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1 Department of Medicine, Division of Gastroenterology, 2 Department of Radiation Oncology, and 3 Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland; 4 Veterans Affairs Maryland Health Care System, Baltimore, Maryland; 5 Department of Medicine, Division of Gastroenterology, Rhode Island Hospital/Brown University, Providence, Rhode Island; and 6 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Materials and Methods |
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Reverse Transcription-Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction.
Total RNA was isolated from AGS cells and cDNA synthesized, and primer sequences and polymerase chain reaction (PCR) product sizes for ß-actin and PAO1 [mouse homologue of SMO(PAOh1)] and PCR conditions for the multiplex reactions were as reported (2)
. Primer sequences for human SMO(PAOh1) were: sense, 5'-GACCACAATCACGACACTGG-3', and antisense, 5'-TTAGCACACCTAGCGACACG-3', yielding a 160-bp product. Real-time PCR was performed using SYBR Green (2)
. For AGS experiments, relative expression of SMO(PAOh1) was determined using ß-actin as the internal control (2)
. In tissue studies, SMO(PAOh1) expression was normalized to 18S rRNA.
SMO(PAOh1) Promoter Activity.
A genomic DNA plasmid library was constructed from human A549 adenocarcinoma cell DNA in the pBluescript SK(–) plasmid and screened with a cDNA probe homologous to exon 1 of SMO(PAOh1). From this library, a clone containing –4479 bp to the transcriptional start site was identified. Deletion constructs were generated by restriction enzyme digestion and subcloned into pGL-2 basic. AGS cells were transiently transfected (2)
with 200 ng of the above constructs and luciferase activity performed according to the manufacturer’s instructions (Promega, Madison, WI).
SMO(PAOh1) Activity.
Lysates of AGS cells were analyzed by a chemiluminescence assay as described previously (2
, 12)
, and the activity was expressed as nanomoles of H2O2 per minute per milligram protein.
Measurement of H2O2.
AGS cells were incubated with 10 µmol/L CM-H2DCFDA and intracellular H2O2 detected by flow cytometry as described previously (2)
. For measurement of H2O2 in supernatants, 5 x 105 cells were plated in 24-well plates. After stimulation, cells were washed and incubated with 50 µmol/L Amplex Red reagent (Molecular Probes, Eugene OR) and 0.1 unit/mL horseradish peroxidase for 30 minutes at 37°C. Plates were read using a microplate reader at 560 nm, and a standard curve with varying dilutions of H2O2 was used (2)
.
Assessment of Apoptosis.
Apoptosis was assayed using an annexin V-fluorescein isothiocyanate apoptosis detection kit (Oncogene Research Products, San Diego, CA) according to the manufacturer’s instructions. Cells (1 x 104) were analyzed by flow cytometry (2)
. Apoptosis was also assessed by enzyme-linked immunosorbent assay of cytoplasmic histone-associated DNA fragments (13)
.
Transient Transfection of SMO(PAOh1).
AGS cells were transfected with 200 ng of pcDNA3.1-SMO(PAOh1) using LipofectAMINE PLUS and optiMEM medium (2)
. Transfection efficiency was determined by fluorescence in cells transfected with 400 ng of pIRES2-EGFP (Clontech, Palo Alto, CA).
Transient Transfection of SMO(PAOh1) Small Interfering RNA.
Small interfering RNA duplexes were used that targeted SMO(PAOh1) nucleotides 468 to 486, numbered from the start codon (sense, 5'-GGACGUGGUUGAGGAAUUC-3'; antisense, 5'-CCUGCACCAACUCCUUAAG-3'). Scrambled small interfering RNA with no sequence homology to any known genes was used as the control. Transfection conditions were as described previously (2)
.
DNA Damage Assays.
DNA damage was assessed by the alkaline single-cell gel electrophoresis (comet assay) method (14)
. AGS cells were stimulated, trypsinized, and embedded into 0.5% low-melting agarose on glass microscope slides. After treatment with alkaline lysis buffer, slides were electrophoresed, stained with propidium iodide, and analyzed by epifluorescence microscopy. DNA damage was measured by the tail moment, defined as product of the length of the tail (in micrometers), which is DNA migrated from the nucleus, and the percentage of DNA in the tail (14)
.
DNA damage was also determined by 8-oxoguanosine binding. In brief, after fixation and permeabilization, cells were washed, blocked, and incubated with 8-oxoguanosine–fluorescein isothiocyanate conjugate (Kamiya Biomedical, Seattle, WA) for 1 hour in the dark. Cells were resuspended in PBS and analyzed by flow cytometry for fluorescence.
