University of Melbourne, Department of Medicine, Western Hospital, Footscray, Victoria 3011, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adaptation of the gastric mucosa to nonsteroidal anti-inflammatory drug-induced injury is a well-documented phenomenon, but the mechanisms are not known. We investigated whether changes in stress protein expression and apoptosis play roles in adaptation of rat stomach to aspirin. RT-PCR and Western blotting techniques were used to analyze mRNA and protein expression of HSP72 and HSP90 and cleavage of caspase 3 protein. Apoptosis was detected by the TUNEL method and quantified. HSP72 mRNA and protein expression was unchanged in adapted mucosa, whereas HSP90 mRNA and protein levels decreased. Caspase 3 protein was activated, and the number of apoptotic cells increased in mucosa after one aspirin dose. However, in adapted mucosa after aspirin, activated caspase 3 and the number of apoptotic cells had returned to basal levels. Induction of the stress response was found not to be a mechanism of mucosal adaptation against multiple doses of aspirin. Our results lead us to propose instead that resistance to aspirin-induced apoptosis plays a role in the protective phenomenon of adaptation.
stress proteins; caspase 3; nonsteroidal anti-inflammatory drugs; gastric mucosa
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NONSTEROIDAL ANTI-INFLAMMATORY drugs (NSAIDs) are used
extensively to treat a wide variety of medical conditions. Aspirin is
also prescribed as a prophylactic treatment against coronary artery
disease and stroke. Although NSAIDs have many beneficial effects, each
dose is known to cause measurable damage to the gastric mucosa (11). A
phenomenon called adaptation has been recognized for more than 25 years
(21, 45) and is characterized by a marked reduction in gastric mucosal
injury after repeated administration of a damaging agent over several
days (45). The mechanisms of adaptation are not known, and many of the
publications reporting changes in NSAID-adapted mucosa are
contradictory. The most consistent findings to date are that adapted
stomachs have increased expression of the growth factor transforming
growth factor (TGF)- (9, 36), increased infiltration of
granulocytes (24, 44, 47), and increased cellular proliferation (10, 24, 26, 49, 51).
The stress (or heat shock) proteins are a large family of proteins that are upregulated in cells after they have been damaged by a variety of agents, including heat, toxic metals, and alcohol. The stress proteins are also expressed constitutively and are known to play a fundamental role in a number of biological processes, including the proper folding and translocation of many cellular proteins. One of the stress proteins, HSP90, is generating much interest as its importance in the control of signal transduction pathways (by assisting shape changes in some kinases and steroid hormone receptors) is unraveled (1, 8, 32, 34, 38).
Prior induction of heat shock proteins has been widely demonstrated to protect cells against damage either by the same agent that induced the stress response or by other damaging agents (22). In a similar way, gastric mucosa that has adapted to NSAIDs is not only protected from damage caused by the same NSAID but is also protected against damage caused by other gastric mucosal toxins (4, 41). For these reasons, induction of the stress response has been suggested to be one of the ways by which the gastric mucosa might protect itself against ongoing damage (14, 20, 31).
We postulated that induction of stress proteins might mediate mucosal adaptation to NSAIDs. This article examines whether any changes occur in the expression of two major members of the stress protein family, HSP72 and HSP90, during gastric adaptation to aspirin in rats.
To date, several studies have reported the contribution of apoptosis to NSAID-induced damage to the gastric mucosa. After a single damaging dose of an NSAID, including aspirin, there is an increase in the number of cells undergoing apoptosis in the stomach (42, 50). We wanted to investigate whether changes in apoptosis occur in the mucosa after adaptation to aspirin. We measured two parameters of apoptosis after single and multiple aspirin doses: 1) TUNEL staining to detect cells with nuclei characteristic of programmed cell death and 2) caspase 3 cleavage from the precursor form to the active cell death effector.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adaptation of rat stomach to aspirin. Male Long Evans rats weighing 220 ± 45 g were maintained on a controlled feeding regimen to enable the animals to be dosed daily in a fasted state. Rats were dosed orally by gavage at 24-h intervals with either 1% methylcellulose vehicle (pH 5.0) or aspirin suspended in 1% methylcellulose vehicle (pH 3.1). The dosing schedule was as follows: 6 daily doses of 1% methylcellulose vehicle (control); 5 daily doses of vehicle and 1 dose of 120 mg/kg aspirin (single-dose); or 6 daily doses of 120 mg/kg aspirin (adapted). Four hours after the last dose, rats were killed under Fluothane anesthesia and the stomachs were immediately removed, opened along the lesser curvature, and pinned out flat. They were gently rinsed with saline and photographed. The fundic (i.e., corpus) portion of each stomach was divided into sections for histology and extraction of RNA and protein.
