Autophagy

ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: https://www.tandfonline.com/loi/kaup20

Sexual dimorphism of autophagy in Syrian

hamster Harderian gland culminates in a
holocrine secretion in female glands

Ignacio Vega-Naredo, Beatriz Caballero, Verónica Sierra, Covadonga

Huidobro-Fernández, David de Gonzalo-Calvo, Marina García-Macia, Delio
Tolivia, María Josefa Rodríguez-Colunga & Ana Coto-Montes

To cite this article: Ignacio Vega-Naredo, Beatriz Caballero, Verónica Sierra, Covadonga
Huidobro-Fernández, David de Gonzalo-Calvo, Marina García-Macia, Delio Tolivia, María Josefa
Rodríguez-Colunga & Ana Coto-Montes (2009) Sexual dimorphism of autophagy in Syrian hamster
Harderian gland culminates in a holocrine secretion in female glands, Autophagy, 5:7, 1004-1017,
DOI: 10.4161/auto.5.7.9610

To link to this article: https://doi.org/10.4161/auto.5.7.9610

Published online: 01 Oct 2009.

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[Autophagy 5:7, 1004-1017; 1 October 2009]; ©2009 Landes Bioscience

Basic Research Paper

Sexual dimorphism of autophagy in Syrian hamster Harderian gland

culminates in a holocrine secretion in female glands
Ignacio Vega-Naredo,1 Beatriz Caballero,1 Verónica Sierra,1,2 Covadonga Huidobro-Fernández,1 David de Gonzalo-
Calvo,1 Marina García-Macia,1 Delio Tolivia,1 María Josefa Rodríguez-Colunga1 and Ana Coto-Montes1,*
1Departamento de Morfología y Biología Celular; Facultad de Medicina; Universidad de Oviedo; Oviedo, Spain; 2Servicio Regional de Investigacion y Desarrollo
Agroalimentario (SERIDA); Villaviciosa, Spain

Abbreviations: CAT, catalase; CMA, chaperone-mediated autophagy; DRAM, damage-regulated autophagy modulator; FOX, ferrous
ion oxidation xylenol orange; HG, harderian gland; HIF-1, hypoxia-inducible factor-1; IKK, IκB kinase; iNOS, inducible nitric oxide
sinthase; LAMP-2, lysosome-associated membrane protein 2; LC3, microtubule-associated protein 1 light chain 3; MCA, aminomethyl-
coumarin; NFκB, nuclear factor-kappaB; NIK, NFκB inducing kinase; PCD, programmed cell death; PCNA, proliferating cell nuclear
antigen; pNA, p-nitroaniline; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate polyacrilamide gel electrophoresis;
SIRT1, sirtuin 1; SOD, superoxide dismutase; SOD2, manganese superoxide dismutase
Key words: oxidative stress, autophagy, holocrine secretion, NFκB, p53, Harderian gland

The Syrian hamster Harderian gland (HG) has a large Introduction

porphyrin metabolism with a sexual dimorphism, showing
male HGs much lower porphyrin concentrations than female
glands. Damage derived from this production of porphy-
rins, displayed by reactive oxygen species, forces the gland to
develop morphological changes that must have a physiological
significance. Thus, oxidative stress is present in two states: mild
oxidative stress in male HGs and extreme oxidative stress in
female HGs. Cathepsins data gave indirect indications about
the presence of programmed cell death affecting the lysosomal
pathway, especially in female HGs, which showed an accu-
mulation of autophagic bodies. Our results showed different
degrees of autophagy in Syrian hamster HGs depending on
sex and probably controlled by the redox-sensitive transcrip-
tion factors: NFkappaB and p53. The discovery of these sexual
dimorphisms in redox signaling and in autophagy corroborates
previous findings and underlines the key role of reactive oxygen
species in the regulation of autophagy. In addition, in this paper
we propose a physiological significance for these phenomena:
male HGs develop a survival autophagy, while in female HGs,
autophagy culminates in a detachment-derived cell death that
plays a central role in its secretory activity, leading to a massive
glandular secretion.
*Correspondence to: Ana Coto-Montes; Departamento de Morfología y Biología
Celular; Facultad de Medicina; Julián Clavería 6; Oviedo 33006 Spain; Tel.:
+34.985.102779; Fax: +34.985.103618; Email: acoto@uniovi.es
Submitted: 01/09/09; Revised: 07/14/09; Accepted: 07/22/09
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/9610
The Harderian gland (HG) is a compound tubuloalveolar
structure located within the orbital cavities of most terrestrial
vertebrates. The Syrian hamster Harderian glands exhibit marked
sexual differences in cell type and porphyrin production. The
glands of male hamsters have two secretory cell types (Types I and
II), while the glands of females consist of a single secretory cell
type (female Type I) and large intraluminal deposits of porphyrins.
Even in male glands, with much lower porphyrin concentrations
than in females, porphyrin production is higher than in the liver.
Moreover, due to the localization of the Harderian gland, porphy-
rins exposed to light produce reactive oxygen species (ROS) by
photo-oxidation. On the basis of data exposed above, the gland is
an excellent model for studying physiological oxidative stress1 since
it exhibits two oxidative levels: mild oxidative stress in male HGs
and extreme oxidative stress in female glands. In a recent publica-
tion, we showed that the survival strategy of the Harderian gland
is based on autophagic processes that are considered a constant
renovation system.2
Macroautophagy is a cellular degradation process responsible
for the turnover of unnecessary or dysfunctional organelles and
cytoplasmic proteins, being a high-capacity process in which
sequestration and degradation of multiple cytosolic constituents
occur simultaneously in the lysosomal lumen.3 Macroautophagy
involves the formation of double-membrane vesicles called autopha-
gosomes which fuse with lysosomes releasing single-membrane
bound autophagic bodies that are degraded by the lysosomal
proteases.4 It should be noted that these cellular processes may be
recruited by an alternative form of programmed cell death (PCD)
called autophagic type II cell death.5 Like apoptotic cell death,
autophagic cell death is an essential part of growth regulation,