H. pylori Gastritis Tissues.
C57BL/6 mice were infected with H. pylori SS1 and gastric tissues harvested 4 months later (13)
. Human gastritis samples were obtained from patients at the Baltimore Veterans Affairs Medical Center, with H. pylori status determined as described previously (15)
. Antral biopsies from patients with H. pylori infection from Uijongbu St. Mary Hospital (Uijongbu, Korea) were evaluated before and 2 months after confirmed H. pylori eradication. Histologic analysis after eradication demonstrated complete resolution of acute inflammation in all cases and significant reduction of chronic inflammation. Laser capture microdissection of approximately 5000 gastric epithelial cells from formalin-fixed, paraffin-embedded endoscopic gastric biopsies was performed using the Autopix automated LCM system (Arcturus, Mountain View, CA). RNA was extracted, and SMO(PAOh1) mRNA was quantified by real-time PCR and normalized for 18S rRNA.
Statistical Analysis.
For quantitative data, values represent the mean ± SE. For comparisons between multiple groups, the Student-Newman-Keuls test was used; and for single comparisons between two groups, Student’s t test was used.
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Results |
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. Stimulation with H. pylori also resulted in a significant increase in SMO(PAOh1) promoter activity with the –1117-bp construct (Fig. 1B)
, indicating that the observed increase in SMO(PAOh1) mRNA is due to infection-induced transcription. There was a time-dependent increase in SMO(PAOh1) enzyme activity (Fig. 1C)
that peaked at 12 hours after H. pylori stimulation. In contrast, there was no induction of activity of the acetyl PAO (ref. 16
; data not shown) when assessed by a specific assay (2)
. To determine whether this increase in oxidase activity was accompanied by apoptosis, the sensitive technique of annexin V and propidium iodide staining of live cells was used to measure apoptosis in a highly quantitative manner. As shown in Fig. 1C
, the time course of the induction of apoptosis closely paralleled that of SMO(PAOh1) activity.
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that was prevented by the inhibition of SMO(PAOh1) by MDL 72527 or by the addition of catalase. We also used the Amplex Red assay, specific for H2O2 in the medium, to demonstrate that H. pylori induced a significant, 2.7 ± 0.1-fold increase in extracellular H2O2 that was inhibited by 79.6 ± 7.1% with MDL 72527 and 100.4 ± 7.8% with catalase (P < 0.01 for H. pylori versus control and for H. pylori + inhibitors versus H. pylori alone; data not shown). Consistent with the H2O2 data, apoptosis induced by H. pylori was significantly attenuated by MDL 72527 and catalase, as shown in Fig. 1E and F
. We also confirmed these findings by analysis of apoptosis by DNA fragmentation enzyme-linked immunosorbent assay (data not shown).
Because MDL 72527 inhibits both PAO and SMO(PAOh1) (8
, 9
, 16) , to determine whether the spermine oxidation-mediated apo-ptosis was specifically due to SMO(PAOh1), we transiently transfected AGS cells with a duplex small interfering RNA specific for SMO(PAOh1). This treatment significantly inhibited H. pylori-stimulated SMO(PAOh1) mRNA expression (Fig. 2A)
and produced a 79.1 ± 8.1% inhibition of SMO(PAOh1) enzyme activity (Fig. 2B)
. This knockdown of SMO(PAOh1) was associated with a 71.6 ± 5.8% inhibition of apoptosis (Fig. 2C)
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H. pylori-Induced Deoxyribonucleic Acid Damage in Gastric Epithelial Cells Is Mediated by SMO(PAOh1).
The comet assay was used to directly visualize DNA damage morphologically. There was a marked increase in the size and intensity of the tail of the DNA fluorescence of the cells in the H. pylori-treated (Fig. 3B)
versus untreated cells (Fig. 3A)
. Treatment of AGS cells with MDL 72527 (Fig. 3C)
or catalase (Fig. 3D)
resulted in reduction of damage. We quantitated the tail moment in >270 cells for each condition and found that there was a 4.4 ± 0.1-fold increase with H. pylori (P < 0.01 versus control) that was inhibited by 90.3 ± 4.1% with MDL 72527 and 87.6 ± 4.2% with catalase (P < 0.01 for both inhibitors versus H. pylori alone). When we assessed 8-oxoguanosine binding by flow cytometry as an indicator of oxidatively damaged DNA, we found that stimulation with H. pylori resulted in increased fluorescence that was significantly attenuated with MDL 72527 or catalase (Fig. 3E)
or transfection with SMO(PAOh1) small interfering RNA (Fig. 3F)
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and human SMO(PAOh1) (Fig. 4B)
in H. pylori gastritis tissues. Levels of SMO(PAOh1) in human gastritis tissues from H. pylori-negative patients (Fig. 4B)
were only modestly increased, whereas tissues from H. pylori-infected patients exhibited consistently higher levels of expression. Real-time PCR analysis in the mouse tissues revealed a 3.8 ± 0.4-fold increase in H. pylori gastritis versus uninfected tissues (P < 0.01), and in human samples, there was a 2.5 ± 0.4-fold increase in H. pylori-negative gastritis and a 4.9 ± 1.5-fold increase in H. pylori-positive gastritis (P < 0.05 versus normal for H. pylori positive only). To confirm that SMO(PAOh1) was expressed in vivo in the gastric epithelium, we used RNA extracted from epithelial cells harvested by laser capture microdissection from H. pylori-infected gastric tissues (Fig. 4C)
. After eradication therapy, SMO(PAOh1) mRNA levels determined by real-time PCR decreased in each patient, with an 85.4 ± 7.5% inhibition compared with levels before treatment (P < 0.001).
