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Experimental Therapeutics |
Cancer Chemoprevention Group Nutrition Sector, [K. S., H. H. Y., K. K., J. Y. W., S. Z. A., S. S. B.], Searle Research and Development [M. G. C.], Monsanto Life Sciences Company, St. Louis, Missouri 63167, and Truman Memorial Veterans Affairs Hospital [S. L. E., Y. W., B. W. M, L. R. F.], Departments of Pharmacology [S. L. E., Y. W., N. S. J., H. D. K., L. R. F.] and Surgery [B. W. M.], Missouri University, Columbia, Missouri 65212
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ABSTRACT |
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50% of control. Our findings suggest
that uroguanylin and guanylin regulate the turnover of epithelial cells
within the intestinal mucosa via activation of a cGMP signaling
mechanism that elicits apoptosis of target enterocytes. The intestinal
R-GC signaling molecules for guanylin regulatory peptides are promising
targets for prevention and/or therapeutic treatment of intestinal
polyps and cancers by oral administration of human uroguanylin. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uroguanylin and guanylin are small peptides that are related both in primary structures and in biological activities (5, 6, 7) . Both of these regulatory peptides are produced at high concentrations throughout the intestinal mucosa. A cell-surface receptor in the intestine that has been identified at the molecular level is R-GCC, which is specifically activated by the guanylin peptides (7 , 8) . R-GCC proteins are localized on the luminal surface of enterocytes in the intestinal tract (7 , 8) . Binding of uroguanylin or guanylin to the extracellular domain of R-GCC stimulates production of the intracellular second messenger, cGMP.2 cGMP activates the CFTR protein that serves as an apical membrane channel governing Cl- and HCO3- efflux from enterocytes lining the intestinal tract (9 , 10) . Activation of CFTR channel proteins and the subsequent enhancement of transepithelial secretion of Cl- and HCO3- is the main driving force for secretion of Na+ and water into the intestinal lumen (7) . Therefore, a growing body of evidence strongly suggests that one of the major physiological functions of the guanylin hormones is to regulate fluid and electrolyte transport in the intestinal tract by serving as local regulators of CFTR activity.
In addition to a role for uroguanylin and guanylin as hormonal modulators of intestinal fluid and electrolyte secretion, there may be other physiological functions for the guanylin family of cGMP-regulating peptides. Recent experiments demonstrate that expression of the R-GCC form of receptors for uroguanylin and guanylin is maintained at normal levels in both primary and metastatic cancers of the colon and rectum (11) . This finding resulted in the suggestion that R-GCC may serve as a specific marker for colon tumors in the body (11) . In contrast, expression of guanylin mRNA is markedly down-regulated in adenocarcinomas of the colon (12) . Loss of guanylin production in the colon may have deleterious effects on tumor growth, but uroguanylin is also produced in the colon mucosa and regulates the activity of R-GCC and intestinal anion and fluid secretion. Thus, we investigated the expression of both uroguanylin and guanylin mRNAs in colon polyps and adenocarcinomas and tested the efficacy of uroguanylin as a potential therapeutic agent in the treatment of colon tumors.
We found that mRNA transcripts for guanylin and uroguanylin are severely decreased in both polyps and adenocarcinomas of human colon relative to the mRNA levels found in the surrounding normal colon mucosa. The mRNA expression of R-GCC was essentially normal in both polyps and cancers of the colon. In addition, we demonstrate for the first time that treatment with uroguanylin leads to induction of apoptosis in human colon carcinoma cells in vitro, and that oral administration of human uroguanylin suppresses the formation and apparent progression of polyps in the Min/+ mouse animal model of colorectal cancer (13) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80%.
Tissue Collection.
Samples of normal colon and tumors were obtained following colon
resections for adenocarcinoma under a human experimentation protocol
that was approved by the Institutional Review Board for Missouri
University School of Medicine/Truman VA Hospital. Mucosa samples from
normal colon tissues adjacent to the colon adenocarcinomas were
isolated from the submucosal tissue by scraping the luminal surface
with a microscope slide to separate mucosa from the underlying tissue.
