Download - Survival mechanisms in a physiological oxidative stress model

©2005 FASEB The FASEB Journal express article 10.1096/fj.04-3595fje. Published online September 26, 2005. Survival mechanisms in a physiological oxidative stress model Cristina Tomás-Zapico, Beatriz Caballero, Verónica Sierra, Ignacio Vega-Naredo, Óscar Álvarez-García, Delio Tolivia, María Josefa Rodríguez-Colunga, and Ana Coto-Montes Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain

Corresponding author: Ana Coto-Montes, Departamento de Morfología y Biología Celular Facultad de Medicina, C/Julián Clavería s/n, 33006 Oviedo, Spain. E-mail: [email protected]

ABSTRACT

The Syrian hamster Harderian gland has as the remarkable feature of an extraordinary rate of porphyrin production, even higher than the liver. The low activity of the last enzyme of the route gives rise to the accumulation of the uncomplex porphyrins in the female glands. Moreover, due to the localization of the Harderian gland, porphyrins exposed to light produce reactive oxygen species and, thus, the gland presents a physiological oxidative stress, with a great number of sings of degeneration, but without compromising the gland integrity. The appearance of abnormal features in this gland was largely described in the past, but the significance is interpreted for the first time in this study. We have found that autophagic processes are the first result of an elevated porphyrin metabolism, as it is observed in both sexes. This mechanism is considered, in this case, as a constant renovation system that allows the normal gland activity to be sustained. Furthermore, there is a second procedure, invasive processes toward connective tissue, which even occasionally reach blood vessels with intravasation of damaged gland components into the bloodstream. This effect is a consequence of a strong oxidative stress environment that is mainly observed in the female gland, resembling to tumoral progression. Both mechanisms, autophagy and invasive processes, have to be implied in the maintenance of the gland integrity.

Key words: autophagy • Syrian hamster • Harderian gland • cathepsins • cytokeratins • apoptosis • invasive process • metastasis

arderian glands (HGs) are large orbital lacrimal glands present in most terrestrial vertebrates. In the Syrian hamster, HGs show a strong dimorphism, which is under androgenic control (1–3). A remarkable feature of the HG is its high porphyrinogenic

activity. Even in male Syrian hamsters, with much lower porphyrin concentration than female HG, this activity is higher than in the liver. In most tissues, protoporphyrin IX is converted to heme by ferrochelatase. However, in the HG, the activity of this enzyme is very low so that the uncomplexed porphyrins can rise to unusually high amounts. These secretory products, as well as precursors such as δ-aminolevulinic acid, give rise to a type of physiological oxidative stress that includes photoreactions of these compounds. Light is entering the gland and, in mice, an excess of light exposure leads to its damage and squamous metaplasia (4). Part of these effects

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may be attributed to protoporphyrinyl cation radicals, superoxide anions, and singlet oxygen generated by excited protoporphyrin IX, another part to hydroxyl radicals deriving from photoreactions of δ-aminolevulinic acid (5–8). Additionally, hydroxyl radicals may be formed via a Fenton-type reaction by copper-porphyrins found in the HG (9).

Thus, the physiological situation in the Harderian gland, with very high levels of oxidative stress (5, 6), resembles very much a pathological one, namely that of porphyria cutanea tarda, in which skin lesions are originated from same photoreactions. In a sense, the HG seems to survive in a state of “permanent porphyria”, however, at the expense of some unavoidable cellular alterations. This cell damage has been described by several authors (11, 12) who have reported great numbers of signs of degeneration in the gland from cells with dilated mitochondria and short and swollen endoplasmic reticulum cisternae, which are called clear cells (12), until invasive activity affects even the blood vessels of the gland (11). However, the death mechanism developed by these harderian cells remains less clear, and attempts to establish the type of programmed cell death (PCD) exhibited by this gland have been unsuccessful (10).

PCD comprises two subtypes, as revealed by electron microscopy. Apoptosis, or type I PCD, is characterized by condensation of cytoplasm and preservation of organelles, essentially without autophagic degradation. Autophagic cell death, or type II PCD, exhibits extensive autophagic degradation of Golgi apparatus, polyribosomes, and endoplasmatic reticulum, which precedes nuclear destruction (13). The mechanisms and implicated molecules are different in each type of cell death: Caspases, a class of proteases, play an essential role in the induction and execution of apoptosis and, when activated, they acquire the ability to cleave key intracellular substrates, which results in the biochemical and morphological changes associated with apoptosis (14). Recent reports have indicated that lysosomal enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol during PCD with acting cathepsin D as a death mediator and its death-inducing activity is usually suppressed by cathepsin B (14, 15). Although the translocation of these enzymes appeared usually to be restricted to apoptosis (16), accumulating evidences suggest that these cysteine and aspartic proteases are not only confined to apoptosis (15, 17, unpublished data from our laboratory).