Assessment of macroscopic damage. Macroscopic gastric mucosal injury was measured using a digital planimeter to trace areas of damage on each photograph (with an observer blinded to treatment groups). Damage was expressed as a percentage of the total fundic area and was defined as hemorrhagic regions of the mucosa that did not clear after rinsing.
Histology. Rat gastric body sections were immersion fixed in phosphate-buffered formalin. The tissues were then dehydrated and embedded in wax. Four-micrometer sections were cut, dewaxed, and stained with hematoxylin and eosin.
Stress protein expression in rat gastric body explant culture. Rat stomachs were taken immediately after death, and small sections (~0.1 cm2) of gastric body were placed immediately into 37°C standard DMEM containing 10% FCS. The stomach samples were incubated at either 37°C (control) or 42°C (heat shock) for 1.5 h. RNA was extracted, and RT-PCR was performed and quantified for HSP72, HSP90, and the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; parameters described below).
RT-PCR. Total RNA was extracted from rat gastric body using the acid-phenol method (5). Three micrograms of total RNA was reverse transcribed into single-stranded cDNA using 200 units Moloney murine leukemia virus (Promega) primed with 0.3 µg oligo(dT) in 40 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 1 mM dNTPs in a 40-µl volume at 37°C. Rat sense and antisense primer sequences were designed on the basis of GenBank entries and are as follows: HSP72, sense 5'-TCGAGGAGGTGGATTAGAG, antisense 5'-GGGATGCAAGGAAAAAAC; HSP90, sense 5'-ACATCATCCCCAACCCTC, antisense 5'- TCCACCAGCAGAAGACTCC; GAPDH, sense 5'-TGTCAGCAATGCATCCTGCA, antisense 5'-CTGTTGAAGTCACAGGAGAC. The optimum number of cycles for PCR of each primer pair was determined by adding fourfold serial dilutions of the cDNA to the PCR reactions until a linear response was obtained. The optimum number of cycles for each primer pair were found to be HSP72, 29 cycles, HSP90, 24 cycles, and GAPDH, 26 cycles. Parameters for each cycle were as follows: denaturation at 95°C for 40 s, annealing at primer-specific temperature (HSP72, 57°C; HSP90, 61°C; GAPDH, 45°C) for 40 s, and extension at 72°C for 1 min. Reaction conditions were 10 mM Tris · HCl, 50 mM KCl, 2 mM MgCl2, 200 µM dNTPs, 25 pmol each of sense and antisense primer, and 1.25 units Taq polymerase (Promega) in a 50-µl volume. Control reactions to ensure that RNA samples and the RT-PCR reagents did not contain contaminating DNA were performed for each sample and primer pair. PCR products were resolved on 2% agarose gels containing ethidium bromide and were visualized under ultraviolet light. Photographs of each gel were scanned, and the intensities of the bands were measured using the ImageMASTER software system (Pharmacia Biotech).
Western blotting. Stomach sections were weighed and homogenized in 10 volumes of ice-cold 1% NP-40 in 50 mM Tris (pH 8.8) buffer. The homogenates were centrifuged, and the supernatants were transferred into sterile tubes. Fifteen-microgram protein aliquots of each sample, as determined by the Coomassie blue method, were resolved on 15% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with BSA before being incubated with primary antibody overnight at 4°C [antibodies used were kind gifts from Dr. Susan Lindquist of University of Chicago (HSP90), Dr. David Walsh of University of Sydney (monoclonal rat HSP72), and Dr. Don Nicholson of Merck-Frosst (caspase 3)]. Membranes were rinsed and then incubated for 1 h with peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using the enhanced chemiluminescence system (Amersham). The films were scanned, and the intensities of the bands were measured using the ImageMASTER software system (Pharmacia Biotech).
In situ detection and quantification of apoptotic cells. Apoptotic cells were detected using a modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Promega DeadEnd colorimetric apoptosis detection system). Horseradish peroxidase-labeled streptavidin was bound to incorporated biotinylated nucleotides, and positively labeled cells were detected using hydrogen peroxide and diaminobenzidine. Nuclei that stained dark brown and showed morphological signs indicative of apoptosis (i.e., condensed chromatin and/or fragmented nuclei) were considered positive. Apoptotic cells were counted (observer blinded to treatment groups) in two separate gastric mucosal compartments: the surface epithelial compartment and the glandular compartment, the junction of which was defined by the highest parietal cell. Ten areas per compartment were counted for each sample and expressed as the number of apoptotic cells per 105 µm2. Only areas of intact surface epithelium were included in the assessment of apoptosis; areas containing lesions were omitted due to the increased density of staining and difficulty of accurately determining whether nuclei were exhibiting characteristics indicative of apoptosis. Cells that were detached from the epithelium were not counted.
