[Cancer Research 61, 6356-6359, September 1, 2001]
© 2001 American Association for Cancer Research
Helicobacter Pylori Membrane Protein 1: A New Carcinogenic Factor of Helicobacter Pylori1
Masami Suganuma,
Miki Kurusu,
Sachiko Okabe,
Naoko Sueoka,
Masaru Yoshida,
Yoshio Wakatsuki and
Hirota Fujiki2
Saitama Cancer Center, Saitama 362-0806, Japan [M. S., M. K., S. O., N. S., H. F.], and Division of Clinico Bio-Regulatory Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8707, Japan [M. Y., Y. W.]
 |
ABSTRACT
|
|---|
Considering a suspected link between Helicobacter pylori
infection and human stomach cancer, a new H. pylori
gene for membrane protein 1 (HP-MP1)
was recently cloned. Because HP-MP1 induces release of inflammatory
cytokines and tumor necrosis factor-
acts as both initiator and
tumor promoter, we studied the possible involvement of HP-MP1 in
carcinogenesis of H. pylori. Two cell lines, BALB/3T3
cells as control and v-Ha-ras-transfected BALB/3T3 cells
(Bhas 42 cells) as putative initiated cells, were each transfected with
HP-MP1, urease B genes, or vector alone.
All of the Bhas/mpl clones showed strong expression of tumor necrosis
factor- gene and produced tumors in 100% of nude mice. Two Bhas/ure
clones showed weak tumorigenicity; the other Bhas and BALB clones
showed none. Results indicate strong carcinogenic activity of HP-MP1 in
cooperation with viral Ras protein and weak activity of urease B.
|
Introduction
|
|---|
HP-MP13
is a Mr 16,000 protein, the gene of
which was cloned recently (1)
from genomic DNA of
H. pylori strain SR 7791. Yoshida et al.
(1)
reported that HP-MP1 protein attached to the inner
membrane of H. pylori as homodimer but that its function in
the bacteria was not clear. We paid special attention to the results
showing that HP-MP1 and urease induced release of various inflammatory
cytokines, such as TNF-, IL-1, IL-8, and macrophage
inflammatory protein 1 from human monocytes (1
, 2) and
also noted that anti-HP-MP1 antibody was detected in the sera of
H. pylori-infected patients (1)
. Considered
together, the results suggested that HP-MP1 might be a new
proinflammatory mediator in stomach infected with H. pylori.
Our study on tumor promotion with TNF--deficient mice had revealed
the TNF- and other inflammatory cytokines stimulate tumor
promotion mediated through activation of nuclear factor 
B (3
, 4)
.
Among various inflammatory cytokines, we demonstrated recently
(3)
that TNF- is the first instigator in tumor
promotion and that the sequence of cytokine network for tumor promotion
appears to be from TNF- through IL-1 and IL-6. On the basis of this,
we believe that among various inflammatory cytokines, TNF- is the
essential tumor promoter (5)
. If so, the TNF--inducing
activity of HP-MP1 would have a function as a cancer mediator. In the
light of this, we asked how could the cytokine-inducing activity of
HP-MP1 be linked to carcinogenesis?
Our first indication of TNF- as an endogenous tumor promoter was
finding that TNF- induced clonal growth of
v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), whereas
it did not induce growth of BALB/3T3 cells without v-Ha-ras
(6)
. Furthermore, we and other investigators found that
Bhas 42 cells were useful tools to demonstrate in vitro the
tumor-promoting activity of some proteins, such as leukemia-related
protein MTG8 (ETO; Ref. 7
) and hepatitis C virus core
protein (8)
. Considering the accumulated results, we
transfected HP-MP1 gene into both Bhas 42 and BALB/3T3
cells. We found that Bhas/mpl clones expressed TNF- gene
more strongly than did BALB/mpl clones, with the Bhas/mpl clones
inducing cell transformation. The carcinogenicity of the transformed
clones was further confirmed by soft agar colony formation and tumor
development in nude mice. Bhas 42 and BALB/3T3 cells were similarly
transfected by urease B gene of H. pylori or
vector alone. Two Bhas/ure clones, which expressed TNF-
gene, showed weak tumorigenicity in nude mice, but none of the other
clones of either cell type showed any significant transformation.