As to biochemical differences, recent evidence suggests that the cytoskeleton exhibited distinct fates during autophagic and apoptotic cell death. In apoptosis, the cell preparatory as well as executional steps include depolymerization or cleavage of actin, cytokeratins, lamins, and other cytoskeletal proteins (17, 18). In contrast, by autophagic death, the cytoskeleton was found to be redistributed but largely preserved (13, 17). Furthermore, Kabeya et al. (19) have suggested the microtubule-associated protein light chain 3 (LC3) as an ideal marker for the autophagic process. LC3 is converted into LC3-I, and part of this modified protein is subsequently converted into LC3-II, localized to autophagosomal membranes. The conversion of LC3-I to LC3-II is induced under starvation conditions, which are well known to induce autophagy.

The aim of our study is to establish a relationship between the high levels of oxidative stress present in the HGs of Syrian hamster, due to the extremely high porphyrin synthesis and accumulation and the degenerative changes observed in control HGs. These findings emphasize in the study the differences between two processes that are described in this article: role of autophagic cell death mechanism and tumoral-like processes in glandular survival.


MATERIALS AND METHODS

Animals

Thirty-two one-month-old male and female Syrian hamsters (Mesocricetus auratus) (Harlan Interfauna Ibérica, Barcelona, Spain) were divided 4 per cage and housed in a 14:10 h light/dark cycle and temperature controlled (22±2°C) room. Animals received tap water and a standard pellet diet ad libitum.

After two months in the animal house, hamsters were killed and Harderian glands were immediately removed, frozen on liquid nitrogen, and stored at –80°C until the experiments were performed.

If not indicated otherwise, 100 mg of each tissue were homogenized with a polytron homogenizer at 4°C in 1 ml of lysis buffer (20 mM HEPES, pH 7.4; 2 mM EDTA; 1% Nonidet P-40; 5 mM dithiothreitol; Na-deoxycholate 0.25%) with proteases inhibitors (1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, and 1 μg/ml aprotinin). The tissue homogenates were then centrifuged for 6 min at 3000 rpm at 4°C. Supernatants were collected and centrifuged again at the same conditions. The protein content of the supernatants was measured by the method of Bradford (20).

Morphological studies

Harderian glands for structural and ultrastructural studies were lightly fixed by immersion in a solution containing 1.5% glutaraldehyde, 2.5% paraformaldehyde in phosphate buffer 0.1 M (pH 7.4). The fixation was prolonged overnight in fresh fixative at 4°C. The tissues were postfixed in 1% OsO4 during 2 h. After dehydration in graded acetone, the pieces were embedded in Taab 812. Semithin sections (1 μm) were stained with toluidine blue, studied, and photographed in a Leitz Ortoplan microscope. Ultrathin sections were collected on cooper grids, stained with uranyl acetate–lead citrate, and examined with a transmission electron microscope Zeiss EM-109 (Zeiss, Germany) operating at 80 kV. Negatives were scanned by hpscanjet 3970, imported by Adobe Photoshop 7.0.1 and incorporated into the figure by Corel Draw 8.0.

Immunohistochemistry

Harderian glands were fixed for 15 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in the same buffer for at least 2 days, and washed in PBS. The specimens were embedded in paraffin by standard methods. The sections were rinsed 3 × 10 min in 0.01 M PBS with 0.1% Triton X-100 and 0.25% bovine serum albumin (BSA) (PBS-TB), and blocked in 30% rabbit serum in PBS-TB for 30 min. This was followed by the incubation in anti-goat cathepsin H (sc-6496, Santa Cruz Biotechnologies, Santa Cruz, CA), anti-goat LC3 (sc-16756, Santa Cruz Biotechnologies) for 24 h at 4°C in a humid chamber. After 3 × 10 min washes in PBS-TB, the sections were incubated in peroxidase conjugate rabbit anti-goat IgG for 1 h at room temperature, washed 3 × 10 min in PBS-TB, and incubated in peroxidase anti-peroxidase complexes, for 1 h at room temperature. Finally, the sections were washed 2 × 10 min in PBS-TB, and 10 min in Tris/HCl buffer (pH 7.6) and incubated 5 to 15 min in 0.05% 3,3′-diaminobenzidine/HCl with 0.005% H2O2 in Tris/HCl, The stained sections were studied by a


light microscope (Leica DMLB, Germany); the images were received by Leica Camera D200, imported by Corel Photo-Paint 8.0, and incorporated into the electronic figure by Corel Draw 8.0

Immunocytochemistry

Harderian glands were fixed for 15 min in 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in the same buffer for at least 2 days, and washed in PBS. Sections of 60 µm were obtained with a vibratome (Campden Instruments, Ltd., UK) and collected in PBS. For the present study, the immunohistochemical procedure was performed using an indirect method, with avidin-biotin-peroxidase complex (21) with minor modifications.