1004 Autophagy 2009; Vol. 5 Issue 7

 Autophagic secretion in Harderian gland
maintenance of homeostasis, cell defense and adaptation to an
adverse environment.
Many macromolecules undergoing degradation inside lysosomes
contain iron that, when released in labile form, makes lysosomes
sensitive to oxidative stress.6 Hence, it has been postulated that the
regulation of autophagy by oxidative stress7 occurs with the redox-
sensitive transcription factors Nuclear Factor-kappaB (NFκB)8
and p53,9 acting as key mediators.
NFκB is composed of homo- and heterodimers of five members
of the Rel family, which usually form p50/Rel heterodimers. The
NFκB dimers are normally sequestered as latent complexes in the
cytoplasm by IκB inhibitors. Accordingly, NFκB induction is
mainly based on inducible IκB degradation. The NFκB transcrip-
tion factor is involved in different aspects of proliferation, cell
survival, and even PCD, playing a role in normal development
and homeostasis.8 NFκB most commonly antagonizes PCD by
activating the expression of antiapoptotic proteins, such as Bcl-2
and other antioxidant molecules.
The tumor suppressor p53 plays important roles in cell cycle
control and cell death. In normal cells, p53 levels are low, but
its expression can be increased in response to stress signals acting
through both transcription-dependent and -independent mecha-
nisms to coordinate cellular responses that either prevent or repair
damage.
Here, for the first time, we describe gender-related differences
in the autophagic-lysosomal pathway in the Syrian hamster HG
that are directly related to oxidative stress status and controlled
through the redox-sensitive transcriptions factors NFκB and p53.
Furthermore, we postulate the mechanism that governs these
gender-related differences. The discovery of this modulation in the
autophagic pathway in vivo corroborates previous findings that
consider the interaction between the different types of lysosomal
mechanisms and reactive oxygen species signaling.10
Results
Sexual dimorphism in oxidative stress. To compare the oxida-
tive status between male and female Syrian hamster HGs, we
determined their content in protein carbonyl and water-soluble
hydroperoxides, and the activities of the antioxidant enzymes
catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC
1.15.1.1). As shown in Figure 1A, female HGs had more protein
damage than males. In addition, the measurement of H2O2 (or
short-chain water-soluble hydroperoxides) with the FOX test
showed that female H2O2 steady-state levels reached almost three
times those of males (Fig. 1B). Respect to the antioxidant enzymes
activities, female HGs showed the highest SOD activity (Fig. 1C).
It may be hypothesized that an imbalance between superoxide
radical formation and hydrogen peroxide degradation might acti-
vate cell death. Our data showed high CAT activity (Fig. 1D) in
females, even though the hydroperoxide levels continued to be
high. These findings indicate that female HGs have a decreased
capacity for detoxifying hydrogen peroxide.
NFκB activation. We examined its activation state using
immunoblot analysis of key proteins in the NFκB pathway: IKKα,
phospho-IκBα (Ser32), IκBα, phospho-NFκB-p65 (Ser536).
www.landesbioscience.com
Activation occurs via phosphorylation of IκBα at Ser32 by IκB
kinase complex (IKK), resulting in the ubiquitin-mediated protea-
some-dependent degradation of IκBα and the release and nuclear
translocation of active NFκB dimmers. IKK expression was higher
in males and the phospho-IκBα (Ser32) immunoblotting was
according to this, leading to IκBα degradation in male HGs, as
shown in Figure 2A. Furthermore, male HGs showed the highest
expression of NFκB p65 phosphorylation at Ser536 which regu-
lates activation, nuclear localization and transcriptional activity
(Fig. 2A). According to this, nuclear extracts were prepared and
subjected to western blotting against NFκB p50 and p65 subunit
antibodies, and the results confirmed higher NFκB translocation
to the nucleus in males than in females (Fig. 2A).
Moreover, nuclear extracts were used in a NFκB p50
Transcription Factor Assay corroborating the former results and
demonstrating that male HGs have higher NFκB p50 DNA
binding activity than female glands. Therefore, we can state that, in
Syrian hamster HG, the NFκB signaling pathway is more activated
in males than in females (Fig. 2B).
Then, we examined the consequence of NFκB activation
in male HGs studying the expression of some transcription
target genes related to its functions in ROS-signaling (SOD2 or
MnSOD), cell death (Bcl-2) and inflammation (iNOS). As shown
in Figure 2C we found that the levels of the antioxidant proteins
SOD2 and Bcl-2 were upregulated in male glands. However, this
upregulation was not coincident with the extraordinary expres-
sion of the pro-inflammatory marker iNOS in female HGs. These
results seem to indicate that NFκB activity in males promotes cell
survival through the modulation of the ROS-signaling pathway,
and that NFκB is not involved in the transcriptional regulation of
iNOS in female glands. These results are relevant to more precisely
understand the role of NFκB in a physiological oxidative stress
situation.
p53 activation. We sought to examine the p53 pathway evalu-
ating the subcellular localization of p53 and levels of p53-related
proteins: damage-regulated autophagy modulator (DRAM),
Sirtuin 1 (SIRT1), proliferating cell nuclear antigen (PCNA) and
hypoxia-inducible factor-1α (HIF-1α).
Thus, western blotting was performed to detect the presence of
p53 in Syrian hamster HGs in nuclear and cytoplasmic extracts.
The nuclear localization is critical for its transcriptional activity
as well as cell death-inducing function. The analysis of the bands
obtained from the nuclear fractions showed higher nuclear trans-
location in female HGs than in male ones. This higher presence
of p53 in female HG nuclei may activate genes that prevent cell
growth and DNA damage. In contrast, the immunoblot analysis of
cytoplasmic extracts revealed that p53 is significantly accumulated
in cytoplasm from male HGs (Fig. 2D).
The phosphorylation of p53 at Ser15 was explored to evaluate
its transactivation function since this phosphorylation promotes
its activation and stabilization. Unexpectedly, male HGs showed
a phosphorylation at Ser15 in p53 (Fig. 2D). To confirm this
data we evaluated the levels of DRAM, a transcription target gene
of p53 involved in autophagy activations11 and, effectively, we
showed elevated levels in male glands. SIRT1, a class III histone
Autophagy 1005
 Autophagic secretion in Harderian gland