Statistical analysis of data. All data are expressed as means ± SE. Comparisons between groups were performed using the one-way ANOVA followed by the Student-Newman-Keuls test or (where parametric tests were inappropriate) the Kruskal-Wallis one-way ANOVA on ranks followed by the Dunnett's method of all pairwise multiple comparison.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macroscopic damage.
Adaptation of the gastric mucosa to aspirin is characterized by a
decrease in the amount of damage to the mucosa after successive doses
of this NSAID. Figure 1 compares the level
of macroscopic damage in the gastric mucosa of control (6 daily doses
of 1% methylcellulose vehicle), single-dose (5 daily doses of vehicle
and 1 dose of 120 mg/kg aspirin), and adapted (6 daily doses of
120 mg/kg aspirin) rats 4 h after the last dose. Both control (0.04 ± 0.03) and adapted rats (0.35 ± 0.17) sustained significantly less
damage than rats exposed to a single dose of aspirin (1.30 ± 0.33). Overall, there was a 73% reduction in damage in the
adapted group (P < 0.05). Two of nine rats in this group
showed moderate adaptation, whereas the remaining seven were
markedly protected (mean reduction 93%).
|
|
RT-PCR of stress proteins in normal and heated stomach explant
cultures.
Stomach explant cultures incubated at either 37°C or 42°C were
used to determine the heat inducibility of HSP72 and HSP90 in rat
gastric mucosa. After RNA extraction and RT-PCR, the products were
resolved on an agarose gel alongside a 100-bp ladder to confirm that
the sizes of the products were as predicted: HSP72, 307 bp; HSP90, 269 bp; and GAPDH, 425 bp (data not shown). Quantification of the HSP72 and
HSP90 bands (standardized to GAPDH) is shown in Fig.
3, and as expected HSP72 gene transcription
increased in the gastric mucosa after incubation at 42°C. No
significant change in HSP90 gene transcription was observed between the
control and heat-shocked samples.
|
Analysis of HSP72 mRNA and protein expression.
Levels of HSP72 mRNA and protein in control, single-dose, and adapted
gastric mucosa 4 h after the last dose of vehicle or aspirin were
assessed by semiquantitative RT-PCR and Western blotting, respectively
(Fig. 4). The level of HSP72 mRNA in
adapted stomach was unchanged compared with the control and single-dose
groups (Fig. 4A). HSP72 protein expression (Fig. 4B) in
adapted stomach was also unchanged compared with the control and
single-dose groups.
|
Analysis of HSP90 mRNA and protein expression.
HSP90 mRNA and protein levels in the gastric mucosa 4 h after the last
dose of either vehicle (control) or aspirin (single dose, adapted) were
assessed using semiquantitative RT-PCR and Western blotting,
respectively (Fig. 5). After a single dose
of aspirin, no change in HSP90 mRNA expression was detected compared with control. Adapted stomachs showed reduced HSP90 mRNA expression (0.64 ± 0.03) compared with both the single-dose stomachs (0.79 ±0.06) and controls (0.89 ± 0.05) (Fig.
5A). In accordance with this, HSP90 protein was markedly
reduced in adapted stomachs after aspirin (426 ± 55) compared with
both single-dose stomachs (921 ± 73) and controls (1067 ± 74) (Fig.
5B).
|
Analysis of intact and cleaved caspase 3 levels.
Concentrations of both the intact (inactive precursor) and cleaved
(active enzyme) products of the apoptosis-associated caspase 3 protein
were measured by Western blotting (Fig. 6).
After aspirin, the amount of the 32-kDa precursor form of caspase 3 in
adapted stomach (373 ± 64) was reduced compared with both single-dose stomachs (885 ± 112) and controls (981 ± 80) (Fig. 6A). The
changes observed in the levels of the 17-kDa active caspase 3 were
quite different from that of the 32-kDa precursor form. The amount of active caspase 3 in normal mucosa given a single dose of aspirin (660 ± 80) increased compared with mucosa given vehicle only (423 ± 79).
However, in adapted stomachs the amount of active caspase 3 after
aspirin (368 ± 60) remained approximately the same as that in control
stomachs that received vehicle alone (Fig. 6B).
|
Quantification of apoptosis.