Ultrathin sections were stained with uranyl acetate-lead citrate and examined with a transmission electron microscope Zeiss EM-109 operating at 80 kV. Negatives were scanned by hpscanjet 3970, imported by Adobe Photoshop 7.0.1 and incorporated into the figure by Corel Draw 8.0.

Caspase-3 activity assay

Caspase-3 activity was determined by using a colorimetric assay according to the manufacturer's instructions (CASP-3-C, Sigma, Germany). In the assay system, the colorimetric substrate (Ac-DEVD-pNA) is hydrolized by caspase-3 and p-nitroaniline (pNA) is released from the substrate. The concentration of the pNA released from the substrate was monitored by a plate reader (ELx800 uv, Bio-Tek) at 405 nm. Data were normalized by protein content of each sample. Results of caspase-3 activity were expressed as μmol pNA per min per milligram of protein.

Cathepsin assays

The cystein-proteinases cathepsin B (EC 3.4.22.1) and cathepsin H (EC 3.4.22.16) were assayed fluorimetrically (CytofluorTM 2350. MILLIPORE) according to Barrett (22) with minor modifications (23) using Z-Arg-Arg-MCA as specific substrate for cathepsin B and Z-Phe-Arg-MCA for cathepsin H (Sigma, Germany). For the assay, 40 μl of tissue homogenates (see above) was diluted with 300 μl of incubation buffer (100 mM sodium acetate, pH 5.5, containing 1 mM EDTA, 5 mM dithiothreitol, and 0.1% Brij-35). Of this dilution, 50 μl was pipetted into 96-well fluorescence microtiter plate. Reaction was started by adding 20 μl substrate solution (40 μM Z-Arg-Arg-MCA or Z-Phe-Arg-MCA in incubation buffer) and incubated at 37°C for 20 min. The reaction was stopped by the addition of 150 μl of stop buffer (33 mM sodium acetate, pH 4.3, and 33 mM sodium chloroacetate.) An excitation wavelength of 360 nm and an emission wavelength of 460 nm were used, with aminomethylcoumarin solutions (MCA) as standards. The cathepsin results were expressed as enzymatic milliunits/ mg protein.

The aspartate-proteinase cathepsin D (EC 3.4.23.5) was assayed spectrophotometrically (UVIKON 930) at 280 nm according to Takahashi and Tang (24), with minor modifications (23) and the use of hemoglobin as substrate. Tissue homogenates (200 μl; see above) were mixed with 500 μl of substrate solution (3% hemoglobin in 200 mM acetic acid) and incubated at 37°C for 30 min. The reaction was stopped by addition of 500 μl of 15% trichloroacetic acid, and samples were kept at 4°C during 30 min following a centrifugation at 12,000 × g for 5 min. The optical densities of the supernatants were read. The cathepsin D results were expressed as enzymatic units/ mg protein.


Western blotting

Samples were fractionated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in phosphate-buffer saline containing 0.05% Tween-20 solution and incubated with the respective primary antibody against anti-mouse cytokeratins (Sigma), anti-goat cathepsin D (sc-6486, Santa Cruz Biotechnologies), anti-goat cathepsin H (sc-6496, Santa Cruz Biotechnologies) and anti-goat LC3 (sc-16756, Santa Cruz Biotechnologies). Development was performed using the Western Blotting Luminol Reagent (Santa Cruz Biotechnologies). Then, membranes were incubated twice in a 0.2 M Glycin/HCl, pH 2.5, buffer containing 0.05% Tween-20 and 100 mM β-mercaptoethanol for 60 min at 60°C. After this step, membranes were blocked and incubated with primary antibody against anti-goat β-actin (sc-1615, Santa Cruz, California) and with its corresponding horseradish peroxidase-conjugated secondary antibody (Sigma, Germany). Detection was effectuated using luminol as substrate.

Statistical analysis

Data are presented as mean values ± SEM calculated from at least three separate experiments, each performed in triplicate. The normality of the data was analyzed by the Kolmogorov-Smirnov test. Statistical comparisons among sexes were made by Student's t test in normal data. The non-parametric test Mann-Whitney was applied in non-normal data. The level of significance accepted was P < 0.05.

RESULTS

Light microscopy studies

The Harderian gland of hamsters consists widely of tubule-alveolar units that, in sections, appear as round, oval, or roughly polygonal profiles smooth in outline, separated by connective strands containing blood vessels and nerve fibers. In males, these units consist of two types of cells (Type I and Type II), which differ in size of lipid droplets: small in Type I and larger in Type II. Moreover, females present porphyrins accretions into acinar lumen and every acinus is integrated by Type I cells only. In both sexes and under control conditions, some damage structures were found in the HG. The cells of the secretory units presented a basophilic basal zone, containing the nucleus and an apical one where lipid droplets are mainly concentrated. Frequently, acinar cells appeared to be in a phase of detachment from the tubule wall, seemingly preparing for being partially or totally released into the lumen of the tubule. Some tubules of the gland were filled with a mass of cellular debris displaying a weak basophilia (Fig. 1A). Sometimes they protruded at the opposite pole toward the connective tissue (Fig. 1B). These characteristics were common for both sexes and stretched along wide glandular zones, distributed at random, and roughly in the same proportion in both sexes.

