the size of AVt and its subcompartments. An important practical consequence of this observation is that host acinar tissue alone seems to serve well as a satisfactory control when VBL-induced autophagic activities of different progressive stages of azaserine-induced carcinogenesis are under study.


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Table I. CVF (% ± SEM) of the autophagic compartment (AVt) and its subcompartments in control and AACs of untreated, VBL- and VBL + CHI-treated rats
 
As has been frequently discussed previously (2,23,3032), accumulation of AV1 may be the consequence of either inhibition of its conversion to AV2 with segregation rate unchanged or, equally, of stimulated segregation provided the autolysosomal compartment is overloaded with substrate. Regression of AVs after application of a segregational inhibitor may be decisive in this problem. CHI caused almost total regression of the VBL-induced AV1 subcompartment (i.e. it really inhibited segregation) in each of the four acinar cell phenotypes studied. It is also obvious from this finding that conversion of AV1 to AV2 was not significantlyly inhibited 90–110 min after VBL treatment. Therefore, the expansion of AV1 must have been caused by stimulation of segregation by VBL. On the other hand, the AV2 subcompartment did not regress considerably after CHI treatment in any of the three control cells, suggesting that conversion of AV2 to AV3 was retarded, or at least slower than AV1/AV2 conversion. Thus, the present results strengthen our proposal (23,30,31,42) for a dual mode of action of VBL on the autophagolysosomal system. In contrast to the control cells, the small AV2 compartment of AACs regressed to a near zero value after the CHI treatment, i.e. their conversion to AV3 was ongoing.

In the AACs, VBL treatment resulted in a 2–3 times larger AVt compartment than in the controls, more than two-thirds of its volume having come from AV1 and only about one-fifth from AV2. This observation indicates highly stimulated segregation. We approached the rate of segregation and the rate of intravacuolar degradation by analysing the expansion and regression data shown in Table IIGo.


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Table II. Volume changes of the autophagic compartment (AVt) and its subcompartments in control and AACs
 
No considerable variations between the control cells in expansion of the AVt and, notably, of the AV1 + 2 subcompartments were found, but there were differences in the regression values. These different regression rates were clearly caused by different AV2/AV3 conversion and, in part, AV3 regression rates. Therefore, the rate-limiting steps of the VBL-induced autophagic wave in control cells of different physiological states might be at AV2/AV3 conversion and/or intravacuolar degradation.

In order to obtain comparable approximate values for the segregation rates we made a simple calculation (for the formulae see Table IIGo) for the AV1 subcompartment based on the assumption that its expansion in unit time is a function of segregation minus regression (42). Thus, segregation was calculated as the sum of expansion and regression per minute values. The result is an average rate (% cytoplasmic volume segregated/min) during the 90 min period of expansion blocked by the application of CHI. The data in Table IIGo show that neither expansion nor regression (conversion to AV2) rates of AV1 varied significantly between the controls. Consequently, their segregation rates also fell within a narrow range (0.011–0.017%/min). Since in untreated steady-state cells segregation and regression rates were in the range 0.003–0.008%/min (37,38,40), our results, once again, verify that VBL is a segregation enhancer.

A similar conclusion was reached previously from data obtained in liver parenchymal (31), pancreatic (23,42) and seminal vesicle exocrine (30) cells of VBL-treated mice and in isolated and perfused rat liver (47). However, VBL did not influence autophagic segregation in isolated normal rat hepatocytes but caused retardation of conversion of AV1 to advanced forms (4850).The latter phenomenon is routinely interpreted as the consequence of inhibited fusion of autophagosomes (AV1) with enzyme-carrying lysosomes. Attributing a fusion-inhibitory effect to the microtubule poison VBL is reasonable as microtubules are known to take part in directed prefusion organelle movements, among others also within the lysosomal system (52,53). It is not improbable that enzyme deficiency (54) and retardation of the conversion of AV2 to AV3 in our present and other (23,30,31,42) systems also involves VBL inhibition of fusion. As to the divergent results regarding the influence on autophagy of VBL in in vivo systems and isolated hepatocytes, we do not believe that VBL has different modes of actions in different mammalian cell types and experimental systems. One possible explanation for the lack of segregational stimulation by the drug in isolated hepatocytes may be that, as a result of the isolation procedure, autophagic segregation in these cells is very high, perhaps maximal (A.L.Kovács, personal communication) at the moment of exposure to VBL.