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Discussion |
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H. pylori infection has been reported to cause production of H2O2 in AGS human gastric epithelial cells, and it has been suggested that this may contribute to the carcinogenic process in H. pylori-induced gastric cancer (18) . However, the source of the H2O2 was not determined. Recently, it has been demonstrated that in glutathione peroxidase 1 and 2 (Gpx1 and Gpx2) knockout mice, there is a high incidence of ileocolitis and microflora-asociated cancers when mice are raised in conventional housing that includes infection with Helicobacter species (19) , but when these mice are raised under germ-free conditions, they do not develop tumors. Because Gpx1 and Gpx2 are major detoxifying enzymes for H2O2, these results indicate that the reactive oxygen species responsible for the inflammation and carcinogenesis is bacterial infection-induced H2O2 production.
Here, we demonstrate that H. pylori exposure leads to increased H2O2 production in AGS cells that can be inhibited by MDL72527, indicating that a polyamine oxidase activity is responsible for the H2O2 production. It should also be noted that our results are not restricted to a single cell line, because we have found similar effects of MDL 72527 in MKN-28 gastric epithelial stimulated with H. pylori (data not shown). The SMO(PAOh1) activity is sufficient to produce DNA-damaging amounts of H2O2, as evidenced by both 8-oxo-guanosine production and comet assay. Although generation of reactive oxygen species in response to H. pylori has been previously linked to DNA damage (6 , 7) , our studies provide a new mechanism for generation of reactive oxygen species in epithelial cells and directly demonstrate that the polyamine oxidation causes both apoptosis and DNA damage by this mechanism. The DNA damage and apoptotic cell death produced by H2O2 was inhibited by MDL 72527, catalase, and most importantly, SMO(PAOh1)-specific small interfering RNA, thus demonstrating that the oxidase in question is, in fact, the spermine oxidase SMO(PAOh1) and not the classical N1-acetylpolyamine oxidase, PAO (16) . These data combined with those from the transient transfection studies demonstrating that SMO(PAOh1) produces the same effects as H. pylori exposure confirm that the oxidation of spermine by SMO(PAOh1) is the source of H2O2 in H. pylori-exposed AGS cells and is directly responsible for the observed downstream effects.
It is important to note the potential that some reactive oxygen species-induced mutations may not be lethal and may lead to growth and/or survival advantages. 8-Hydroxydeoxyguanosine, a common adduct downstream from H2O2 production, is known to produce G>T transversion mutations that are commonly found in tumor suppressor genes and oncogenes (20 , 21) .
The in vitro results are consistent with an association between activation of SMO(PAOh1) and H. pylori-induced pathogenesis. The results from both the C57BL/6 mouse model of gastritis and human gastritis patients clearly demonstrate an association between H. pylori infection and expression of SMO(PAOh1) (or its mouse homologue). Importantly, when human H. pylori infection is eradicated by antibiotic treatment, there is a significant decrease in SMO(PAOh1) mRNA expression in the gastric epithelium. These data demonstrate that H. pylori-induced SMO(PAOh1) expression extends beyond in vitro systems and has in vivo relevance.
In summary, the results presented here demonstrate that H. pylori infection leads to the increased expression of an important polyamine catabolic enzyme, the spermine oxidase, SMO(PAOh1). This enzyme oxidizes spermine producing the DNA-damaging reactive oxygen species, H2O2. Because reactive oxygen species have been directly linked to the etiology of multiple cancers including H. pylori-induced gastric cancer, these data are entirely consistent with the hypothesis that H. pylori-induced SMO(PAOh1) activity is responsible for the genotoxic insult that results in tumorigenic transformation of affected gastric epithelial cells.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Keith T. Wilson, University of Maryland School of Medicine, 22 South Greene Street, Room N3W62, Baltimore, MD 21201. Phone: 410-706-1471; Fax: 410-706-1573. E-mail: kwilson{at}umaryland.edu
Received 9/29/04. Accepted 10/13/04.
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, individual patients; 
, mean ± SE for each group; ***, P < 0.001 versus HP + before eradication by paired Student’s t test.