Portions of the tumors were collected and processed as an intact
tissue. Tissues from 11 subjects between the ages of 48 and 82 years
representing 2 female and 9 male patients were used in this study.
Isolation of RNA.
RNA was extracted from tissue using a combination of the TRI reagent
method (Molecular Research Center, Inc., Cincinnati, OH) and the
RNAeasy kit (Qiagen, Valencia, CA). The tissue was homogenized in TRI
reagent following the manufacturer’s protocol. After phase separation
with chloroform, the aqueous supernatant phase containing total RNA was
removed and mixed with an equal volume of 70% ethanol and lysis buffer
without ß-mercaptoethanol. The resulting mixture was loaded onto the
RNAeasy columns and then processed following the protocol provided by
the manufacturer.
Northern Assays.
Total RNA (20 µg) isolated from colon tissues was subjected to
electrophoresis in formaldehyde-agarose gels and then transferred to
nylon membranes (Zeta-Probe; Bio-Rad Laboratories, Inc., Hercules, CA).
The membranes were prehybridized for 2 h at 65°C in ExpressHyb
solution (Clontech, Palo Alto, CA) and then hybridized with human
guanylin, uroguanylin, and GC-C cDNAs overnight at 65°C. All cDNA
probes were labeled with 32P by random priming
(Boehringer Mannheim, Indianapolis, IN). RNA blots were then washed
twice with 2x SSC-0.1% SDS for 5 min at room temperature, followed by
a 15-min wash at 60°C with 0.2x SSC-0.1% SDS. Exposure to X-ray
film was performed at -80°C with intensifying screens.
RT-PCR-Southern Assays.
Oligo(deoxythymidine)18 primed cDNAs were
synthesized from 3 µg of total RNA using reverse transcriptase
(Superscript II; Life Technologies, Gaithersburg, MD). Two PCR primers,
5' primer (5'-GAACCCAGGGAGCGCGAT-3') and 3' primer
(5'-CTGGTGGGCTCAGGGTACC-3'), were designed from regions flanking the
open reading frame of human pre-pro-uroguanylin cDNA (14)
.
A PCR product of the expected size of 384 bp was amplified from colon
cDNAs after 25 cycles at 93°C for 1 min, 56°C for 1 min, and 72°C
for 1.5 min using Taq DNA polymerase (United States Biochemical Corp.,
Cleveland, OH). The pair of primers for RT-PCR of pre-pro-guanylin were
5' primer (5'-AACTCAGGAACTTTGCAC-3') and 3' primer
(5'-CGTAGGCACAGATTTCAC-3'). These primers produced 174-bp cDNAs for
human guanylin using the PCR conditions of 25 cycles at 93°C for 1
min, 59°C for 1 min, and 72°C for 1.5 min. The primers for
amplification of GC-C cDNAs were 5' primer (5'-CAAATACGACAAAAAGCGAG)
and 3' primer (5'-GAATGTGCCATCAGGGTAG). These primers produced a 235-bp
GC-C product using the PCR conditions of 35 cycles at 93° for 1 min,
57° for 1 min, and 72° for 1.5 min. The PCR-generated cDNA products
were subjected to electrophoresis on 1% agarose gels in TAE buffer (40
mM Tris acetate, 1 mM EDTA, pH 8.5) containing
ethidium bromide and then transferred to nylon membranes. Southern
hybridization was carried out using uroguanylin and guanylin cDNA
probes. Prehybridization was for 1 h at 65°C with either
ExpressHyb or PerfectHyb Plus (Sigma Chemical Corp., St. Louis, MO)
solutions, and then hybridization was for 3 h at 65°C. Blots
were washed as described above and exposed to X-ray films at -80°C
with intensifying screens.
Cell Proliferation Assays.