Stimulation of segregation in our system was most pronounced in the AACs, where the 0.057%/min segregation rate is the highest ever recorded in any cell type. This rate is 6–20 times higher than those measured in steady-state cells. It is noteworthy that in the AACs the same segregation rate could be calculated from the expansion and regression of either AVt, AV1 or AV1 + 2, which clearly shows that unlike in the controls, AV2/AV3 conversion is not retarded in AACs. Therefore, the high gross regression of AVt in AACs is a measure of the cytoplasmic volume that was degraded inside AVs during the 20 min after application of CHI. The rate of this intravacuolar degradation was 0.040%/min, very high.

The most important conclusion of this study is that the autophagic capacity of carcinogen-induced premalignant AACs is far higher than that of corresponding control cells. In response to the autophagy inducer microtubule poison VBL, these cells are capable of increasing their segregation rate (rate of formation of new AVs) to one at least 3–5 times higher than that of control cells. It seems resonable to think, therefore, that they have an increased segregational capacity compared with the controls.

One can only speculate at present that regulatory factor(s) of the cell segregational capacity may be related to the microtubular cytoskeleton, to the availability of a pool(s) for the segregating membrane or to some other unknown factor(s). Microtubules probably play a stabilizing anti-segregational role, since depolymerizing agents other than VBL (55,56) are also known to induce and the microtobule stabilizing drug taxol to inhibit (57) autophagy. At present neither the source(s) of the segregating membranes nor the mechanism of their formation is known satisfactorily (discussed more deeply in ref. 23).

The other component of autophagic capacity is degradative capacity, which is also unprecedently high in AACs. Intravacuolar degradation in these cells during the 20 min after application of CHI was 5–13 times faster then autophagic degradation rates measured in control or steady-state cells (37,38,40). It is not known at present if AACs have an increased pool of lysosomal hydrolases or whether the increased digestion is due to some other type of up-regulation.

Anyway, cells of such high autophagic capacity may be prone to prompt up-regulation of their self-digesting activity in response to different extracellular signals or drugs.

In contrast to our present results, Seglen and co-workers (2022) reported reduced autophagy in premalignant and malignant liver cells. Their measurements were based on the assumption that cytosolic enzymes are evenly distributed in the cytoplasm and hence non-selective uptake is taken as a measure of overt autophagic segregation. They applied an assay for LDH in hepatocytes isolated from livers in various stages of carcinogenesis and, in fact, observed a progressive reduction in the amount of segregated enzyme down to ~20% of normal in fully malignant cells. These results are convincing, but it is still difficult to compare them with ours and state that the two findings are in clear contradiction. Apart from differences in the in vitro and in vivo systems, means of stimulation of segregation and cell types used in the two studies and the stages of tumour progression of liver and pancreas may differ from each other. According to our immunocytochemical studies in progress, as well as indications in the literature (58,59), there exist more than two premalignant stages in the lengthy progression of azaserine-induced pancreatic carcinoma. As the present study was restricted to 6-month-old AACN and autophagic capacity may vary with age of the lesion, extension of similar investigations to earlier and later stages is expected to clarify this contradiction.


    Acknowledgments
 
We would like to thank Prof. János Kovács for critically reading the manuscript and Attila L.Kovács for meaningful discussions, as well as Sarolta Pálfia, Ágnes Keserû and Mariann Saródy for their excellent technical assistance. This study was partly supported by a Copernicus Contract (CIP-ACT 930210) of the European Union.


    Notes
 
1 To whom correspondence should be addressed Email: grez{at}cerberus.elte.hu Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received January 19, 1999; revised May 4, 1999; accepted June 21, 1999.





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