Moreover, HG from female hamsters exhibited their own damage characteristics that were very rarely present in males HG. In some areas of the gland, acini lost their tubule-alveolar morphology and became interstitial bulks of a material very similar to intratubular one described above. This material also contained lipid droplets and cytoplasmic debris, and nuclei in general were euchromatic and very often showed a picnotic aspect and an irregular profile, although less euchromatic nuclei were also present (Fig. 1C). Occasionally, this material flowed along the intertubular strands of connective tissue, expanding and progressively filling in the whole


available space among the tubule-alveoli. Some tubules reached by this invasive mass lost their morphologic identity and fused with it, increasing the volume of the invasive material. These damage structures were proportionately scarcer than autophagic processes and can coexist with them (Fig. 1C).

Ultrastructural analysis

Studies by electron microscopy revealed characteristics of autophagy in male and female HG. The first detectable ultrastructural alteration was the presence of cytoplasm filled of swollen Golgi complexes, rich in mitochondria, some of them with clear cristae and profusion of late autophagosomes. These autophagosomes are a single delimiting membrane with unrecognizable content (Fig. 1D). Not infrequently, endoplasmic reticulum was surrounding mitochondria (Fig. 1E). During later stages of cell death, cellular debris have been secreted into the lumen of the gland, even nuclei, showing differences in electrodensity with normal secretory cells (Fig. 2A).

Almost exclusively in female HG, since the probability to find in males is extremely low, bulk images of acinar cells were also observed by electron microscopy. The cells implicated in these processes frequently appear as secreting part of the cell into the connective tissue (Fig. 2B). Additionally, collagen fibrils are organized around these cellular pieces and between them and their cells of origin (Fig. 2B). These cellular fragments could contain autophagic vacuoles and mitochondria (Fig. 2B), confirming that the same cells may suffer both processes at the same time. Of particular interest were images of these invasive masses inside blood vessels as happen in tumoral situations (Fig. 2C).

Caspase-3 activity assay

Caspase-3 activity was measured colorimetrically for 48 h. Although different tissue concentrations were assayed, caspase-3 activity was not detectable in all cases (data not shown). At same time, as positive control, human recombinant caspase-3, was measured showing increased colorimetrical values along time reaching 654 nmol pNA released per minute per milligram of protein.

Cathepsin studies

Cathepsin H, B, and D protease activities were assayed by means of their corresponding substrates in the Harderian gland of both sexes. Cathepsin H has been recently related to invasive processes, and in our results it showed significant higher activity in females than in males (P≤0.001; Fig. 3A) a result that was corroborated by Western blot analysis, were cathepsin H expression was greater in females (Fig. 3B). Immunohistochemical analysis for cathepsin H showed that cathepsin H-immunoreactivity was mainly circumscribed to Type I cells; thereby all acinar cells from female Harderian gland exhibited immunopositivity occupying the entire cells but without staining into lipid droplets (Fig. 3C), while male Harderian glands showed immunostaining in Type I cells, but not in Type II cells (Fig. 3E). These results agree with obtained data from Western analysis and enzymatic activity. In both sexes, some cells showed higher immunostaining than others (Fig. 3C), and this immunostaining was presented also in lumen and into invasive masses in connective tissue (Fig. 3C, D).


Cathepsins D and B are lysosomal proteases considered an active part on cell death mechanism. In the case of the Syrian hamster Harderian gland, they did not exhibit significant differences between both sexes with regard to their activities (Fig. 4A, B) as well as in the protein expression pattern for cathepsin D (Fig. 4C), results that point out to the development of autophagy to the same extent in male and female HG.

LC3 analysis

Microtubule-associated protein light chain 3, LC3, is an autophagosomal ortholog of yeast Atg8. LC3-modification is essential for the autophagic process, since the protein LC3-II is localized to preautophagosomes and autophagosomes and is considered as an autophagosomal marker.

We have developed a series of analysis of LC3 starting for protein expression. Western blot showed two bands corresponding to LC3-I and LC3-II (18 and 16 kDa, respectively) with no differences between male and female Harderian glands (Fig. 5A). LC3-immunoreactivity by optic microscopy was not restricted to any particular cell type. Positive immunoreaction was observed at the level of membranes that sequester cytoplasmic portions (Fig. 5B). Likewise, immunopositivity was observed as punctuated labeling distributed in the lumen, glandular cells, and even invasive masses into connective tissue (Fig. 5B). Some cells showed higher immunostaining than others, and no differences between males and females were seen. In a similar way, our observations under electron microscopy showed a clear labeling at the vesicle level, showing punctate staining within vesicles in addition to immunopositivity at membranal level (Fig. 5C, D). Immunoreactivity was also observed at cytosolic level and surrounding the vesicles (Fig. 5C, D).