Figure 1. Comparison of protein damage (A), hydroperoxides levels (B) and the activities of the antioxidant enzymes superoxide dismutase (C) and
catalase (D), between sexes of Syrian hamster Harderian glands. Data are represented as mean ± s.d from three independent experiments. Significant
differences between sexes: *p < 0.05; **p < 0.01; ***p < 0.001.

deacetylase, has the ability to deacetylate p53 decreasing its tran-
scriptional activity. Our data also showed a higher level of this
deacetylase in male glands (Fig. 2E).
p53 inhibits replication of the genome blocking cell cycle
progression at G1/S check point in response to DNA damage
and PCNA is a highly conserved cellular protein that functions
in DNA replication and repair. Thus, it was described that p53
tightly regulates DNA replication and repair by modulating
the levels of PCNA.12 The immunoblot analysis against PCNA
showed that female glands which presented higher p53 nuclear
translocation have the highest expression (Fig. 2E). Likewise,
HIF-1 is a transcription factor which regulates cellular energy
metabolism in response to oxidative stress13 but besides HIF-1,
p53 is accumulated and activated under conditions of prolonged/
severe stress. Previous observations suggest that activated p53
decreases HIF-1α protein levels by accelerated proteasome-depen-
dent degradation, but DNA damage is an essential prerequisite.14
Therefore, we developed an immunoblot for HIF-1α and levels
of this transcription factor were only detected in male HGs.
Probably, female HGs could be terminating HIF-1 responses and
initiating PCNA-dependent DNA repair (Fig. 2E) by p53-medi-
ated actions.
These results suggest that the sexual dimorphism in oxidative
stress triggers complex regulatory events in the p53 pathway which
present gender-related divergences promoting different functions
depending on sex.
Apoptosis. The TUNEL reaction was developed in parallel with
a proapoptotic agent-treated HG as a positive control to identify

1006 Autophagy 2009; Vol. 5 Issue 7

 Autophagic secretion in Harderian gland

Figure 2. Western blot analysis for studying the NFκB pathway. (A) expression of IKKα, phospho-IκBα (Ser32), IκBα, phospho-NFκB-p65 (Ser536) and
nuclear expression of NFκB p65 and p50 in male and female Harderian gland extracts from a representative experiment. The NFκB p50 colorimetric
transcription factor assay (B) was run with 5 μg protein/well from nuclear extracts of both sexes and confirmed that male Harderian glands present
the highest NFκB DNA binding activity. The immunoblot analysis of NFκB transcription target genes. (C) Manganese superoxide dismutase (SOD2),
B-cell lymphoma 2 (Bcl-2) and inducible nitric oxide synthase, (iNOS) showed in male glands the highest levels for the antioxidants proteins SOD2 and
Bcl-2 but the lowest for iNOS. Western blot analysis for study the p53 pathway: nuclear and cytoplasmic localization and transactivation by the rate of
phospho-p53 (Ser15) (D) in male and female Harderian gland extracts from a representative experiment. The levels of p53-related proteins. (E) damage-
regulated autophagy modulator (DRAM), Sirtuin 1 (SIRT1), proliferating cell nuclear antigen (PCNA) and hypoxia-inducible factor-1α (HIF-1α) showed
sexual divergences in p53 regulation. The activity of these redox transcription factors had strong sexual differences.