Approximately 10,000 cells (T-84; CaCo-2) were inoculated in each well
of 96-well plates. After an incubation period of 3 days, the various
concentrations of human uroguanylin were added to the media, and cells
were allowed to grow until they formed semiconfluent monolayers.
Subsequently, BrdUrd labeling agent (BrdUrd in PBS) was added
(final concentration, 100 µM), and cells were
reincubated for an additional 24 h. Monolayers of cells were
washed, and the incorporation of BrdUrd was measured following the
manufacturer’s instructions (Boehringer Mannheim Corp.).
cGMP Accumulation Bioassays with T84 Cells.
The human uroguanylin peptide (NDDCELCVNVACTGCL) was custom
synthesized by Multiple Peptide Systems (San Diego, CA). Biological
activity of the synthetic peptide was assayed as reported previously
(6)
. Briefly, the confluent monolayers of T84 cells in
24-well plates were washed twice with 250 µl of DMEM
containing 50 mM HEPES (pH 7.4), preincubated at 37°C for
10 min with 250 µl of DMEM containing 50 mM HEPES
(pH 7.4) and 1 mM IBMX, followed by incubation with
uroguanylin (0.1 nM to 10 µM) for 30
min. The medium was aspirated, and the reaction was terminated by the
addition of 3% perchloric acid. After centrifugation and
neutralization with 0.1 N NaOH, the supernatant was used
directly for measurements of cGMP by using the ELISA kit (Caymen
Chemical, Ann Arbor, MI).
Ussing Chamber Assays.
The seromuscular layer of human colon mucosa was removed by blunt
dissection, and one to four mucosal sheets from each specimen (1
cm2
) were used. To isolate intestinal mucosa from
mice, animals were sacrificed by CO2, and the
proximal portion of the colon distal to the cecum was dissected from
the intestinal tract. Intestinal tissue was placed in ice-cold,
oxygenated KRB solution, opened along the mesenteric border and then
pinned with the luminal-side down on a pliable silicone surface. The
outer muscle layers were striped by shallow dissection with a scalpel
and fine forceps. Mouse proximal colon and human colon tissues,
consisting of only mucosa and submucosa, were mounted between two
Ussing half-chambers and bathed on both sides with KRB solutions in a
manner similar to that reported previously (10)
.
Electrical measurements were monitored with an automatic voltage clamp,
and direct-connecting voltage and Isc were recorded. Tissues
were equilibrated under short-circuit conditions until Isc
had stabilized, and the potential difference across the epithelium was
measured intermittently.
Apoptosis Assays.
T84 cells were grown in 35-mm dishes for 7 days. The cells were washed
once with serum-free DMEM and incubated with the same media containing
different concentrations of human uroguanylin for the indicated times.
After this incubation, cells were quickly collected by trypsinization,
and the cell pellet was washed twice with PBS. Cells were resuspended
in PBS at a concentration of 108 cells/ml. For
demonstration of nucleosomal ladders, the apoptotic DNA was isolated
from these cells by following the instructions of the DNA fragmentation
analysis kit (Boehringer Mannheim Corp.). The apoptotic DNA was
separated on a 1.8% agarose gel electrophoresis, followed by staining
with ethidium bromide. Induction of apoptosis by uroguanylin was
further demonstrated by using the TUNEL assay with human CaCo-2 colon
adenocarcinoma cells as per the instructions of the In Situ Cell Death
Detection kit (Boehringer Mannheim Corp., Indianapolis, IN).
Min/+ Mouse Model.