Apoptotic cells in the control, single-dose, and adapted stomachs were
detected using a modified TUNEL assay and quantified as outlined in
MATERIALS AND METHODS. In all three sample groups, the
majority of apoptotic cells were located in the surface epithelial compartment of the gastric mucosa. Apoptotic cells found in the glandular compartment of the mucosa were located mainly in the area
immediately below the first parietal cell or at the base of the glands.
Sections showing cells with morphological signs indicative of apoptosis
are shown in Fig. 7. Quantification
revealed that at 4 h after a single dose of aspirin the number of
apoptotic cells (per 105 µm2) increased in
both the surface epithelial (111 ± 11) (Fig.
8A) and glandular compartments (4.3 ± 0.5) (Fig. 8B) compared with controls (76 ± 14 and 2.8 ± 0.2, respectively). In adapted mucosa the number of apoptotic cells
detected in both the surface epithelial (66 ± 5) and glandular
compartments (3.5 ± 0.3) was very similar to that of controls and
substantially lower than after a single aspirin dose, particularly in
the surface epithelial compartment.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of the studies described here was to ascertain whether changes in expression of the stress proteins HSP72 and HSP90 occur in rat stomach after single or multiple oral doses of the NSAID aspirin and therefore contribute to the cytoprotective phenomenon of adaptation. The stress (or heat shock) proteins are both constitutively and inducibly expressed in all tissues. Their inducibility is dependent on many factors, including cell type, tissue environment, type of stressor, and the level of basal constitutive expression. The expression and inducibility of the HSP72 stress protein also varies considerably between in vitro preparations of cells and whole tissue in vivo. In this study we have demonstrated that inducible HSP72 is expressed in rat stomach under basal conditions and that its expression can be modestly increased (almost twofold) by heat shock. This result conforms with other in vivo experiments in which induction of HSP72 after various forms of stress, such as whole body hypo/hyperthermia and water restraint, results in only a modest two- to fourfold increase in HSP72 expression in a variety of tissue types, including gut (7, 14, 28, 46). This is in contrast with most in vitro cell culture studies (and our unpublished data), which demonstrate almost no basal expression and large increases in expression of HSP72 after stress. We found that constitutive expression of HSP90 in the stomach is also high but it is not inducible after a short exposure to heat. The detection of high tissue concentrations of both HSP72 mRNA and protein in undamaged gastric mucosa suggests that the rat stomach has an augmented and sustained inducible HSP70 response compared with other tissues that lack an ongoing harsh extracellular environment, such as that of the gastric lumen. This conclusion is supported by previous studies in our laboratory, which have shown that a major site of HSP72 expression in the stomach is the gastric epithelium (50).
Although basal HSP72 expression is high in the stomach, and can be modestly elevated by heat shock, no increase in gastric HSP72 mRNA or protein was detected in adapted animals or those given a single damaging dose of aspirin, compared with controls. Although pretreatment with an NSAID has been shown to augment the stress response after heat shock (2, 12), it has been demonstrated in systems as diverse as yeast and fruit flies (17, 23, 48) that salicylate itself does not increase HSP72 gene transcription or protein levels. Our observations suggest that the same may be true for the stomach.
In contrast to HSP72 expression, single or multiple (adapting) doses of
aspirin resulted in a progressive reduction in expression of HSP90 mRNA
and protein, so that steady-state synthesis was reduced by ~60% in
the adapted group. Little is known of the factors that regulate HSP90
gene expression, and to our knowledge this is the first report of
inhibition of HSP90 in an in vivo system. HeLa cells transfected with
the heat shock transcription factor (HSF4) were found to synthesize
less HSP90 while maintaining HSP72 expression, suggesting that HSF4 may
contribute to the transcriptional control of this stress protein (30).
HSP90 inhibition in a monoblastoid cell line has implicated it in cell
cycle control and differentiation (15), and these same cells were shown
to be resistant to TNF--induced apoptosis (16). Aspirin-induced
damage in the gastric mucosa has been shown to be TNF-
-dependent,
with both types of TNF-
receptors, R1 and R2, independently involved
in modulating caspase activity (13). Since HSP90 is known to play an
important role in receptor signaling, with reduced HSP90 levels
inhibiting the activation of steroid receptors (32), it is possible
that the decreased HSP90 level in adapted stomach acts to inhibit
activation of the TNF-
receptors, thereby reducing subsequent
caspase cleavage and apoptosis.