Cytokeratin pattern

Cytokeratin pattern was analyzed in male and female Harderian gland under control conditions (Fig. 6A). The study was developed using a broad-range anti-cytokeratin antibody, which recognizes various cytokeratins that include type II neutral-to-basic cytokeratin subfamily and type I acidic subfamily.

Four bands corresponding to cytokeratins 6, 8, 18, and 19 were detected. There were not significant differences between sexes in the cytokeratin pattern. Cytokeratin 6 (CK 6) is a “hyperproliferation” cytokeratin expressed in tissues with natural or pathological high turnover. The simple epithelial CK 8 and CK 18 cytokeratins are present in a variety of adult epithelial cell types, where they are the only type of intermediate filament protein. Cytokeratin peptide 19 (CK 19) is a type I keratin, which can be expressed alone in both simple and stratifying epithelia at specific sites, and it is highly expressed in both sexes, compared with the others in our system. Males and females presented a band at 33 kDa below the molecular weight expected for cytokeratin.

The immunoblot analysis for β-actin was also developed to demonstrate that this protein did not degrade, a characteristic of autophagy (Fig. 6B).

DISCUSSION

The Syrian hamster Harderian gland has as remarkable feature: high porphyrinogenic activity. It has been largely shown that porphyrin accumulation in the female lumen acini can result in cell


damage, due to porphyrin’s ability to produce reactive oxygen species (ROS) when they are exposed to light. Recent studies developed by our group have shown that, even in control conditions, the Syrian hamster HG presents a physiological oxidative stress (5, 6). The particular characteristic of this gland is that, even in porphyrin-dependent oxidative stress conditions, it can survive without compromising the gland integrity. The efforts in the past sought a possible explanation about these results were in vain. In the present study, we have found that the Syrian hamster Harderian gland follows two possible pathways to eliminate damaged cells and/or organelles. These routes coexist in the control female HG and in minor proportion in male HG.

Physiological oxidative stress determines Harderian gland survival mechanisms

Female Syrian hamster HG shows higher cellular damage than males, since its porphyrin pathway along the circadian rhythm presents two points of maximum activity vs. one point in males (6). Although female hamsters respond to the oxidative stress increasing antioxidant enzymes, they still present cellular damage such as the appearance of clear cells and acini disorganization (11, 12). Clear cells are considered damaged cells that could be secreted as a whole into the lumen of the gland. The presence of these cells is in good relation with porphyrin production, since their percentage in castrated males increases in the same extent than porphyrins do (10). On the contrary, in males, which produce high concentration of porphyrins but hundred times minor than female HGs, these cells appear in constant and low number (10, 12).

This huge porphyrin production causes dramatically effects in liver, as in erythropoietic protoporphyria (25), in skin, and in porphyria cutanea tarda (26). However, HG survives without apparent physiological effects. Therefore, this gland must have developed some mechanism(s) for releasing its excess products and damaged structures. In the light of the obtained results, one of these routes is the released of damage components by detachment to connective tissue, previously describe as invasive processes.

Invasive Processes

Our co-workers first described acini disorganization as a release of cells into connective tissue (11). A deeper study of these morphological observations leads us to think about these processes as tumoral-like ones. Invasive masses described in the present article emulate every step in the process of metastasis (27): Cells grow as a benign tumor in epithelium, followed by breaking through basal lamina, and eventually invasion of capillaries. These processes are normal in the physiology of the female HG and sporadical in male HG, as both of them are under control conditions. On the other hand, similar morphological damage, although less evident, was observed in the uterine vessels by the trophoblast (28, 29). Previous descriptions of these morphological features restrict their appearance to female HG. However, a profound study of the HG from both sexes shows that these processes are also present in the male HG to a lesser extent. Due to the distribution of these processes at random within the gland, and the fact that they appear scarcely in the male HG, this fact has remained unknown.

Several reports have shown an increased expression of cathepsin H in tumor cells and tissues (30–34), and it was even postulated that higher levels of cathepsin H, possibly the secreted form, in carcinoma cells may contribute to tumor invasiveness (35). In view of the tumoral-like characteristics observed in the Syrian hamster HG, we have studied cathepsin H as a possible protease involved in the degradation of extracellular matrix observed in invasive processes. The


activity of this enzyme was significantly higher in females than in males and this result was confirmed by Western blotting, which showed less cathepsin H protein expression in males than in females. These data were overall attested by an immunohistochemical study, which demonstrated only positive reaction to anti-cathepsin H in Type I cells and no in Type II cells. The percentage of Type II cells in male Harderian gland has been estimated between 42% (36) and 50% (37), thereby the correspondence with obtained enzymatic values was almost exact. Moreover, as the acini disorganization appears more frequently in female Harderian gland of females than male ones, the possibility of the cathepsin H implication arouses. Waghray et al. (38) compared the gene expression profiles in normal and tumor prostate tissues by serial analysis. Cathepsin H was one of the genes that were overexpressed. Further analyses revealed that these cells expressed a truncated, but enzymatically active, form of cathepsin H (35). Obviously, we could not discard the implication of other proteases so further studies are needed.