Male C57BL/6J-ApcMin/+, a strain
containing a fully penetrant dominant mutation in the Apc
gene, were obtained at 4–5 weeks of age from The Jackson Laboratory
(Bar Harbor, ME). All mice were fed a high-fat AIN-93G diet (Research
Diets, Inc., New Brunswick, NJ), tap water to drink ad
libitum, and housed in a humidity- and temperature-controlled room
with a 12-h light-dark cycle. After 1 week of quarantine period, the
animals were randomly divided into three groups of 10 animals each and
ear tagged. Animals were fed the same diet containing different
concentrations of human uroguanylin (0, 10 and 20 µg of
uroguanylin/5 g of diet). The mice were also given additional amounts
of either 10 or 20 µg of human uroguanylin or vehicle (0.2 ml
of PBS containing 20% polyethylene glycol) by oral gavage twice a
week. Food consumption and body weight of these animals were monitored
weekly. At the end of 17 weeks, animals were sacrificed by
CO2 asphyxiation, and the GI tracts were removed.
After flushing with PBS, the GI tract was divided as sections of
duodenum (5 cm from the stomach), jejunum (middle portion, 10–13
cm), ileum (5 cm proximal to the cecum), and the entire colon.
Tissues were opened longitudinally, washed with the Streck fixative
(Streck Laboratories, Inc., Omaha, NE), and placed between two layers
of blotting paper in a tray containing the tissue fixative. Polyps and
other tumors were counted independently by four different observers.
The results are expressed as a mean of the total number of polyps for
each individual animal as recorded by the four observers. Analysis of
the data obtained from all observers revealed a statistically
nonsignificant, interobserver variance. Sections of these tissues were
viewed under constant magnification (x10) to measure the diameter of
intestinal polyps in situ.
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RESULTS |
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99% purity and used for this study. To ensure
that the synthetic form of human uroguanylin had efficacy in both mouse
and human colon mucosa, its biological activity was examined using
mouse and human colonic mucosa mounted in Ussing chambers. Fig. 1
compares the short circuit current (Isc) responses of colon
mucosa to 1 µM uroguanylin after pretreatment
with tetrodotoxin and amiloride, which reduces and stabilizes the
Isc in both human and mouse colon (10)
.
Addition of uroguanylin to the luminal reservoir stimulates a rapid
increase in Isc, which was sustained for the course of the
experiments. Isc is a measurement of the electrogenic
secretion of Cl- and
HCO3- across the colon mucosa
when amiloride is used to inhibit the electrogenic absorption of
Na+. These results confirm that synthetic human
uroguanylin is effective in stimulating the transepithelial secretion
of Cl- and
HCO3- in the mucosa of both
human and mouse intestine. Therefore, we used the synthetic human
uroguanylin for all of the subsequent experiments that are presented in
this communication.
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70% inhibition of growth at
10 µM peptide (Fig. 2)
. In contrast, a biologically inactive variant of this peptide neither
inhibits cell growth nor stimulates the production of cGMP (data not
shown), suggesting that the growth inhibition by active uroguanylin is
mediated by intracellular cGMP.
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. On the other hand, DNA from uroguanylin-treated cells
exhibited relatively extensive DNA fragmentation. The action of
uroguanylin to elicit DNA fragmentation in T84 cells was also detected
by 2 h after treatment, and this action of uroguanylin was greatly
augmented by the addition of an inhibitor of cGMP hydrolysis, IBMX
(Fig. 3B)
. Induction of apoptosis by uroguanylin treatment
of human colon cancer cells was further investigated using CaCo-2 cells
and the TUNEL assay for apoptosis. Uroguanylin treatment significantly
increased the generation of apoptotic cells as compared with vehicle
treated CaCo-2 cells (Fig. 4)
. About 25% of the CaCo-2 cells exhibited positive reaction for
apoptosis induced by treatment with uroguanylin, as detected using the
TUNEL method. These results confirm and extend the finding that
uroguanylin induces apoptosis in human colon cancer cells in
vitro and suggest that uroguanylin induces apoptosis in human
colon cancer cells by a cGMP-dependent signaling mechanism.
