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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 26, 2005 as doi:10.1096/fj.04-3595fje. |
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Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain
1Correspondence: Departamento de Morfología y Biología Celular Facultad de Medicina, C/Julián Clavería s/n, Oviedo 33006, Spain. E-mail: acoto{at}uniovi.es
SPECIFIC AIMS
The Syrian hamster Harderian gland (HG) has been studied largely with regard to its porphyrin accumulation, the photoreactions where these compounds are implicated and the oxidative stress generated in consequence. Even in porphyrin-dependent oxidative stress conditions, this gland is able to survive without compromising its gland integrity. The aim of this study was to establish a relationship between the high levels of oxidative stress present in the HGs of Syrian hamster and the degenerative changes observed in control HGs.
PRINCIPAL FINDINGS
1. Invasive processes
Female Syrian hamster HG shows higher cellular damage than males due to their phorphyrin accumulation in the lumen acini. These accumulated porphyrins contribute to a greater extent to the oxidative stress found in the gland. Thus, acini disorganization, described in the female Syrian hamster HG as a release of cells into connective tissue, is normal in this sex and sporadic in males, which do not accumulate porphyrins (Fig. 1
). A deeper analysis of the HG morphology has revealed that these invasive masses emulate the steps observed in the process of metastasis.
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We have studied the activity of cathepsin H in the HG of both sexes. The results have shown higher cathepsin H activity in female than in male HG, which is in good correspondence with the invasive masses observed in this sex. These data were attested by Western blot analysis and immunohistochemical studies, which demonstrated only positive reaction to anti-cathepsin H in type I cells and none in type II cells. The percentage of type II cells in the Harderian gland of male Syrian hamsters has been estimated between 42 and 50% whereas in females it is 100%; therefore the correspondence with obtained enzymatic values and Western blot analysis was nearly exact.
Another factor related to metastatic behavior is the expression of cytokeratin 19 (CK19). The analysis of the cytokeratin pattern in the Syrian hamster HG manifested that this cytokeratin is highly expressed in the gland, a fact that is considered by some authors as a high degree of invasiveness.
2. Autophagic processes
The habitual presence of glandular tubules filled with a mass of cellular debris is evidence of massive cellular elimination during the physiological oxidative stress which HG suffers (Fig. 1)
. The cellular damage observed in the gland led to considering apoptotic programmed cell death as the most probable death mechanism of the Harderian gland cells. The analyses made in the past and the determination of caspase-3 activity developed in our laboratory discount this mechanism. However, electron microscopy data showed abundant mitochondria, endoplasmic reticulum, and a wide variety of vacuoles in cells with normal-appearing nuclei corresponding to extensive autophagy in the HG epithelial cells. The negative results obtained for caspase activity confirms two important facts: first, the elevated oxidative stress present in the HG of both sexes gives rise to an oxidized environment, which in turn inhibits caspase activation since they present an active site cysteine nucleophile; second, these results strongly suggest that autophagic cell death may be assigned to the caspase-independent type of programmed cell death. The conversion of LC3-I to LC3-II observed in Harderian gland from both sexes by Western blot analysis have confirmed that autophagy is an active process in adult control HGs (Fig. 2
).
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The next step was to determinate the activity levels of lysosomal enzymes cathepsins D and B. The cathepsin activities obtained determinate high levels of cathepsin D and low levels of cathepsin B, as expected in the autophagic process, with no differences between sexes, corroborating ultrastructural studies.
Autophagy cell death needs an intact cytoskeleton, although it could be redistributed. Therefore, we also studied the cytokeratin pattern in the HG using a broad range anti-cytokeratin antibody and we detected four bands corresponding to cytokeratins 6, 8, 18, and 19. The last three are considered the most abundant cytokeratins in malignant cancer.
CONCLUSIONS AND SIGNIFICANCE
A remarkable feature of the Syrian hamster Harderian gland is high porphyrinogenic activity. It has been 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. Efforts in the past to look for a possible explanation about these results were in vain. In the present study, we found that the Syrian hamster HG 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.
In light of our findings, autophagic processes in the Syrian hamster HG are the first result of an elevated porphyrin metabolism observed in both sexes. In this case, autophagy is not a cell death mechanism per se but a constant renovation system that allows normal gland activity to continue. On the other hand, invasive processes, resembling tumoral progression, are a consequence of a strong oxidative stress environment that is observed mainly in female Syrian hamster HG that presents intraluminal porphyrins (Fig. 3
).
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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: 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. 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. Thus, the presence of autophagy in the Syrian hamster HG, far from being a malignant process, allows the gland to continue 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 future analysis of the mechanism(s) by which this gland triggers the survival system to counteract its high ROS production.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3595fje;
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). Thus, photoreactions of these compounds are produced. As a consequence, the generation of reactive oxygen species (ROS) provokes an increase of oxidative stress which gives rise to cellular damage. This process is produce 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 those gland-damaged components to be released into the bloodstream. As both processes can coexist, the possibility of an interconnection between them is opened.