Apoptosis, or programmed cell death, plays an important role in
maintaining normal tissue homeostasis in most organs. In the stomach,
apoptotic processes serve to maintain cell number by ensuring that cell
division is balanced by cell death (19). The pathogenesis of gastric
mucosal injury induced by damaging agents such as NSAIDs, ethanol, and
Helicobacter pylori, has been correlated with increased
apoptosis (18, 27, 29, 33, 40, 42, 52), and induction of apoptosis has
been proposed as the mechanism by which NSAIDs prevent colorectal
tumorigenesis (3, 40). Conversely, attenuation of gastric mucosal
injury has been shown to be accompanied by decreased apoptosis (18, 33,
42, 52). In addition, it is known that induction of TGF- correlates with reduced programmed cell death (35, 37, 39), and we and others have
shown that the epidermal growth factor receptor ligand TGF-
is
induced in the adapted gastric mucosa (9, 36). Together, these data
suggest that acute damage produces increased apoptosis, and adaptation
might lead to inhibition of apoptotic processes.
To proceed, programmed cell death requires the activation of the
intracellular protease family of caspases (6). These proteolytic enzymes participate in a cascade in which upstream members cleave and
activate those immediately downstream, which in turn cleave substrate
proteins, leading to the biochemical and morphological changes
characteristic of apoptosis. Caspase 3 (also known as CPP32 or apopain)
is one of the more downstream caspases of the apoptotic cascade and is
activated by the cleavage of an inert 32-kDa precursor to yield a
17-kDa active enzyme. Increased expression of the active enzyme
correlates with an elevated incidence of apoptotic cell death. In the
stomach, immunohistochemical analysis of caspase 3 expression in vivo
has shown that caspase 3 is highly expressed by foveolar cells lining
the neck region of the gastric pits, but only a low level of caspase 3 expression was found in the parietal and chief cells (25). Recent
studies have reported that aspirin-induced apoptosis in the gastric
mucosa is mediated by TNF--dependent activation of caspases,
including caspase 3 (13, 43). Our results revealed that the level of
active 17-kDa caspase 3 enzyme is increased almost twofold 4 h after a
single damaging dose of aspirin, whereas the level of the 32-kDa
zymogen is modestly decreased. However, in adapted stomachs after
aspirin, there was no increase in the level of active caspase 3, whereas expression of the inactive zymogen was found to have depleted by ~70%. This observation suggests that synthesis of the caspase 3 precursor is chronically inhibited in aspirin adaptation, perhaps contributing to increased resistance to apoptosis.
To further test whether adaptation may induce resistance to apoptosis, the numbers of apoptotic nuclei in gastric mucosal sections from control, single-dose, and aspirin-adapted rats were quantified by TUNEL staining and cell morphology. Quantification of apoptotic cells was carried out on randomly chosen regions of multiple sections; however, areas of frank erosion were excluded due to the difficulty in distinguishing between cells undergoing programmed cell death or necrosis and because apoptotic cells were likely to have been lost from the eroded surface. It is possible therefore that the magnitude of the increase in apoptotic areal density after a single aspirin dose was actually underestimated in this study. However, in adapted stomachs in which erosions were few, any such underestimation would have been much less. By TUNEL staining, the majority of apoptotic nuclei were located in the surface/pit compartment, consistent with the reported morphological analysis of caspase 3 expression (25) and with the fact that surface and pit epithelial cells have a faster turnover rate than any other cell in the gastric epithelium. In both the surface epithelial and glandular compartments, substantially more apoptotic cells were observed 4 h after a single aspirin dose compared with controls. This result is consistent with reports that gastric epithelial cell apoptosis after NSAIDs is readily detectable within several hours of dosing using a number of detection methods, including DNA laddering (13, 43).
In adapted mucosa after aspirin, the number of apoptotic cells was equivalent to the number in control mucosa that received vehicle alone (correlated with the return of active caspase 3 to the control level). The reduction in apoptosis after aspirin in adapted stomach was more marked in the surface epithelial compartment.
The onset and offset of adaptation in rats has been shown to take ~3-5 days (41, 45), which correlates closely with mammalian surface epithelial cell turnover time. It has been proposed that adaptation may be attributable to the generation of new surface epithelial cells that appear after damage and may have developed the capacity to resist further damage (21, 41). The results of the quantification of apoptotic cells in adapted mucosa presented here suggests that this hypothesis is credible since the most significant decrease in apoptosis after aspirin in adapted stomach was demonstrated in the surface epithelial compartment.