This study shows that TNF- protein, induced by both HP-MP1 and
urease B, resulted in cell transformation in cooperation with viral Ras
protein, strongly suggesting a new carcinogenic mechanism of human
stomach cancer development associated with H. pylori
infection.
|
Materials and Methods
|
|---|
Plasmids and Transformed Clones.
HP-MP1 gene cloned from H. pylori SR 7791 and
urease B gene from H. pylori NCTC 11637 were
subcloned into the His tag expression vector, pET28c(+) (Novagen,
Madison, WI), as reported previously (1)
. The
HP-MP1 and urease B genes were separately
inserted into the mammalian expression vector pcDNA3.1/hygro(+)
(Invitrogen, Carlsbad, CA), which contains human cytomegalovirus
immediate-early promoter and hygromycin-resistant gene,
resulting in the expression plasmids, pcDNA-mpl and pcDNA-ure. These
two plasmids were each transfected into both
v-Ha-ras-transfected BALB/3T3 cells (Bhas 42; Ref.
9
) and BALB/3T3 A31-1-1 cells (JCRB0601) by lipofection
protocol using DIMRIE-C reagent (Life Technologies, Inc., Rockville,
MD). The two types of cells were provided by the Japanese Cancer
Research Resources Bank (Tokyo, Japan). After 48-h culture, hygromycin
B (Sigma Chemical Co.) at a concentration of 400 µg/ml was added to
the medium, and this was further cultured for 2 weeks. As a negative
control, pcDNA3.1/hygro vector alone was transfected into both types of
cells. Twenty hygromycin B-resistant foci were picked from each set of
transfections and were then grown separately to study the properties of
transformed clones. Finally, three groups of Bhas clones, Bhas/mpl,
Bhas/ure, and Bhas/vec, and three groups of BALB clones, BALB/mp1,
BALB/ure, and BALB/vec, were obtained.
Expression of TNF- Gene.
Total RNA was isolated from five clones randomly selected from the
three Bhas groups and five clones from the three BALB groups by Isogen
reagent (10)
. TNF- mRNA was determined using a
semiquantitative RT-PCR method, as reported previously
(11)
. PCR amplification was conducted in the presence of
[-32P]dCTP, and
glyceraldehyde-3-phosphate dehydrogenase gene was
used as a control. The primers used were
5'-AGCCCACGTCGTAGCAAACCACCAA-3' (sense) and
5'-ACACCCATTCCCTTCACAGAGCAAT-3' (antisense) for TNF-
gene, and 5'-TGGCATTGTGGAAGGGCTCATGAC-3' (sense) and
5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' (antisense) for
glyceraldehyde-3-phosphate dehydrogenase gene. TNF- mRNA
was expressed as relative fold expression in comparison with that of
nontransfected controls, Bhas 42 and BALB/3T3 cells. All of the
experiments were repeated twice.
Determination of TNF- Protein.
Each (3 x 106 cells) of the Bhas
and BALB clones in growing stage was extracted with lysis buffer [20
mM Tris-HCl (pH 8.0), 150 mM NaCl, 4
mM EDTA, 10 mM sodium
PPi, 2 mM sodium vanadate, 0.1% SDS,
1% sodium deoxycholate, 1% Triton X-100, 10 µg/ml aprotinin, 5
µg/ml leupeptin, and 2 mM phenylmethylsulfonyl
fluoride]. After 14,000 rpm centrifugation for 15 min, each
supernatant (about 350 mg) was subjected to TNF- ELISA kit (Genzyme
Co., Cambridge, MA). The concentration of TNF- in cell lysates was
expressed as pg/mg protein. Experiments were duplicated.
Expression of HP-MP1 and Urease B
Genes in Bhas and BALB Clones.