www.landesbioscience.com Autophagy 1007

 Autophagic secretion in Harderian gland
the apoptotic cells. The results were negative in both sexes, indi-
cating that apoptotic processes are not occurring in HG cells.
Lysosomal cell death. In order to characterize the lysosomal-
related processes present in the Syrian hamster Harderian gland, we
have followed the guidelines and methods proposed by Klionsky et
al.15 and Vega-Naredo et al.16
Cathepsin B and D protease activities were assayed in the
HGs of both sexes and compared with their values in the liver
because lysosomal viability is related to high cathepsin B and low
cathepsin D activities. Figure 3A shows that cathepsin B activity
was higher in the liver than in the HGs, and the opposite result
was found for cathepsin D activity (higher in the HGs than in the
liver). Furthermore, Table 1 lists the cathepsin D/B activity ratios,
showing a lower cathepsin D/B activity ratio in the liver than in
HGs from both sexes. Taking this into account, we are able to
affirm that the HG develops lysosomal-related cell death processes,
as opposed to another porphyrinogenic tissue, the liver.
In relation to sex divergences, the lower cathepsin D activity
in female HGs compared to that in males is an important result
to consider. In fact, cathepsin D western blot analysis (Fig. 3B)
confirmed that in the female HG, the mature form of cathepsin
D undergoes limited proteolytic processing, leading to an accu-
mulation of pro-cathepsin D (46-kDa) and, consequently, to a
reduction in its activity.
To complete the study, cathepsin D immunohistochemistry
was developed. Male HG immunohistochemistry seemed to show
high cathepsin D expression, mainly in type II cells, displaying a
defined transmembrane stain around vesicles. Cathepsin D immu-
nohistochemistry from female HG sections showed a gradual loss
of expression towards basal layers (Fig. 3C). Therefore, we hypoth-
esize that pro-cathepsin D may be secreted at the apical side of
female type I cells, as it was described in mammary glands.17
Lysosome-associated membrane protein 2 (LAMP-2) is
commonly considered a marker of lysosomes and late endosomes.
In order to deepen the study of the lysosomal pathway in Syrian
hamster HGs, a LAMP-2 immunoblot analysis was carried out
showing a LAMP-2 deficiency in female glands (Fig. 4A).
LAMP-2 gene undergoes alternative splicing, rendering three
different mRNA species that encode the LAMP-2A, LAMP-2B
and LAMP-2C variants of this protein. LAMP-2A is the only
isoform shown to participate in CMA; therefore, it is used as a
marker of CMA activity.18 To evaluate if these types of degradation
processes are occurring in Syrian hamster HGs, we have performed
a reverse transcription-polymerase chain reaction (RT-PCR) assay,
showing LAMP-2A transcription rates in males, as well as in
females (Fig. 4B).
We have performed western blot analysis using antibodies
against Beclin 1, LC3 and beta-actin, in order to further study
lysosomal-related processes, such as macroautophagy, in the Syrian
hamster Harderian gland.
Beclin 1, a class III phosphatidylinositol 3-kinase-interacting
protein, plays an essential role in promoting autophagy. The
immunoblot analysis showed slight differences between both
sexes with regard to protein expression (Fig. 4C), although female
HGs showed the highest expression, indicating that autophagy is
upregulated in female HGs.
1008
In turn, the microtubule-associated protein 1 light chain 3
(LC3) is an autophagosomal ortholog of yeast Atg8. LC3 modifica-
tion is essential for the macroautophagic process, since the protein
LC3-II is localized to preautophagosomes and autophagosomes,
and it is considered an autophagosomal marker. Western blot
analysis showed two bands corresponding to LC3-I and LC3-II
(18 and 16 kDa respectively) in HGs of both sexes (Fig. 4C) but
with higher expression in female than in male glands. Therefore,
female HGs had an increased accumulation of vacuoles carrying
the autophagic marker, but this data at punctual moment does not
indicate the total autophagic flux. Curiously, the ratio of LC3-II/
LC3-I in our experiment was higher in males (Fig. 4D) which may
due to an increased turnover of LC3 in male glands as a result of
robust and maintained activation of autophagy.
Recently, it was proposed that the autophagic machinery is
linked with the actin cytoskeleton.19 In this study, the beta-actin
western blot analysis revealed a significant loss of beta-actin expres-
sion in female HGs (Fig. 4C).
Morphological study. Through electron microscopy, it was
revealed that Syrian hamster HG cells from both sexes undergo
autophagy. HG cells in males had a cytoplasm filled with vacuolar
structures that were localized around the perinuclear region and
contained cytoplasmic components with an indistinguishable mate-
rial (Fig. 5A). This morphology corresponds with an autophagic
process, and in this way, these autophagic vacuoles seem to be
late autophagosomes. Furthermore, it was usual to observe female
HG cells containing whorls of membranous material (Fig. 5B).
These membranes appeared to be made from smooth endoplasmic
reticulum. Likewise, female HG cells had autophagic vacuoles and
endoplasmic reticulum surrounding abundant organelles (Fig. 5C).
The hallmark characteristic of normal glands is the presence
of a hollow lumen (Fig. 6A), but in Syrian hamster HGs, some
acinar cells showed protuberances towards the lumen (Fig. 6B).
These protuberances seemed to grow until being released into the
lumen. Consequently, some tubules in the gland appeared filled
with a mass of cellular debris and nuclei (Figs. 3D, 6B and C),
even leading to their collapse (Fig. 6D). And then, when both the
nuclear envelope and the plasma membrane lost their integrity, the
cells exhibited intense vacuolization of the cytoplasm, especially
in females, as observed by optic microscopy (Fig. 6D), and more
profusely in zones where intertubular-syncytial masses coexist (Fig.
6E). These alterations of the cell structure and the disruption of
cell-matrix and cell-cell interactions (data not shown) lead to cell
detachment in female Harderian gland. These findings, the pres-
ence of cell death processes and the release of granules, together
with cellular debris and some nuclei in the lumina, suggest a holo-
crine type of secretion for female HGs.
Discussion
On the basis of our previous studies20,21 physiological autophagy
is a constant renovation system that allows cells to maintain vital
functions and adapt to environmental stress, being a prominent
feature associated with sexual organ adjustment. Our results, in
HGs, confirm the presence of autophagic processes in both, male
and female HG, but with important differences between them.
Autophagy 2009; Vol. 5 Issue 7
 Autophagic secretion in Harderian gland

Figure 3. The activities of the lysosomal proteases (A) cathepsins B and D were determined in Harderian glands (HG) and livers from male and female
Syrian hamsters, using the substrates Z-Arg-Arg-AMC and hemoglobin, respectively. Cathepsin B activity was higher in the liver than in HGs (*p <
0.05), meanwhile, cathepsin D activity was higher in HGs (**p < 0.05). For HGs, there were no significant differences between sexes in cathepsin B
activity, but on the contrary, the cathepsin D activity was lower in female HGs than in males (ap < 0.05). Values are mean ± s.d. Immunoblot analysis of
cathepsin D (B) only showed a band of 46 kDa corresponding to pro-cathepsin D in female Harderian glands. The pictures of male (♂) and female (♀)
Harderian glands from the cathepsin D immunolocalization assay (C) showed a defined staining around vesicles (arrows), predominantly in type II cells.
In fact, the scarce female type II cells were intensely immunostained (rounded). Female glands showed a gradual expression of cathepsin D from the
apical zone (+) of the acinar cells to the basal zone (-). Note the space caused by the detachment of material, leaving a slim acinar layer (arrowhead)
and the presence of cellular content in some acinar lumens (asterisks).