To date, investigation into the possible mechanisms of gastric adaptation to injury has focused on changes in growth factor expression and the subsequent enhancement of wound healing. However, it is becoming increasingly clear that gastric mucosal defense and repair mechanisms are complex, and while enhanced wound healing undoubtedly contributes to adaptation, changes in the expression and associations of intracellular proteins like the stress proteins and caspases are also likely to be involved. Combining the studies reported here with what we and others have previously reported on adaptation, we conclude that a major contributor to mucosal adaptation to chronic aspirin application may be the development of resistance to programmed cell death.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a grant from the National Health and Medical Research Council of Australia.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. S. Giraud, Dept. of Medicine, Western Hospital, Footscray, Victoria, 3011 Australia (E-mail: a.giraud{at}medicine.unimelb.edu.au).
Received 2 September 1999; accepted in final form 5 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aligue, R,
Akhavan-Niak H,
and
Russell P.
A role for HSP90 in cell cycle control: wee1 tyrosine kinase activity requires interaction with HSP90.
EMBO J
13:
6099-6106,
1994[Abstract].
2.
Amici, C,
Rossin A,
and
Santoro MG.
Aspirin enhances thermotolerance in human erythroleukemic cells: an effect associated with the modulation of the heat-shock response.
Cancer Res
55:
4452-4457,
1995[Abstract].
3.
Barnes, CJ,
Cameron IL,
Hardman WE,
and
Lee M.
Non-steroidal anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis.
Br J Cancer
77:
573-580,
1998[ISI][Medline].
4.
Brzozowski, T,
Konturek PC,
Konturek SJ,
and
Stachura J.
Gastric adaptation to aspirin and stress enhances gastric mucosal resistance against the damage by strong irritants.
Scand J Gastroenterol
31:
118-125,
1996[ISI][Medline].
5.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
6.
Cohen, GM.
Caspases: the executioners of apoptosis.
Biochem J
326:
1-16,
1997[ISI][Medline].
7.
Cullen, KE,
and
Sarge KD.
Characterization of hypothermia-induced cellular response in mouse tissues.
J Biol Chem
272:
1742-1746,
1997
8.
Dittmar, KD,
Demady DR,
Stancato LF,
Krishna P,
and
Pratt WB.
Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery.
J Biol Chem
272:
21213-21220,
1997
9.
Doljanin, K,
Skeljo MV,
Yeomans ND,
and
Giraud AS.
Adaptation of the gastric epithelium to injury is maintained in vitro and is associated with increased TGF- expression.
J Gastroenterol Hepatol
11:
259-263,
1996[ISI][Medline].
10.
Eastwood, GL,
and
Quimby F.
Effect of chronic aspirin ingestion on epithelial proliferation in rat gastric body, antrum and duodenum.
Gastroenterology
82:
852-856,
1982[ISI][Medline].
11.
Elliott, SL,
Yeomans ND,
Buchanan RRC,
and
Smallwood RA.
Efficacy of 12 months' misoprostol as prophylaxis against NSAID-induced gastric ulcers.
Scand J Rheumatol
23:
171-176,
1994[ISI][Medline].
12.
Fawcett, TW,
Xu Q,
and
Holbrook NJ.
Potentiation of heat-stress-induced hsp70 expression in vivo by aspirin.
Cell Stress Chaperones
2:
104-109,
1997[ISI][Medline].
13.
Fiorucci, S,
Antonelli E,
Santucci L,
Morelli O,
Miglietti M,
Federici B,
Mannucci R,
Del Soldato P,
and
Morelli A.
Gastrointestinal safety of nitric oxide-derived aspirin is related to inhibition of ICE-like cysteine proteases in rats.
Gastroenterology
116:
1089-1106,
1999[ISI][Medline].
14.
Fukudo, S,
Abe K,
Hongo M,
Utsumi A,
and
Itoyama Y.
Brain-gut induction of heat shock protein (HSP) 70 mRNA by psychophysiological stress in rats.
Brain Res
757:
146-148,
1997[ISI][Medline].
15.
Galea-Lauri, J,
Latchman DS,
and
Katz DR.
The role of the 90kD hsp in cell cycle control and differentiation of the monoblastoid cell line, U937.
Exp Cell Res
226:
243-254,
1996[ISI][Medline].
16.
Galea-Lauri, J,
Richardson AJ,
Latchman DS,
and
Katz DR.
Increased heat shock protein 90 (HSP90) expression leads to increased apoptosis in the monoblastoid cell line U937 following induction with TNF- and cycloheximide.
J Immunol
157:
4109-4118,
1996[Abstract].
17.
Giardina, C,
and
Lis JT.
Sodium salicylate and yeast heat shock gene transcription.
J Biol Chem
270:
10369-10372,
1995
18.