Analogously to cathepsin H analyses, many studies have showed cytokeratins as tumoral markers. Even they are used for the clinical detection in a variety of carcinomas, one of the most important cytokeratin 19 (CK19) commonly associated with metastatic behavior (39–41). Uenish et al. (42) have observed that CK19-positive hepatocellular carcinoma exhibit a tendency toward hepatic metastasis, suggesting a high degree of invasiveness, a study corroborated by Ding et al. (41). In the female hamster HG, it is common the presence of invasive masses inside blood vessels. Western blot analyses have revealed that in this gland the production of CK19 is excessive. These results open interesting possibilities about the fate of the cells released to the blood vessels.

Autophagic processes

In the past decade, several articles have described abnormal structures into HG of both sexes (10–12). However, the attempts to associate these characteristics with any type of known physiological process were unsuccessful. Even the possibility of apoptosis process was nonconsistent (10). In this article, first biochemical and molecular features of autophagy in HG are described, verifying that morphological structures described belong to with autophagic process.

Thus, electron microscopy data showed abundant mitochondria, endoplasmic reticulum, and a wide variety of vacuoles into cells with normal-appearing nuclei that correspond to extensive autophagy in HG. Autophagic cell death has been observed in physiological states of development (during insect metamorphosis, mammalian embryogenesis) and adulthood (mammary gland post-weaning, ovarian attretic follicles, human endometrium) (17, 43, 44). Therefore, autophagy in HG as androgen-dependent structure seems reasonable. Even clear cells, widely described in the HG literature (10, 12, 45) without interpretation until now, have been reported as characteristic of progressive autophagy by degradation of cytoplasmic components resulting in progressive loss of electrondensity (17). Like apoptosis, autophagic cell death has been described as being completed by phagocytosis (46, 47). In line with the general function of macroautophagy, namely being the major inducible pathway for degradation of cytoplasmic components, including whole organelles, autophagic PCD predominantly appears to be activated when the developmental program or in adulthood, homeostatic mechanisms demand massive cell elimination; in all cases, the bulk of cytoplasm is removed by autophagy before nuclear collapse ensues (17). The situation of physiological oxidative stress, which HG suffers, requests massive


cellular elimination and the habitual presence of glandular tubules filled with a mass of cellular debris is the evidence.

As in numerous biological systems, the cell´s suicide program has been found to involve the autophagic/lysosomal compartment. As it was demonstrated by Antolin et al. (10), the apoptosis process in HG was ruled out. However, because autophagic cell death and apoptosis are not mutually exclusive phenomena, we have studied the caspase-3 activity in the gland since it is considered as one of the main apoptosis executors. The negative results confirm the observations of Antolin et al. (10) and indicate that the cellular turnover has to be performed by other mechanism. Caspases contain an active site cysteine nucleophile, which is prone to oxidation or thiol lakylation (4850), so activity of caspases is optimal under reducing environments. Hampton and Orrenius (51) demonstrated that hydrogen peroxide suppresses both the activation and the activity of caspases. In our system, with high levels of oxidative stress, the inactivation of caspases it is not surprising, since the redox status in this gland is altered due to the free radical formation as a consequence of the porphyrin metabolism (52–58).

These results strongly suggest that autophagic cell death may be assigned to the caspase-independent type of programmed cell death. The lysosomal cystein proteases, cathepsins, have been implicated in the activation of caspases and apoptosis (59–62) and, more recently, autophagy (15, 63). Some authors have studied these lysosomal proteases in PC12 cells following serum deprivation (15, 63, 64) and in sympathetic neurons (65); both of these cell lines showed autophagic features. Isahara et al. (15) have suggested a novel pathway of active cell death, which is regulated by those lysosomal proteinases wherein cathepsin D acts as a death factor and its death-inducing activity can be usually suppressed by cathepsin B. These data are corroborated by the studies done by Broker et al. (66) in non-small cell lung cancer cells, where the inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against microtubule stabilizing agents, which are known to induce caspase-independent cell death.

In the light of those findings, we have studied cathepsin B and D activity in Syrian hamster HG. No significant differences were found between male and female activities, corroborated by morphological studies that have shown roughly same proportion in both sexes, slightly superior in female HG. Likewise, the obtained cathepsin activities determined that cathepsin D was high and cathepsin B was low compared with control values achieved from Syrian hamsters brains (data not shown), displaying that the relation between the autophagic process and the elevated cathepsin D/B ratio is evident in the HG of both sexes. Thus, the present data are in agreement with those obtained by other authors previously (15). By last, the conversion of LC3-I to LC3-II observed in Harderian gland from both sexes by Western blotting have confirmed that autophagy is an active process in adult control HGs.