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. The levels of R-GCC mRNAs were similar in both the adenocarcinomas
and normal mucosa. Similar expression patterns for uroguanylin and
guanylin mRNAs were found when RNA preparations from 11 additional
specimens of colon mucosae and tumors were analyzed by highly sensitive
RT-PCR-Southern assays. Guanylin and uroguanylin transcripts were
markedly reduced in 10 of 11 specimens of colon adenocarcinomas as
compared with the levels of mRNA in the adjacent normal mucosa (data
not shown). These finding suggest a possible link between colon cancer
and expression of these cGMP-regulating peptides in the intestinal
epithelium.
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. The levels of R-GCC mRNAs were similar in both polyps and normal
mucosa tissues. These findings raise the possibility that loss of
uroguanylin and guanylin production by enterocytes may be an early
event that plays a fundamental role in the process of tumor formation
and/or tumor progression in the colon. Continued expression of R-GCC
provides an appropriate target for replacement therapy with
uroguanylin.
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At the end of the study, all animals were sacrificed, and the number
and distribution of polyps in the small intestine and colon tissues
were measured (Table 1)
. Intestinal tracts of the vehicle-treated control group contained
48.3 ± 7.7 (mean ± SE) polyps/animal.
Most of the polyps were located throughout the small intestine, and
only a few polyps were found in the colon. We did not measure the
precise size of each polyp. However, the sizes of polyps in control
Min/+ mice were in the range of approximately 3–5 mm in
diameter and were clearly visible. In addition, three animals in the
control group also developed globular tumors in the duodenum that were
similar in appearance to that of carcinomas. Oral administration of
uroguanylin reduced the total number of polyps by 50% from
48.3 ± 7.7 per animal to 23.3 ± 3.1
(P < 0.05). Furthermore, this peptide also
appeared to inhibit the progression of polyps because the majority of
polyps in these animals were not clearly visible and were very small
(e.g., <2.0 mm in diameter). In addition,
uroguanylin-treated Min/+ mice did not develop globular
tumors in the duodenum and had no polyps in the colon. The appearance
of polyps in the colon and carcinoma-like tumors in the small intestine
of Min/+ mice usually occurs at the later and more severe
stages of disease. The absence of carcinomas in the small intestine and
polyps in the colon of uroguanylin-treated mice suggests that this
peptide may inhibit both the formation of tumors and the progression of
polyps into more advanced tumors. Taken together, our results strongly
suggest that oral administration of uroguanylin substantially reduces
the formation and/or growth of polyps in the Min/+ mouse
model of colon cancer.
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DISCUSSION |
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Another potential physiological role for guanylin and uroguanylin that was revealed in the present study concerns the activation of a cGMP signal transduction pathway that may help to regulate the turnover of epithelial cells and maintain homeostasis of the intestinal mucosa. The biochemical processes that influence the longevity of cells in the intestinal mucosa coupled with those processes involved in the regulation of cell proliferation and differentiation function together in an integrated fashion to maintain homeostasis of a normal intestinal epithelium. Abnormalities in the processes of programmed cell death and/or renewal of the epithelium as characterized by increased cell proliferation and/or suppressed apoptosis may lead to the formation of tumors within the intestinal tract (19) . Our findings clearly demonstrate that the expression of mRNAs encoding uroguanylin and guanylin are markedly suppressed in both adenocarcinomas and polyps of human colon, and that treatment with uroguanylin to activate R-GCC receptors induces apoptosis in human T84 and CaCo-2 colon carcinoma cells by a cGMP-dependent mechanism. Furthermore, oral administration of uroguanylin to the Min/+ mouse model of colorectal cancer not only inhibited the formation of polyps, but this cGMP-regulating peptide apparently delayed the progression of intestinal polyps during the course of treatment. Taken together, these findings suggest that down-regulation of uroguanylin and guanylin gene expression may be one mechanism underlying the derangement of programmed cell death in the intestinal mucosa that is associated with the formation of polyps in the intestine.