Hahm, KB,
Leek J,
Kim YS,
Kim JH,
Cho SW,
Yim H,
and
Joo HJ.
Quantitative and qualitative usefulness of rebamipide in eradication of Helicobacter pylori.
Dig Dis Sci
43 Suppl 9:
192S-197S,
1998[ISI][Medline].
19.
Hall, PA,
Coates PJ,
Ansari B,
and
Hopwood D.
Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis.
J Cell Sci
107:
3569-3577,
1994
20.
Hirakawa, T,
Rokutan K,
Nikawa T,
and
Kishi K.
Geranylgeranylacetone induces heat shock proteins in cultured guinea pig gastric mucosal cells and rat gastric mucosa.
Gastroenterology
111:
345-357,
1996[ISI][Medline].
21.
Hurley, JW,
and
Crandall LA.
The effect of salicylates upon the stomachs of dogs.
Gastroenterology
46:
36-43,
1964[ISI].
22.
Jaattela, M.
Heat shock proteins as cellular lifeguards.
Ann Med
31:
261-271,
1999[ISI][Medline].
23.
Jurivich, DA,
Sistonen L,
Kroes RA,
and
Morimoto RI.
Effect of sodium salicylate on the human heat shock response.
Science
255:
1243-1245,
1992[ISI][Medline].
24.
Konturek, JW,
Dembinski A,
Stoll R,
Domschke W,
and
Konturek SJ.
Mucosal adaptation to aspirin induced damage in humans. Studies on blood flow, gastric mucosal growth, and neutrophil activation.
Gut
35:
1197-1204,
1994[Abstract].
25.
Krajewska, M,
Wang HG,
Krajewski S,
Zapata JM,
Shabaik A,
Gascoyne R,
and
Reed JC.
Immunohistochemical analysis of in vivo patterns of expression of CPP32 (caspase-3), a cell death protease.
Cancer Res
57:
1605-1613,
1997[Abstract].
26.
Levi, S,
Goodland RA,
Lee CY,
Walport MJ,
Wright NA,
and
Hodgson HJE
Effect of nonsteroidal anti-inflammatory drugs and misoprostol on gastrointestinal epithelial proliferation in arthritis.
Gastroenterology
102:
1605-1611,
1992[ISI][Medline].
27.
Mannick, EE,
Bravo LE,
Zarama G,
Realpe JL,
Zhang XJ,
Ruiz B,
Fontham ETH,
Mera R,
Miller MJS,
and
Correa P.
Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants.
Cancer Res
56:
3238-3243,
1996[Abstract].
28.
Marber, MS,
Walker JM,
Latchman DS,
and
Yellon DM.
Myocardial protection after whole body heat stress in the rabbit is dependent on metabolic substrate and is related to the amount of the inducible 70-kDa stress protein.
J Clin Invest
93:
1087-1094,
1994[ISI][Medline].
29.
Moss, SF,
Calam J,
Agarwal B,
Wang S,
and
Holt PR.
Induction of gastric epithelial apoptosis by Helicobacter pylori.
Gut
38:
498-501,
1996[Abstract].
30.
Nakai, A,
Tanabe M,
Kawazoe Y,
Inazawa J,
Morimoto RI,
and
Nagata K.
HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator.
Mol Cell Biol
17:
469-481,
1997[Abstract].
31.
Nakamura, K,
Rokutan K,
Marui N,
Aoike A,
and
Kawai K.
Induction of heat shock proteins and their implication in protection against ethanol-induced damage in cultured guinea pig gastric mucosal cells.
Gastroenterology
101:
161-166,
1991[ISI][Medline].
32.
Picard, D,
Khursheed B,
Garabedian MJ,
Fortin MG,
Lindquist S,
and
Yamamoto KR.
Reduced levels of HSP90 compromise steroid receptor action in-vivo.
Nature
348:
166-168,
1990[ISI][Medline].
33.
Piotrowski, J,
Piotrowski E,
Skrodzka D,
Slomiany A,
and
Slomiany BL.
Gastric mucosal apoptosis induced by ethanol: effect of anti-ulcer agents.
Biochem Mol Biol Int
42:
247-254,
1997[ISI][Medline].
34.
Pratt, WB,
and
Toft DO.
Steroid receptor interactions with heat shock protein and immunophilin chaperones.
Endocr Rev
18:
306-360,
1997
35.
Reinartz, J,
Bechtel MJ,
and
Kramer MD.
Tumor necrosis factor-alpha-induced apoptosis in human keratinocyte cell line (HaCat) is counteracted by transforming growth factor-alpha.