www.landesbioscience.com Autophagy 1009


Table 1 Cathepsin D/Cathepsin B activity ratios
Males
Harderian Gland 174967 ± 2556***
Liver 11499 ± 984
The cathepsin D/B activity ratio provides indirect information relating to lysosomal viability. The
comparison of cathepsin D/B activity ratio in Harderian glands and liver from Syrian hamsters indicated
a higher ratio in the Harderian gland (***p < 0.001).
In contrast to the classical view that lysosomes are one of the
main causes responsible for cellular damage during oxidative stress,
recent evidence supports a protective role for the lysosomal system
in the early phases of the oxidative insult.10
Chaperone-mediated autophagy (CMA) allows the selective
lysosomal degradation of oxidized proteins22 and differs from the
other lysosomal degradation pathways in that vesicular traffic is
not involved.22 Thus, the presence of CMA in the Syrian hamster
HG supports again the role of this gland as a physiological oxida-
tive stress model.
The cathepsins data provided indirect information relating to
the stabilization of the lysosomal system. The magnitude of gener-
ated lysosomal destabilization determines if reparative autophagy,
apoptosis or necrosis will follow. Our results showed that each
gender presents its own peculiarities in the lysosomal pathway.
Female HGs showed a deficiency in LAMP-2 expression. It was
described that the half-life of early and late autophagic vacuoles is
prolonged in LAMP-2 deficient cells. Thus, the endosomal/lyso-
somal constituents are delivered to the autophagic vacuoles that
carry the LC3 marker and are accumulated in the cytoplasm.23
Then, the cells succumb to cell death with characteristics of type 1
and type 2 programmed cell death.24
Effectively, the female Syrian hamster HG seems to show
this phenotype of programmed cell death. Therefore, it is not
surprising that female HGs showed a collapse in trafficking of pro-
cathepsin D from the trans-Golgi network to the late endosomes
and lysosomes, leading to an accumulation of pro-cathepsin D.
In addition, cathepsin D immunohistochemistry indicated higher
expression in male glands, particularly in type II cells, which is in
agreement with the activity data. The percentage of type I cells in
the HGs of male Syrian hamsters has been estimated to be between
42% and 50%, meanwhile in female HGs appear in a percentage
near 100%.25 Then, a question was raised: Would it be possible
for male type II cells to complete cathepsin D processing as long as
type I cells present the lysosomal trafficking altered? This hypoth-
esis could explain why female glands develop a huge accumulation
of autophagic bodies carrying the LC3-II marker, and why some
treatments cause evident morphological changes.26,27
Beclin 1 and LC3, as well as ultrastructural data, point out that
female HGs have extensive macroautophagic processes, while male
autophagy is different and more controlled by survival signals, such
as NFκB, Bcl-2, SIRT1 and HIF-1α. This discrepancy between
sexes provokes other remarkable differences. The first one appears
when studying the cytoskeleton. Actin dynamics are important for
1010
Autophagic secretion in Harderian gland
the maintenance of cell-shape and for trafficking from the trans-
Golgi network to the lysosomes,28 and therefore, the loss of actin
Females in female glands, although surprising, agrees with data concerning
62679 ± 1786*** cathepsin D and LAMP-2. Furthermore, dynamic changes in the
5196 ± 553
abundance and localization of actin were described in autophagic
programmed cell death in the salivary gland of Drosophila.29 In
fact, classical cell death includes depolymerization or cleavage of
actin, cytokeratins, lamins and other cytoskeletal proteins,4 leading
to cell shape changes. This is in accordance with our ultrastructural
data showing morphological changes in female HGs, such as the
presence of intratubular-syncitial masses and detached cells. The
attachment of epithelial cells to the extracellular matrix, as well as
cell-cell anchorage, critically regulates survival, so that inadequate
contacts would lead to a detachment-mediated programmed cell
death.30 It is well known that cells in the glandular lumen die
due to extracellular matrix deprivation, and recently, Fung et al.31
have discovered that detachment strongly induces autophagy.
Therefore, our results suggest that female HGs could develop a
detachment-derived cell death promoting luminal clearance, as has
been described in mammary glands.32 Meanwhile, in male HG,
autophagy would have a cell-repair component. Now, we question
what triggers this cell death in female HGs.
When oxidative stress ensues the redox-active transcription
factors organize and promote cellular responses.33 In turn, stress
stimuli can activate NFκB and the p53 pathways with a broad
range of actions, some of them related to survival and cell death
processes. In general, p53 exerts a pro-death influence in response
to extreme oxidative and genotoxic stress; meanwhile, NFκB plays
a pro-survival role.34 Recently, evidence has emerged suggesting
that autophagy is another mechanism involved in the control of
cell death35 and therefore, oxidative stress should be influencing
the autophagic mechanism. The way in which autophagy is
controlled requires the identification of these signaling pathways
that impinge on autophagy in specific ways.
The p53 protein appears to sense multiple types of DNA
damage and coordinate multiple options for cellular response.
p53 activity largely depends on its ability to activate or repress
transcription, so that, p53 fluctuates between latent and active
(DNA-binding) conformations and is differentially activated
through post-translational modifications including phosphorila-
tions and acetylations. On the other side, nonsequence-specific
DNA binding may involve other p53 actions and in this way, p53
may govern an apoptosis checkpoint through competition with
DNA repair proteins for nonsequence-specific binding to exposed
single-stranded regions in the DNA duplex.36
This work demonstrated gender-related differences in the p53
pathway promoting different responses depending on sex. We
have to consider that the direct effects of the sexual dimorphism in
oxidative stress may also affect the p53 fate. In fact, redox modifi-
cations of p53 affect its stability and can be a potential mechanism
to select the target genes conferring differential binding affinity
to the response elements.33 According to our present findings,
p53 was constitutively phosphorylated at Ser-15 in male glands
promoting its functional activation. Hence, the transactivation
function of p53 in male glands induced the expression of some
Autophagy 2009; Vol. 5 Issue 7
 Autophagic secretion in Harderian gland

Figure 4. The representative immunoblot of the lysosomal marker LAMP-2 (A) in male (♂) and female (♀) Harderian glands showed a decrease in LAMP-2
expression in female glands with respect to that in males. The isoform LAMP-2A participates in chaperone-mediated autophagy. The reverse transcription-
polymerase chain reaction assay (B) showed LAMP-2A transcription rates in male and female glands, and the rates in liver from female hamsters were
used as negative control (-C). M, 100-bp DNA ladder. Arrowhead indicates the expected size of the PCR products. Expression of macroautophagic
markers: Beclin 1 and LC3 (C) were determined in tissue extracts of both sexes by western blotting. Unexpectedly, re-blotting membranes with actin (C)
showed defects in actin cytoskeleton in female glands in comparison to males. Ponceau staining was used for loading control and bar charts (D) show
optical densities of blot bands from three separate immunoblots. The results demonstrated the presence of these markers in Syrian hamster Harderian
glands, especially in female glands.