It is important to note that expression of uroguanylin and guanylin mRNAs is virtually eliminated in the specimens of adenocarcinomas and polyps that were examined in this study. It is noteworthy that even the ultrasensitive RT-PCR-Southern assays detected only very low levels of transcripts for uroguanylin and guanylin in some of the polyps and adenocarcinomas. Our results are in agreement with a recent report suggesting that expression of guanylin mRNA is markedly reduced in human colorectal adenocarcinoma as revealed by in situ hybridization histochemistry (12) . Both guanylin and uroguanylin genes have recently been mapped to chromosome 4 of mice and to a syntenic position on human chromosome 1p34–35 (20 , 21) . This chromosomal region is frequently associated with loss of heterozygosity in human colon carcinoma (22) . In the Min/+ mouse tumor model, adenoma multiplicity and growth rates are regulated by tumor suppresser Apc genes (23) . This gene is mutated with loss of function of the gene product in the vast majority of colorectal cancers in the human (24) . The principal function of Apc genes is to regulate growth and apoptosis of epithelial cells via the wnt signal transduction cascade (25) . Because uroguanylin and guanylin are essentially lost in both adenomatous polyps and adenocarcinomas of human colon, it is tempting to speculate that these enteric hormones may be involved mechanistically at early stages of colon carcinogenesis. This possibility is consistent with our results in the Min/+ mouse model of colorectal cancer, where oral administration of uroguanylin inhibits both the formation and apparent progression of polyps. Although the biochemical mechanism(s) for this chemopreventive property of uroguanylin is not known, it is plausible that uroguanylin and guanylin both participate in a cGMP-mediated signaling mechanism in target enterocytes that regulates mucosal cell turnover by initiating programmed cell death. Therefore, diminished expression of uroguanylin and guanylin genes in cells within the intestinal mucosa that have inactivating mutations of Apc genes may lead to an inhibition of programmed cell death and contribute to the formation of tumors in the colon and rectum.
Several lines of evidence have implicated a potential role of K+ efflux in the induction of apoptosis (26, 27, 28, 29, 30, 31, 32) . The question that naturally arises concerns the existence of a putative relationship between uroguanylin/cGMP-induced apoptosis on the one hand and K+ and Cl- transport in colon carcinoma cells on the other. Uroguanylin and guanylin stimulate transepithelial Cl- secretion (10) and K+ efflux (7) by activation of receptors with intrinsic guanylate cyclase activity. Other cGMP-regulating peptides, such as ANP, have also been shown to activate K+ conductance in rat mesangial cells and induce apoptosis in cardiac myocytes by a cGMP-dependent mechanism (33 , 34) . Furthermore, pretreatment of rat endothelial cells with either ANP or 8-bromo-cGMP causes a marked accumulation of the nuclear phosphoprotein, p53, a tumor suppresser protein known to induce apoptosis in many cell types (35) . Therefore, it seems possible that uroguanylin and guanylin produced in the intestinal mucosa and released locally may initiate apoptosis of epithelial cells lining the GI tract mucosa by activating R-GCC and/or other intestinal receptors that produce cGMP upon activation by the peptides.
Hormones and neurotransmitters that activate cGMP signal cascades, such as uroguanylin, ANP, and nitric oxide, are all capable of producing apoptosis in target cells (33, 34, 35) . Apoptotic cells are distinguishable from living cells by a number of biochemical and morphological features. Although cell shrinkage has been invariably observed in cells undergoing programmed cell death, the underlying mechanism by which apoptotic cells achieve this feat is poorly understood. The inhibition of K+ loss by raising external K+ concentrations (36 , 37) or by exposing cells to K+ channel blockers (38) results in an abrogation of apoptosis. Our proposed model suggests that uroguanylin may stimulate intestinal fluid secretion by activation of an intracellular cGMP signaling pathway, which promotes K+ recycling across the basolateral plasma membranes of enterocytes and a concomitant activation of anion channels at the apical surface of the uroguanylin target cells (10) . Thus, it seems plausible that the same cellular signaling machinery that is activated by cGMP to regulate intestinal fluid secretion may also be used for the induction of programmed cell death within the intestinal epithelium.