Exp Cell Res
228:
334-340,
1996[ISI][Medline].
36.
Romano, M,
Lesch CA,
Meise KS,
Veljaca M,
Sanchez B,
Kraus ER,
Boland CR,
Guglietta A,
and
Coffey RJ.
Increased gastroduodenal concentrations of transforming growth factor alpha in adaptation to aspirin in monkeys and rats.
Gastroenterology
110:
1448-1455,
1996[Medline].
37.
Santoni-Rugiu, E,
Jensen MR,
and
Thorgeirsson SS.
Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha.
Cancer Res
58:
123-134,
1998[Abstract].
38.
Segnitz, B,
and
Gehring U.
The function of steroid hormone receptors is inhibited by the HSP90-specific compound geldanamycin.
J Biol Chem
272:
18694-18701,
1997
39.
Seki, S,
Sakai Y,
Kitada T,
Kawakita N,
Yanai A,
Tsutsui H,
Sakaguchi H,
Kuroki T,
and
Monna T.
Induction of apoptosis in human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-alpha.
Virchows Arch
430:
29-35,
1997[ISI][Medline].
40.
Shiff, SJ,
Kuotsos MI,
Qiao L,
and
Rigas B.
Nonsteroidal antiinflammatory drugs inhibit the proliferation of colon adenocarcinoma cells: effects on cell cycle and apoptosis.
Exp Cell Res
222:
179-188,
1996[ISI][Medline].
41.
Skeljo, MV,
Cook GA,
Elliott SL,
Giraud AS,
and
Yeomans ND.
Gastric mucosal adaptation to diclofenac injury.
Dig Dis Sci
41:
32-39,
1996[ISI][Medline].
42.
Slomiany, BL,
Piotrowski J,
and
Slomiany A.
Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine.
Scand J Gastroenterol
32:
638-642,
1997[ISI][Medline].
43.
Slomiany, BL,
Piotrowski J,
and
Slomiany A.
Role of caspase-3 and nitric oxide synthase-2 in gastric mucosal injury induced by indomethacin: effect of sucralfate.
J Physiol Pharmacol
50:
3-16,
1999[ISI][Medline].
44.
Stachura, J,
Konturek JW,
Dembinski A,
and
Domachke W.
Do infiltrating leukocytes contribute to the adaptation of human gastric mucosa to continued aspirin administration.
Scand J Gastroenterol
29:
966-972,
1994[ISI][Medline].
45.
St. John, DJB,
Yeomans ND,
Mcdermott FT,
and
de Boer WGRM
Adaptation of the gastric mucosa to repeated administration of aspirin in the rat.
Am J Dig Dis
18:
881-886,
1973[ISI][Medline].
46.
Stojadinovic, A,
Kiang J,
Smallridge R,
Galloway R,
and
Shea-Donohue T.
Induction of heat-shock protein 72 protects against ischemia/reperfusion in rat small intestine.
Gastroenterology
109:
505-515,
1995[ISI][Medline].
47.
Wallace, JL,
McKnight GW,
and
Bell CJ.
Adaptation of rat gastric mucosa to aspirin requires mucosal contact.
Am J Physiol Gastrointest Liver Physiol
268:
G134-G138,
1995
48.
Winegarden, NA,
Wong KS,
Sopta M,
and
Westwood JT.
Sodium salicylate decreases intracellular ATP, induces both heat shock factor binding and chromosomal puffing, but does not induce hsp 70 gene transcription in Drosophila.
J Biol Chem
271:
26971-26980,
1996
49.
Yeomans, ND.
Electron microscopic study of the repair of aspirin induced gastric erosions.
Dig Dis Sci
21:
533-541,
1976.
50.
Yeomans, ND,
Poulsom R,
Cook GA,
Giraud AS,
and
Wright NA.
Distribution of heat shock protein mRNAs in rat gastric mucosa (Abstract).
J Gastroenterol Hepatol
10 Suppl 2:
A33,
1995.
51.
Yeomans, ND,
St. John DJB,
and
de Boer WGRM
Regeneration of gastric mucosa after aspirin induced injury in the rat.
Am J Dig Dis
18:
773-780,
1973[ISI][Medline].
52.
Zhu, GH,
Yang XL,
Lai KC,
Ching CK,
Wong BC,
Yuen ST,
Ho J,
and
Lam SK.
Non-steroidal anti-inflammatory drugs could reverse Helicobacter pylori-induced apoptosis and proliferation in gastric epithelial cells.
Dig Dis Sci
43:
1957-1963,
1998[ISI][Medline].