The autophagy process depends to a greater extent on the cytoskeletal proteins, considering that the sequestration of parts of cytoplasm requires intermediate filaments (cytokeratin and vimentin), and the movement and fusion of lysosomes with the late autophagosomes requires the microtubular system. So, autophagic death would need a preserved cytoskeleton, although it could be redistributed (13, 17). Thus, we have also studied the cytokeratin pattern in the HG as it was described by Bursch et al. (13), who indicated that the fate of the cytoskeleton is different between apoptosis and autophagy.


We have used a broad range anti-cytokeratin antibody and have detected four bands corresponding to cytokeratins 6, 8, 18, and 19. The preservation study developed in in vivo tissues is difficult because of the absence of controls. Thus, all experiments headed toward the study of the preservation of cytoskeleton by autophagy were developed in vitro, using cell culture before cell death induced as control. Nevertheless, after studying autophagy in in vivo different tissues (data not shown) and comparing with Bursch et al. (13) results, we can state that cytokeratins 8 and 18 become essential for the initial formation of autophagosomes or their subsequent fusion with lysosomes depends on microtubule, since they are always present in cytokeratin pattern of autophagic process.

In turn, the appearance of CK8 and 18 in this cytokeratin analysis could have other implications. Thus, cytokeratins 8, 18, together with CK19, are considered as the most abundant cytokeratins in malignant cancer, at least in humans (37, 67, 68). The cytokeratin pair 8 and 18 is normally expressed in all simple epithelia. Recently, Raul et al. (69) have observed in squamous cell carcinomas derived from stratified epithelia the anomalous expression of this CK pair. These authors concluded that increased expression of CK8 in some way changes the phenotypic characteristics of stratified epithelial cells, resulting in malignant transformation. All these return us to invasive processes described above and reinforce our hypothesis that both processes are implicated.

In the light of our findings, autophagic processes in the Syrian hamster HG are the first result of an elevated porphyrin metabolism that is observed in both sexes. In this case, autophagy is not a cell death mechanism per se but a constant renovation system that allows to continuing with the normal gland activity. On the other hand, invasive processes, resembling to tumoral progression, are a consequence of a strong oxidative stress environment that is mainly observed in female Syrian hamster HG, the ones that present intraluminal porphyrins (Fig. 7).

The processes described above have no effects on the rest of the organs of the animal and even maintain the own gland survival. Tumor progression is a complex biologic process for which cells must possess a series of traits that enable them to complete the multiple and sequential steps involved: detachment and emigration from the primary site, invasion of surrounding tissues, entrance into blood or lymphatic vessels (intravasation), escape from the microvasculature (extravasation), seeding, and metastatic growth at distant target sites (70, 71). Recently, it was described that there is often an inverse relationship between autophagic activity and malignant potential, raising the possibility that defects in cellular autophagy contribute to the development of cancer (72, 73). Thus, the presence of autophagy in the Syrian hamster HG, far from being a malignant process, allows to the gland to continuing with its normal activity. The malignancy of the invasive process in the gland is not established yet, since the metastatic behavior of intravasated cells remains unknown. However, the presence of both processes in the control Syrian hamster HG facilitates the future analysis of the mechanism(s) by which this gland triggers the survival system to counteract its high ROS production.

ACKNOWLEDGMENTS

We thank Fernando Jañez and Gloria Menéndez for their excellent technical assistances and Jose Manuel García Fernández for his help in pictures production. This work was partially performed with grants Cajal 01-07 from the Ministerio de Ciencia y Tecnología, Spain, FIS GO3/137 from the Instituto de Salud Carlos III and CAL03-074-C2 from INIA, Spain. A C-M is a researcher


from the Ramón y Cajal Program, Ministerio de Ciencia y Tecnología/Universidad de Oviedo, Spain; C T-Z is a F.P.U. predoctoral fellow from the Secretaria de Estado de Educación y Universidades, Spain.

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Received January 17, 2005; accepted August 4, 2005.


Fig. 1

Figure 1. A) Toluidine blue-stained semi-thin section of a Harderian gland from a male hamster. The typical morphological features, including numerous Type I cells (cI) and Type II cells (cII), are clearly observed. Several cellular debris have been secreted into the lumen of the glands (black stars), even nuclei (arrow). Original magnification: ×410; (B) Semithin sections stained with toluidine blue showing female HG with porphyrin deposits (p) and Type I cells (cI). Some secretory cells are released into the lumen (open arrows), and some tubules appear filled with cellular debris (stars) or protruding towards the connective tissue (arrow). Original magnification: ×410; (C) Stained semithin sections showing interstitial material filling most of the space among the tubules (asterisk), flowed into connective tissue. Some tubules appear fused with invasive mass (open arrows). Picnotic nuclei were immersed into this glandular mass (black arrowheads), as well as less euchromatic nuclei (white arrowheads). Acini with secretory cells in a phase of detachment from the tubule wall coexist with this mass (stars). Original magnification: ×410; (D) Electron micrograph showed late autophagosome (a) with unrecognizable content close to abundant mitochondria (m) and Golgi saccules (G). Original magnification: ×18000; (E) ultrastructural appearance of portion of cytoplasm showing recognizable Golgi saccules (G) and endoplasmic reticulum (arrow), which surround some mitochondria (m). Original magnification: ×12530.