It should be emphasized that uroguanylin and guanylin were discovered because these hormonal peptides are the endogenous agonists for intestinal R-GCC that is also activated by heat-stable toxin (ST) peptides, which are secreted by enteric bacteria that cause diarrhea (7) . The ST peptides are uroguanylin-like in both primary structures and profiles of biological activities. Thus, it is possible that chronic and/or periodic infections with ST-secreting bacteria in the intestine elicit beneficial therapeutic actions for people living in the developing nations, where a relatively low incidence of colorectal cancer is found (39) . Regular exposure and enteric infection with enterotoxigenic Escherichia coli or other intestinal microbes that secrete ST peptides into the colon could provide a uroguanylin-like agonist that stimulates cGMP production in epithelial cells containing mutations in Apc or other genes involved in tumor formation. This action of the ST peptides could elicit apoptosis in cells at relatively early stages of tumor formation. Stimulation of cGMP production by ST in benign tumors may also inhibit the progression of polyps to adenocarcinomas of the colon. Although the hypothesis is speculative, we offer this suggestion to reinforce the historical importance of E. coli ST peptides in the discovery of guanylin and uroguanylin and once again connect the pathophysiology of bacterial ST peptides to the physiology of guanylin regulatory peptides in the intestinal tract (5, 6, 7) .
To summarize, this study demonstrates for the first time that uroguanylin induces apoptosis in human colon carcinoma cells in vitro, and oral uroguanylin inhibits the formation of polyps in the Min/+ mouse animal model of colorectal cancer in vivo. We conclude with the suggestion that uroguanylin and other members of the guanylin peptide family may have therapeutic efficacy for prevention and/or treatment of colon cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Pharmacology, M-515 Medical Sciences
Building, School of Medicine, Missouri University, Columbia, MO 65212.
Phone: (573) 882-1537; Fax: (573) 884-4558; E-mail: lrf@missouri.edu. or shailu{at}royal.net 
2 The abbreviations used are: cGMP, cyclic GMP;
CFTR, cystic fibrosis transmembrane conductance regulator; R-GC,
receptor-guanylate cyclase; R-GCC, uroguanylin-R-GC; BrdUrd,
5-bromo-2'-deoxyuridine; IBMX, isobutylmethylxanthine; KRB,
Krebs-Ringer-bicarbonate; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling; GI,
gastrointestinal; RT-PCR, reverse transcription-PCR; ANP, atrial
natriuretic peptide; ST, heat-stable toxin.
Received 11/11/99. Accepted 7/14/00.
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A. Deguchi, S. W. Xing, I. Shureiqi, P. Yang, R. A. Newman, S. M. Lippman, S. J. Feinmark, B. Oehlen, and I. B. Weinstein Activation of Protein Kinase G Up-regulates Expression of 15-Lipoxygenase-1 in Human Colon Cancer Cells Cancer Res., September 15, 2005; 65(18): 8442 - 8447. [Abstract] [Full Text] [PDF]
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 S. Kazerounian, G. M. Pitari, F. J. Shah, G. S. Frick, M. Madesh, I. Ruiz-Stewart, S. Schulz, G. Hajnoczky, and S. A. Waldman Proliferative Signaling by Store-Operated Calcium Channels Opposes Colon Cancer Cell Cytostasis Induced by Bacterial Enterotoxins J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1013 - 1022. [Abstract] [Full Text] [PDF]
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M. Kikuchi, S. Fujimoto, H. Fukae, H. Kinoshita, T. Kita, M. Nakazato, and T. Eto Role of Uroguanylin, a Peptide with Natriuretic Activity, in Rats with Experimental Nephrotic Syndrome J. Am. Soc. Nephrol., February 1, 2005; 16(2): 392 - 397. [Abstract] [Full Text] [PDF]