Expression of HP-MP1 and urease B genes was
determined by RT-PCR. Briefly, total RNA (1 µg) was reverse
transcribed by murine leukemia virus reverse transcriptase (Roche
Molecular Systems, Inc., Branchburg, NJ) using
oligodeoxythymidylate16. PCR amplification
was performed with AmpliTaq DNA polymerase in the PCR reaction mixture
for 30 cycles (95°C for 30 s, 55°C for 30 s, and 72°C
for 60 s). The PCR products were subjected to electrophoresis in
1.0% agarose gel and stained with ethidium bromide. Primers used in
this experiment were 5'-TTGCAAAATCGCAGTGCGTAT-3' (sense) and
5'-GGCTATAGG GACTTTCGCATCGC-3' (antisense) for HP-MP1 gene
and 5'-CGGTGAAGGTTTGATCGTAACG-3' (sense) and
5'-CAAAGTGTCTGTGTGGATAGCGACT-3' (antisense) for urease B
gene.
Soft Agar Colony Formation of Bhas and BALB Clones.
Five clones from the three Bhas groups (Bhas/mpl, Bhas/ure, and
Bhas/vec) and five from the three BALB groups (BALB/mpl, BALB/ure, and
BALB/vec) were tested for anchorage-independent growth in soft agar.
Each clone (5 x 102 cells) was
cultured in MEM containing 0.22% soft agar in a 35-mm diameter dish in
duplicate (12)
. The numbers of large colonies (>0.4 mm in
diameter) determined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were
counted 3 weeks after plating. The parental Bhas 42 and BALB/3T3 cells
were similarly cultured.
Tumorigenicity of Bhas/mp1 and Two Other Bhas Clones in Nude
Mice.
Three clones (each 5 x 106 cells)
from the three Bhas groups (Bhas/mpl, Bhas/ure, and Bhas/vec) and the
parental Bhas 42 cells were implanted s.c. into two sites/one nude male
mouse (5 weeks of age; BALB/cAJcl-nu; The Jackson Laboratory; Ref.
7
). Thus, one Bhas clone was implanted into a total of six
sites for the three nude mice. Tumor development was measured once a
week for 4 weeks. Average tumor volume was expressed as mean ± SD of tumors by calculation as (short
axis)2 x (long axis)/2.
|
Results and Discussion
|
|---|
Expression of TNF- Gene and Determination of
TNF- in Bhas and BALB Clones.
Three groups of Bhas clones and three groups of BALB clones were
obtained by transfection with pcDNA3.1/hygro vector containing
HP-MP1 gene, urease B gene, or vector alone. Then
five clones were randomly selected from each group and named as
follows: Bhas/mp1: 52, 53, 54, 57, and 63; Bhas/ure: 52, 55, 57, 61,
and 65; or Bhas/vec: 53, 57, 68, 74, and 75; and BALB/mp1: 9, 11, 13,
16, and 19; BALB/ure: 2, 7, 9, 17, and 19; or BALB/vec: 3, 9, 12, 17,
and 18. Expression of TNF- gene in Bhas and BALB clones
was determined using a semiquantitative RT-PCR method. All of the five
Bhas/mp1 clones significantly expressed TNF- gene, with
expression levels ranging from 12.2–27.0-fold higher than basal levels
of the parental Bhas 42 cells (Fig. 1A)
. Two Bhas/ure clones (61 and 65) of five expressed
TNF- gene 15.5-fold and 15.1-fold higher than basal
levels did, but the three other clones did not express it. The
expression levels of all of the five Bhas/vec clones were at basal
levels. Four BALB/mp1 clones expressed TNF- gene from
3.3-fold to 6.3-fold higher than basal levels of the parental BALB/3T3
cells, but the expression level of one clone, BALB/mp1-13, was less
than that of the parental cells (Fig. 1B)
. The expression
levels of BALB/ure and BALB/vec clones were marginal. The results
indicate that, compared with its transfection into BALB/3T3 cells,
transfection of HP-MP1 gene into Bhas 42 cells strongly
induced TNF- mRNA expression in cooperation with v-Ha-ras
gene, with expression levels varying slightly among the Bhas/mp1
clones. Only two Bhas/ure clones (61 and 65) expressed
TNF- gene significantly; the other three did not express
it even in the presence of v-Ha-ras gene.