Fig. 2

Figure 2. A) In a final state, cellular debris including nuclei (n) from glandular cells discharged into lumen of the gland. Note low electrodensity structures. Original magnification: ×4200; (B) cell releasing part of its material, with abundance of vesicles (asterisks) inside, into the conjunctive tissue, which is almost totally filled with cellular debris (start). Note the appearance of collagen fibrils around this mass and between it and the cell (arrows). Original magnification: ×4400; (C) Endothelium (arrowheads) of a blood vessel reached by the invasive material. Vacuoles (v) and mitochondria (arrows) within invasive materials are seen. The intravascular mass present morphological features almost identical to those of the intratubular ones (asterisk). Original magnification: ×4400.


Fig. 3

Figure 3. A) Cathepsin H activity was studied in male ■ and female □ Harderian gland, using Z-Phe-Arg-MCA as substrate. Values are means ± SEM ***P ≤ 0.001. B) Immunoblot analysis of cathepsin H in female (F) and male (M) Harderian gland. Total extracts of tissue were subjected to Western blotting with an anti-Cathepsin H antibody. The result shows different protein expression between sexes, with females displaying higher levels of cathepsin H, as shown in the activity pattern. C) Immunohistochemical demonstration of cathepsin H in Type I cells of Harderian gland from female hamster. Note the stronger labeling in some cells (arrows) regarding the others and the continuation of immunostaining also into invasive masses in connective tissue (asterisk). Original magnification: ×410; (D) immunohistochemical demonstration of cathepsin H also in glandular lumen (start) of Harderian gland from female hamster. Original magnification: ×410; (E) Optic micrograph showing a negative reaction at the level of Type II cells (arrows) of Harderian gland from male hamster while Type I cells are positive to the anti-cathepsin H treatment. Original magnification: ×725.


Fig. 4

Figure 4. A) Cathepsin D activity was determined using hemoglobin as substrate in male ■ and female □ Harderian gland. Values are means ± SEM. B) Cathepsin B activity was determined in male ■ and female □ Harderian gland, using Z-Arg-Arg-AMC as substrate. Values are means ± SEM. C) Immunoblot analysis of cathepsin D in female (F) and male (M) Harderian gland. Total extracts of tissue were subjected to Western blotting with an anti-Cathepsin D antibody.


Fig. 5

Figure 5. A) Immunoblot analysis of LC3 in male (1) and female (2) Harderian gland. Total tissue extracts were subjected to Western blotting with an anti-LC3 antibody. B) Optic micrograph of Harderian gland from female hamster showing LC3-immunoreactive cells. The punctuate staining was present into the glandular cells, in lumen (asterisk) and in invasive masses (arrowheads). Note that some cells showed higher immunostaining than others (arrows). Original magnification: ×410; (C) Electron micrograph of part of the Harderian gland showing immunoreactive membranes (arrows), which delimit an unrecognizable material together with punctuate staining (arrowheads). The punctuate staining (open arrows) in addition to diffuse pattern (asterisk) are also present in the cytoplasm. Original magnification: ×20000; (D) epon-embedded sections showing LC3-immunoreactive membranes (arrows), punctuate structures (arrowheads) localized in positive vesicles and also in cytoplasm (arrowheads) together with diffuse staining (asterisk). Original magnification: ×12000


Fig. 6

Figure 6. Immunoblot analysis of cytokeratins in male and female Harderian gland. Total extracts of tissue (25 µg) were subjected to Western blotting with an anti-pan cytokeratin antibody. A) Male (lane 1) and female (lane 2) Harderian glands display the same cytokeratin pattern without differences in the protein expression. B) β-actin immunoblot was also developed to show no protein degradation. These blots are representative of three different experiments.


Fig. 7

Figure 7. Survival mechanisms in the Harderian gland. The low activity of the last enzyme of the porphyrinogenic pathway leads to the accumulation of protorporphyrin IX. The yuxtaorbital localization of the Harderian gland makes the gland accessible to light (hν). Thus, photoreactions of these compounds are produced. As a consequence, the generation of reactive oxygen species (ROS) provokes an increase of oxidative stress that gives rise to cellular damage. This process is produced at the same extent in both sexes, and both of them respond by means of autophagic mechanisms. On the other hand, porphyrins accumulated in the female Harderian gland produce additional ROS and a strong oxidative stress. In this case, the cellular damage is engaged with invasive processes that allow release of those gland-damaged components into the bloodstream. As both processes can coexist, the possibility of an interconnection between them is opened.