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A. Deguchi, W. J. Thompson, and I. B. Weinstein Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells Cancer Res., June 1, 2004; 64(11): 3966 - 3973. [Abstract] [Full Text] [PDF]
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 D. E. Corpet and F. Pierre Point: From Animal Models to Prevention of Colon Cancer. Systematic Review of Chemoprevention in Min Mice and Choice of the Model System Cancer Epidemiol. Biomarkers Prev., May 1, 2003; 12(5): 391 - 400. [Abstract] [Full Text] [PDF]
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 S. L. Carrithers Diarrhea or colorectal cancer: Can bacterial toxins serve as a treatment for colon cancer? PNAS, March 18, 2003; 100(6): 3018 - 3020. [Full Text] [PDF]
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G. M. Pitari, L. V. Zingman, D. M. Hodgson, A. E. Alekseev, S. Kazerounian, M. Bienengraeber, G. Hajnoczky, A. Terzic, and S. A. Waldman Bacterial enterotoxins are associated with resistance to colon cancer PNAS, March 4, 2003; 100(5): 2695 - 2699. [Abstract] [Full Text] [PDF]
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 J. Lyons-Weiler, S. Patel, and S. Bhattacharya A Classification-Based Machine Learning Approach for the Analysis of Genome-Wide Expression Data Genome Res., March 1, 2003; 13(3): 503 - 512. [Abstract] [Full Text] [PDF]
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K. A. Steinbrecher, S. A. Wowk, J. A. Rudolph, D. P. Witte, and M. B. Cohen Targeted Inactivation of the Mouse Guanylin Gene Results in Altered Dynamics of Colonic Epithelial Proliferation Am. J. Pathol., December 1, 2002; 161(6): 2169 - 2178. [Abstract] [Full Text] [PDF]
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L. A. Santana-Blank, E. Rodriguez-Santana, F. Vargas, H. Reyes, P. Fernandez-Andrade, S. Rukos, and K. E. Santana-Rodriguez Phase I Trial of an Infrared Pulsed Laser Device in Patients with Advanced Neoplasias Clin. Cancer Res., October 1, 2002; 8(10): 3082 - 3091. [Abstract] [Full Text] [PDF]
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J. A. Rudolph, J. A. Hawkins, and M. B. Cohen Proguanylin secretion and the role of negative-feedback inhibition in a villous epithelial cell line Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G695 - G702. [Abstract] [Full Text] [PDF]
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H. Kulaksiz, E. Rehberg, W. Stremmel, and Y. Cetin Guanylin and Functional Coupling Proteins in the Human Salivary Glands and Gland Tumors : Expression, Cellular Localization, and Target Membrane Domains Am. J. Pathol., August 1, 2002; 161(2): 655 - 664. [Abstract] [Full Text] [PDF]
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 A. Deguchi, J.-W. Soh, H. Li, R. Pamukcu, W. J. Thompson, and I. B. Weinstein Vasodilator-stimulated Phosphoprotein (VASP) Phosphorylation Provides a Biomarker for the Action of Exisulind and Related Agents That Activate Protein Kinase G Mol. Cancer Ther., August 1, 2002; 1(10): 803 - 809. [Abstract] [Full Text] [PDF]
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L. Liu, H. Li, T. Underwood, M. Lloyd, M. David, G. Sperl, R. Pamukcu, and W. J. Thompson Cyclic GMP-Dependent Protein Kinase Activation and Induction by Exisulind and CP461 in Colon Tumor Cells J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 583 - 592. [Abstract] [Full Text] [PDF]
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G. M. Pitari, M. D. Di Guglielmo, J. Park, S. Schulz, and S. A. Waldman Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells PNAS, July 3, 2001; 98(14): 7846 - 7851. [Abstract] [Full Text] [PDF]
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 R. Potthast, E. Ehler, L. A. Scheving, A. Sindic, E. Schlatter, and M. Kuhn High Salt Intake Increases Uroguanylin Expression in Mouse Kidney Endocrinology, July 1, 2001; 142(7): 3087 - 3097. [Abstract] [Full Text] [PDF]
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