Although production of TNF- protein in Bhas/mp1 clones was
relatively low, TNF- protein was detected in three of five Bhas/mp1
clones. Specifically, Bhas/mp1-54, -57, and -63 clones produced TNF-
with concentrations of 32.1, 18.9, and 12.2 pg/mg protein,
respectively. However, TNF- protein was under detectable levels in
cell lysates of all of the Bhas/ure clones, including 61 and 65, and
Bhas/vec clones.
Transformation of Bhas/mp1 Clones.
Five Bhas/mp1 clones induced morphological changes of transformation,
such as spindle shape and multilayer/criss-cross formation (Fig. 2A)
, whereas Bhas/ure and Bhas/vec clones did not induce
morphological changes. Before going on to additional experiments, we
first confirmed expression of HP-MP1 and urease B
genes in Bhas/mp1, Bhas/ure, or Bhas/vec clones. Fig. 2B
shows that all of the five Bhas/mp1 clones expressed HP-MP1
gene but not urease B gene, whereas the five Bhas/ure clones
expressed urease B gene but not HP-MP1 gene. The
five Bhas/vec clones did not express either HP-MP1 or
urease B. Furthermore, the presence of HP-MP1 protein in
Bhas/mp1 clones and that of urease B protein in Bhas/ure clones was
confirmed by Western blotting using anti-HP-MP1 antibody and
anti-urease B antibody (data not shown).
View larger version (90K):
[in this window]
[in a new window]
|
Fig. 2. Morphological changes of Bhas/mpl clone compared with
those of Bhas/ure and Bhas/vec clones and parental Bhas 42 cells
(A). All of the five Bhas/mpl clones induced
morphological changes of transformation. Confirmation of
HP-MP1 and urease B gene expressions in
Bhas/mpl, Bhas/ure, and Bhas/vec clones (B).
|
|
Next, we tested the transforming activity by anchorage-independent
growth in a semisolid medium of soft agar. Five Bhas/mp1 clones
produced large colonies in soft agars, with an average colony number of
20 ± 10.1 (Table 1)
, whereas those of Bhas/ure clones, Bhas/vec clones, and the parental
Bhas 42 cells were 4.1 ± 6.1, 2.3 ± 1.5, and 2.0, respectively. Moreover, none of the BALB clones
(BALB/mp1, BALB/ure, and BALB/vec) and none of the parental BALB/3T3
cells induced any significant colony formation. These results clearly
indicated that HP-MP1, a TNF--inducing protein of H.
pylori, transformed BALB/3T3 cells only in cooperation with viral
Ras protein.
Tumorigenicity of Bhas/mp1 Clones in Nude Mice.
To examine malignant phenotypes of Bhas/mp1 clones, three Bhas/mp1
clones (52, 57, and 63) were implanted s.c. into six sites of three
nude mice, two sites/mouse. Fifteen days after implantation, the three
Bhas/mp1 clones produced tumors at 17 of 18 injected sites (94.4%) and
reached 100% (18 of 18) within 20 days (Fig. 3A)
. Tumors of Bhas/mp1 clones grew rapidly, with average
tumor volume reaching 4.3 ± 1.8
cm3
after 28 days (Fig. 3B)
. In
contrast, tumors developed in only 33.3% of sites in the three
Bhas/ure clones 28 days after implantation, and one clone (57)
developed no tumors. In the other two clones (61 and 65), tumors were
found at six sites, with an average tumor volume of 2.4 ± 5.6 cm3
. Thus, these experiments with
Bhas groups showed that the clones that had expressed
TNF- gene significantly were clearly tumorigenic in nude
mice within 28 days after implantation.
Furthermore, Bhas/vec clones and Bhas 42 cells did not induce any
tumors within 28 days. Tumors of Bhas/mp1 clones strongly showed
malignant phenotypes, such as large tumors with rapid growth (Fig. 3B)
, apparent invasion from s.c. tissue into peritoneum, and
strong angiogenesis.