target genes such as DRAM, a critical lysosomal protein required
for the ability of p53 to induce autophagy. Furthermore, it was
found that DRAM is downregulated in some cancers indicating
that there is a selective pressure to lose DRAM during invasive
processes37 that agrees with our hypothesis about female glands.
On the other side, p53 was stabilized and accumulated in the
cytoplasm from male HGs. Previous studies suggest that the cyto-
plasmic retention of p53 is able to repress autophagy,9 that this
retention could be mediated by acetylation since p53 acetylation
reduces its ubiquitination levels, and that the DNA ­damage-induced
phosphorylation of p53 at Ser15 results in ubiquitination disrup-
tion.38 Although the precise molecular mechanisms behind this
remain unclear, it is likely that p53 deacetylases, such as SIRT1,
might be upregulated in males to mediate ubiquitination and
degradation of p53. SIRT1 has a versatile role in the maintenance
of cell and organism survival in response to a variety of stressors by
activating autophagic degradation.39 It seems that SIRT1, in male
HG, is able to regulate the p53-mediated autophagy.
PCNA participates in DNA replication and repair and is readily
detected after genotoxic stress. p53 binds the PCNA promoter and

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 Autophagic secretion in Harderian gland

Figure 5. Electron micrographs of male secretory cells (A) showed secretory vacuoles (v) containing electron-dense material (asterisks) around the peri-
nuclear region. The cell numerous mitochondria (m), loose smooth endoplasmic reticulum (SEr) throughout the cytoplasm, and a nucleus with peripheral
condensation of chromatin (arrows). Scale bar, 1 μm. The electron micrograph of female secretory cells (B) showed autophagic vacuoles (v) close to
smooth endoplasmic reticulum (SEr) and mitochondria (m). The cytoplasmic vacuoles showed the characteristic multilamellar structure. Scale bar, 0.56
μm. Electron micrograph of female acinar cell (C) showed autophagic vacuoles (v) close to smooth endoplasmic reticulum (SEr) and mitochondria (m).
Some cytoplasmic vacuoles contained electron-dense material. Scale bar, 1 μm.

transactivate PCNA in a concentration-dependent manner; lower
levels activate, whereas higher levels do not.12 Although female
HGs showed higher nuclear translocation of p53, actually they had
low levels of active p53 which may directly control DNA replica-
tion and repair by modulating the levels of PCNA. Thus, the
present work supports that in male HGs, p53 activation induces
autophagy as a survival response to ROS, protecting against cell
damage. In such a physiological situation, the inactivation of
p53 becomes crucial and appears another cellular signaling that
increases cell survival.
According to this, our results demonstrated the presence of
SIRT1, HIF-1α and NFκB activation in male glands. The pres-
ence of HIF-1α in males confirms this survival signaling since it
modulates the induction of autophagic proteins such as Beclin 1.40
Beside these actions in male HGs, NFκB promotes cell survival,
decreases the sensitivity of cells to programmed cell death34 and
maintains autophagy at basal levels, increasing expression of
caspase-inhibitory proteins and other anti-apoptotic and anti-
autophagic proteins, such as Bcl-2.41
We cannot forget that female gland integrity is compromised by
the excessive oxidative pressure. It has been previously shown that
NFκB inactivation mediates autophagy through a mechanism that
involves ROS production42 and our results consolidate this role
of NFκB in the regulation of ROS signaling and autophagic rate
in female and male HGs. Thus, the NFκB inactivation in female
glands leads to an imbalance in the antioxidant system, an accu-
mulation of ROS and an increase of damaged proteins and lipids,
favoring death signaling.
Whereas low levels of ROS are important for cellular function
and survival signaling, excessive ROS (females) can lead to cell
death, but at extraordinary high levels of oxidative stress, the cell
cannot maintain a reducing environment, and caspases will not
function due to their sensitivity to oxidative inactivation at their
active cystein group.43 Thus, apoptosis is inhibited in female glands,
and cell death must proceed through other pathways. Female HGs
seem to develop detachment-related cell death processes with
intermediate characteristics between apoptotic and autophagic
cell death: autophagic morphology, collapse of the cytoskeleton,
caspase-3 independence, NFκB inactivation, p53-independent cell
death, absence of DNA fragmentation but presence of DNA repair
processes. In accordance with this hypothesis, recent studies have
shown that detachment of cells from the matrix inhibits NFκB
activity and induces autophagy,31 and that the inhibition of NFκB
activity by overexpression of nonphosphorylable IκB increases
detachment-induced cell death.44 Thus, these high levels in ROS
lead to persistent oxidative stress, which causes selective pressure
for characteristics such as invasion.43 Figure 7 summarizes the
hypothesis proposed in this work, showing how oxidative stress,
through p53 and NFκB, modulates autophagy, and consequently,
orchestrates the Harderian gland physiology.
It is well known that the porphyrinogenic activity of the
Harderian gland fluctuates throughout the oestrous cycle, pregnancy
and lactation, suggesting that there is a link between HG activity
and reproductive function,45 that the gland dimorphism is under
androgenic control46 and that steroids are important regulators of
programmed cell death in animals.47 Autophagy in Syrian hamster
HG plays a dynamic role in adapting to or even regulating its secre-
tory activity by removing part of steroid-producing organelles. This
hypothesis is strongly supported by the fact that the intensity of
autophagy varies depending on sex. In fact, the largest histological
effect of the extreme oxidative stress in female glands was the release
of granules, together with cellular debris and some nuclei in the
lumina, suggesting the presence of holocrine and apocrine types
of secretion. Are female processes therefore a mechanism ascribed
to massive release of glandular secretion? Our observations and the
absence of a morphologically specialized duct system within the

1012 Autophagy 2009; Vol. 5 Issue 7

 Autophagic secretion in Harderian gland

Figure 6. The images from toluidine blue-stained semithin sections of a Harderian gland from a female hamster showed areas (A) with a single layer of
cells and a hollow lumen and areas (B) with protuberances from acinar cells towards the acinar lumen (arrows) and cellular content in the acinar lumen
(asterisk). In this case, some acini seemed to show an epithelium with several layers of cells (stars). Some semithin sections (C) showed cellular content
in the acinar lumen with nuclei inside (arrowheads). The micrograph (D) from a female Syrian hamster Harderian gland showed an acinus filled with
a mass formed by secretory vacuoles and cytoplasmic fragments (asterisk). Note the presence of myoepithelial cells (arrows) surrounding the mass and
compare the size of the mass with the size of a cell (rounded). A stained semithin section of Harderian gland from a female hamster (E) showed an
intertubular-syncitial mass (SM) containing irregular and heterochromatic nuclei (arrows). The adjacent acini are disorganized and filled with cellular
debris and cells occupying the luminal space (asterisks). Some cells are detached from the basal layer (rounded). Note the marked vacuolization.