The potent tumorigenicity of the three Bhas/mp1 clones in nude mice
correlated well with the potency of anchorage-independent growth (Table 1)
, indicating that cooperation of HP-MP1 with viral Ras protein,
specifically, induces transformation of BALB/3T3 cells. The potency of
HP-MP1 for tumorigenicity was much stronger than that of urease B, and
the qualitative difference of carcinogenic features between Bhas/mp1
and Bhas/ure clones is now under investigation.
HP-MP1 protein is structurally not related to virulence factors of
H. pylori, such as urease, the vacuolating cytotoxin (VacA),
and the cytotoxin-associated gene A antigen (CagA)
(13, 14, 15, 16)
. And using viral Ras protein, we were able to
show carcinogenic activity of HP-MP1 in BALB/3T3 cells. Putting these
together, we find that transformation is caused by two main
carcinogenic mechanisms: TNF- is induced by HP-MP1 and consequently
activates nuclear factor-B (17)
; and the presence of
putative activated Ras protein activates mitogen-activated
protein kinase (18)
. Moreover, the expression of
viral Ras protein in v-Ha-ras-transfected BALB/3T3 cells can
be functionally replaced by enhanced c-Ha-ras p21 expression
in the stomach cancers of humans, based on evidence that levels of
c-Ha-ras p21 in extracts of human stomach adenocarcinomas
were higher than those from extracts of their normal counterparts and
also higher than those of c-Ki-ras and c-Na-ras
(19)
. Thus, we think that this carcinogenic process
resembles multistage carcinogenesis in humans. In the light of this,
the crucial inquiry into the link between stomach cancer and H.
pylori infection (20)
should be investigated looking
at expression of HP-MP1 gene and coexpression of
TNF- gene.
|
ACKNOWLEDGMENTS
|
|---|
We thank Dr. Kei Nakachi, Radiation Effects Research
Foundation, for his fruitful discussion.
|
FOOTNOTES
|
|---|
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.
1 Supported by the following Grants-in-Aid: for
Scientific Research on Priority Areas for Cancer Research from the
Ministry of Education, Science, Sports and Culture, Japan; for a
Comprehensive 10-Year Strategy for Cancer Control Cancer Research, and
Comprehensive Research on Aging and Health from the Ministry of Health
and Welfare, Japan; and by the Smoking Research Fund. 
2 To whom requests for reprints should addressed,
at Saitama Cancer Center, Ina, Kitaadachi-gun, Saitama 362-0806, Japan.
E-mail: hfujiki{at}cancer-c.pref.saitama.jp
3 The abbreviations used are:
HP-MP1, Helicobacter pylori gene for
membrane protein 1; TNF-, tumor necrosis factor ; IL,
interleukin; RT-PCR, reverse transcription PCR.
Received 5/29/01.
Accepted 7/18/01.
|
REFERENCES
|
|---|
-
Yoshida M., Wakatsuki Y., Kobayashi Y., Itoh T., Murakami K., Mizoguchi A., Usui T., Chiba T., Kita T. Cloning and characterization of a novel membrane-associated antigen protein of Helicobacter pylori.. Infect. Immun., 67: 286-293, 1999.[Abstract/Free Full Text]
-
Harris P. R., Mobley H. L. T., Perez-Perez G. I., Blaser M. J., Smith P. D. Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production.. Gastroenterology, 111: 419-425, 1996.[Medline]
-
Suganuma M., Okabe S., Marino M. W., Sakai A., Sueoka E., Fujiki H. Essential role of tumor necrosis factor (TNF-) in tumor promotion as revealed by TNF--deficient mice.. Cancer Res., 59: 4516-4518, 1999.[Abstract/Free Full Text]
-
Moore R. J., Owens D. M., Stamp G., Arnott C., Burke F., East N., Holdsworth H., Turner L., Rollins B., Pasparakis M., Kollias G., Balkwill F. Mice deficient in tumor necrosis factor- are resistant to skin carcinogenesis.. Nat. Med., 5: 828-831, 1999.[Medline]
-
Fujiki H., Suganuma M., Okabe S., Sueoka E., Suga K., Imai K., Nakachi K. A new concept of tumor promotion by tumor necrosis factor-, and cancer preventive agents (-)-epigallocatechin gallate and green tea–a review.. Cancer Detect. Prev., 24: 91-99, 2000.[Medline]
-
Komori A., Yatsunami J., Suganuma M., Okabe S., Abe S., Sakai A., Sasaki K., Fujiki H. Tumor necrosis factor acts as a tumor promoter in BALB/3T3 cell transformation.. Cancer Res., 53: 1982-1985, 1993.[Abstract/Free Full Text]
-
Sueoka E., Sueoka N., Okabe S., Komori A., Suganuma M., Kozu T., Fujiki H. Tumorigenicity of MTG8, a leukaemia-related gene, in concert with v-Ha-ras gene in BALB/3T3 cells.. Br. J. Haematol., 101: 737-742, 1998.[Medline]
-
Tsuchihara K., Hijikata M., Fukuda K., Kuroki T., Yamamoto N., Shimotohno K. Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli.. Virology, 258: 100-107, 1999.[Medline]
-
Sasaki K., Mizusawa H., Ishidate M. Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoylphorbol-13-acetate.. Jpn. J. Cancer Res., 79: 921-930, 1988.[Medline]
-
Okabe S., Suganuma M., Sueoka N., Fujiki H. Modulation of gene expression by (-)-epigallocatechin gallate in PC-9 cells using cDNA expression array.. Biol. Pharm. Bull., 24: 883-886, 2001.[Medline]
-
Suganuma M., Okabe S., Sueoka E., Iida N., Komori A., Kim S-J., Fujiki H. A new process of cancer prevention mediated through inhibition of tumor necrosis factor expression.. Cancer Res., 56: 3711-3715, 1996.[Abstract/Free Full Text]
-
Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes.. Nature (Lond.), 304: 596-602, 1983.[Medline]
-
Solnick J. V., O’Rourke J., Lee A., Tompkins L. S. Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.. Infect. Immun., 62: 1631-1638, 1994.[Abstract/Free Full Text]
-
Cover T. L., Tummuru M. K., Cao P., Thompson S. A., Blaser M. J. Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.. J. Biol. Chem., 269: 10566-10573, 1994.[Abstract/Free Full Text]
-
Odenbreit S., Püls J., Sedlmaier B., Gerland E., Fischer W., Haas R. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.. Science (Wash. DC), 287: 1497-1500, 2000.[Abstract/Free Full Text]
-
Parsonnet J., Friedman G. D., Vandersteen D. P., Chang Y., Vogelman J. H., Orentreich N., Sibley R. K. Helicobacter pylori infection and the risk of gastric carcinoma.. N. Engl. J. Med., 325: 1127-1131, 1991.[Abstract]
-
Lin X., Mu Y., Cunningham E. T., Jr., Marcu K. B., Geleziunas R., Greene W. C. Molecular determinants of NF-B-inducing kinase action.. Mol. Cell. Biol., 18: 5899-5907, 1998.[Abstract/Free Full Text]
-
Bar-Sagi D., Hall A. Ras and Rho GTPases. A family reunion. Cell, 103: 227-238, 2000.[Medline]
-
Ohuchi N., Hand P. H., Merlo G., Fujita J., Mariani-Costantini R., Thor A., Nose M., Callahan R., Schlom J. Enhanced expression of c-Ha-ras p21 in human stomach adenocarcinomas defined by immunoassays using monoclonal antibodies and in situ hybridization.. Cancer Res., 47: 1413-1420, 1987.[Abstract/Free Full Text]
-
Correa P. Helicobacter pylori as a pathogen and carcinogen.. J. Physiol. Pharmacol., 48: 19-24, 1997.
Top
ABSTRACT
Introduction
), and Bhas/vec clones and Bhas 42 cells (
).
Tumor-bearing mouse implanted with Bhas/mp1-63 clone 28 days after
(C).