www.landesbioscience.com Autophagy 1013

 Autophagic secretion in Harderian gland

Figure 7. The scheme proposes how redox signaling controls Syrian hamster Harderian gland physiology through a NFκB and p53 signaling. A situa-
tion of mild oxidative stress (which occurs in male Harderian glands) activates survival and reparative signaling. However, an extreme oxidative stress
environment (occurring in female Harderian glands) activates a massive-holocrine secretion. ECM: extracellular matrix.

gland48 point out a massive secretion of autophagic bodies. Anyway,
additional studies are required in order to elucidate the role played
by the redox transcription factors in autophagy regulation and
to deepen the knowledge of the ­relationship between autophagy,
the cell death process and the secretion mode occurring in female
HG.
Material and Methods
Animals. Twenty-four one-month-old male and female Syrian
hamsters (Mesocricetus auratus) (Harlan Interfauna Ibérica,
Barcelona, Spain) were housed 4 per cage under a 14:10 h
light:dark cycle at 22 ± 2°C. Animals received tap water and stan-
dard pellet diet ad libitum.
The Oviedo University Local Animal Care and Use Committee
approved the experimental protocol. All experiments were carried
out according to the Spanish Government Guide and the European
Community Guide for Animal Care (Council Directive 86/609/
EEC).
After one month in the animal house, hamsters were sacrificed
and Harderian glands and livers were immediately removed, frozen
on liquid nitrogen, and stored at -80°C until the experiments were
performed.
If not indicated otherwise, HGs (0.1 g) and livers were
homogenized with a Polytron homogenizer at 4°C in 1 ml of lysis
buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl) with protease
inhibitors (1 mM Na3VO4, 1 μg/ml aprotinin). The tissue homo-
genates were then centrifuged for 6 min at 3,000 xg at 4°C, and
supernatants were collected and centrifuged again under the same
conditions. Nuclear and cytoplasmic fractions were prepared using
the sucrose gradient method.
The amount of protein in the supernatants was measured by the
method of Bradford.49
Protein oxidative damage. Protein carbonyl concentrations
were determined following the method developed by Levine,50
with modifications of Coto-Montes and Hardeland.51 Data are
presented as nmol protein carbonyl/mg protein.
Hydroperoxide determination. The ferrous ion oxidation
xylenol orange (FOX) method of ROOH estimation is based on
the oxidation of Fe+2 to Fe+3 by sample-oxidizing agents, which
then bind with xylenol orange (XO) to give a color complex with
an absorption maximum at 560 nm. The Fe+3-XO color complex
formation is strictly pH sensitive. In our work, FOX-1 has been
used for the estimation of ROOH concentration.52 Results are
expressed as nmol hydroperoxides/mg protein.
Antioxidant defence. Supernatants were used for assaying
superoxide dismutase (SOD, EC 1.15.1.1)53 and catalase (CAT,
EC 1.11.1.6).54 Data were presented as Units SOD/mg protein for
SOD and μmol H2O2/mg protein Min-1 for CAT.
Detection of apoptosis. Tunel assays were performed using the
in situ apoptosis detection kit, AP (Roche, 11 684 809 910).
Activities of cathepsins. The cysteine-proteinase cathe-
psin B (EC 3.4.22.1) was assayed fluorometrically (Millipore,
CytofluorTM 2350) according to the method of Barrett,55 with
minor modifications,56 using Z-Arg-Arg-MCA as the specific
substrate (Sigma). For the assay, 40 μl of tissue homogenates
(see above) were diluted with 300 μl of incubation buffer (100

1014 Autophagy 2009; Vol. 5 Issue 7

 Autophagic secretion in Harderian gland
mM sodium acetate, pH 5.5, containing 1 mM EDTA, 5 mM
dithiothreitol and 0.1% Brij-35). Of this dilution, 50 μl were
pipetted into a 96-well fluorescence microtiter plate. The reac-
tion was started by adding 20 μl of the substrate solution (40 μM
Z-Arg-Arg-MCA in incubation buffer), and was then incubated
at 37°C for 20 min. The reaction was stopped by the addi-
tion 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 B results are expressed as enzymatic milliUnits/mg
protein.
The aspartate-proteinase cathepsin D activity (EC 3.4.23.5) was
assayed spectrophotometrically (Kontron Instruments, UVIKON
930) at 280 nm according to the method of Takahashi and Tang,57
with minor modifications56 and the use of hemoglobin as the
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 for 30 min, followed by centrifugation at 12,000
xg for 5 min. The optical densities of the supernatants were deter-
mined. The cathepsin D results are expressed as enzymatic units/
mg protein.
Immunoblotting. Tissue samples were denatured in sample
buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,
0.01% Bromophenol Blue) at 100°C for 5 min and separated by
12% SDS-PAGE at 100 V. After separation, protein was transferred
to PVDF membranes at 350 mA. Ponceau S staining was used to
ensure equal loading. The membranes were blocked for 1 h at room
temperature in 5% skim milk in PBS containing 0.05% Tween-20
(PBS-T), and then probed with antibodies against IKKα, IκBα,
Phospho-IκBα (Ser32), NFκB p65, Phospho-NFκB-p65 (Ser536)
(Cat. No. 9936) from Cell Signaling Technology; NFκB p50
(sc-114), Bcl-2 (sc-7382), SOD2 (sc-18504), iNOS (sc-651), p53
(sc-6243), SIRT1 (sc-15404), PCNA (sc-56), HIF-1α (sc-53546),
cathepsin D (sc-6486), LAMP-2 (sc-8100), Beclin-1 (sc-10086)
from Santa Cruz Biotechnology, Phospho-p53 (Ser15) (BioVision,
3515), DRAM (Rockland, 600-401-A70), LC3 (MBL, PD014)
and actin (Sigma, A 5441), each previously diluted 1:1,000
in blocking buffer. After three 5 min-washes in PBS-T, the
membranes were incubated with a dilution (1:10,000 in blocking
buffer) of a corresponding horseradish peroxidase-conjugated
secondary antibody (Santa Cruz Biotechnology) for 1 h at room
temperature, and again washed three times for 5 min in PBS-T.
The membrane was developed using the western Blotting Luminol
Reagent (Santa Cruz Biotechnology, sc-2048) according to the
manufacturer’s protocol. All data presented are representative from
at least three separate experiments. The levels of Beclin 1, LC3-I
and LC3-II were quantitatively analyzed with Quantity One 5.5.1.
The results were normalized to loading control.
Determination of NFκB p50 DNA binding activity. NFκB
p50 subunit was detected in the nuclear protein extract from
Syrian hamsters HGs using the NFκB p50 EZ-TFA Transcription
Factor Assay kit (Millipore, 70-515). This assay combines the
www.landesbioscience.com Autophagy
principles of the electrophoretic mobility shift assay (EMSA) with
a 96-well-based ELISA. During the assay, the capture probe, a
double-stranded biotinylated oligonucleotide containing the DNA
binding consensus sequence for NFκB (5'-GGG ACT TTC C-3'),
was mixed with the nuclear extract. The active form of NFκB
contained in the nuclear extract bound to its consensus sequence.
The extract/probe/buffer mixture was then directly transferred to
a streptavidin-coated plate. The active NFκB protein was immo-
bilized on the capture probe bound to the streptavidin plate well,
and inactive unbound material was washed away. The bound
NFκB p50 subunit was detected with a specific primary antibody.
A highly sensitive horseradish peroxidase-conjugated secondary
antibody was then used for detection. This provides sensitive colo-
rimetric detection that can be read in a spectrophotometric plate
reader at 450 nm. The wild-type consensus oligonucleotide (not
biotinylated) was used as a specific competitor for NFκB binding
to monitor the specificity of the assay.
RNA extraction and PCR assay. Total RNA was purified from
HG extracts of Syrian hamsters by a single extraction with TRI
Reagent (Sigma, T9424) following the manufacturer’s recom-
mendations, and was stored at -80°C until use. LAMP-2A cDNA
was synthesized using the Titan One Tube RT-PCR System
(Roche, 11888382001): 25 μl of purified total RNA (0.1 μg/μl)
were added to 25 μl of a RT-PCR mixture containing 0.2 pmol
of primers, according to the manufacturer’s instructions. The
amplification protocol included retrotranscription at 48°C for 30
min, denaturation at 94°C for 2 min, followed by 30 cycles at
94°C for 10 s, 55°C for 30 s and 68°C for 1 min, and one final
cycle at 68°C for 10 min. The PCR products were analyzed using
2% agarose-Trisborate-EDTA gel electrophoresis, stained with
ethidium bromide, and examined with UV light. The primer pairs
used (LAMP-2A, 5'-GCA GTG CAG ATG AAG ACA AC-3',
5'-AGT ATG ATG GCG CTT GAG AC-3') were designed for
amplification of a 120-bp fragment of the Mus musculus lysosomal-
associated membrane protein 2 (LAMP-2) transcript variant 1
mRNA (NM 001017959).
Ultrastructural studies. Harderian glands for morphological
studies were lightly fixed by immersion in a solution containing
1.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phos-
phate buffer (pH 7.4). Fixation was continued overnight at 4°C
using fresh fixative. The tissue was then postfixed in 1% OsO4 for
2 hours. After dehydration in a graded acetone series, the tissue
fragments were embedded in Taab 812, and semithin sections
(1 μm) were stained with toluidine blue. Ultrathin sections were
collected on cooper grids, stained with uranyl acetate-lead citrate,
and examined using a Zeiss EM-109 transmission electron micro-
scope (Zeiss, Oberkochen Germany) operating at 80 kV. Negatives
were scanned by an HP Scanjet 3970, and imported by Adobe
Photoshop 7.0.1; the images were incorporated into figures using
MS PowerPoint.
Immunohistochemistry. Tissues assigned to immunohis-
tochemistry were fixed and embedded in paraffin by standard
methods. The sections were rinsed three times for 10 min in 0.01
M PBS with 0.1% Triton X-100 and 0.25% bovine serum albumin
(PBS-TB) and blocked in 30% serum in PBS-TB for 30 min. This
1015
 Autophagic secretion in Harderian gland
was followed by incubation with antibodies against cathepsin D
(Santa Cruz Biotechnology, sc-6486) for 24 h at 4°C in a humid
chamber. After washing with PBS-TB, the sections were incubated
with a horseradish peroxidase-conjugated secondary antibody, and
afterwards in peroxidase anti-peroxidase complexes (Sigma), for
1 hr at room temperature. Finally, the sections were washed and
developed with 0.05% 3,3'-diaminobenzidine/HCl with 0.005%
H2O2 in Tris/HCl. The stained sections were studied under a
light microscope (Olympus España, CAST2). The images were
imported and incorporated into electronic figures by Corel Draw
X13 and MS PowerPoint.
Statistical analysis. Data are presented as mean values ± SD
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 the Student’s t test with normal data. The nonpara-
metric Mann-Whitney test was applied to non-normal data. The
accepted level of significance was p < 0.05.
Acknowledgements
We thank the INPROTEOLYS network. This work was partially
performed with funding from grants FISS-06-RD06/0013/0011
from the Instituto de Salud Carlos III (Ministerio de Sanidad
y Consumo) and INIA-07-RTA2007-00087-C02-02 from the
Ministerio de Ciencia e Innovación; I.V.-N. grant sponsor: FPU
predoctoral fellow from the Ministerio de Ciencia e Innovación
and Fondo Social Europeo; S.V. grant sponsor: INIA predoctoral
fellow from the Ministerio de Ciencia e Innovación; C.H.-F. grant
sponsor: FIS fellow from Ministerio de Ciencia e Innovación;
D.D.G.-C. grant sponsor: FICYT predoctoral fellow from the
Gobierno del Principado de Asturias. Financial support (grant:
UNOV-09-EDO-11) given by the University of Oviedo is
acknowledged.
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