1 John D. Dingell Veterans Affairs Medical Center and Departments of 2 Internal Medicine, 3 Pathology, and 5 Biochemistry and Molecular Biology, and 4 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Although
accumulating evidence suggests a chemopreventive role for folic acid in
colon cancer, the regulation of this process in unknown. We hypothesize
that supplemental folic acid exerts its chemopreventive role by
inhibiting mucosal hyperproliferation, an event considered to be
central to the initiation of carcinogenesis in the gastrointestinal
tract. The present investigation examines the effect of supplemental
folic acid on proliferation of Caco-2 and HCT-116 colon cancer cell
lines. Furthermore, because certain tyrosine kinases, particularly
epidermal growth factor receptor (EGFR), play a role in regulating cell
proliferation, we also examined the folic acid-induced changes in
tyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116
cells, maintained in RPMI 1640 medium containing 1 µg/ml folic acid,
we observed that the supplemental folic acid inhibited proliferation in
a dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lines
with supplemental folic acid (1.25 µg/ml) completely abrogated transforming growth factor- (TGF-
)-induced proliferation in both
cell lines. Tyrosine kinase activity and the relative concentration of
EGFR were markedly diminished in both cell lines following a 24-h
exposure to supplemental folic acid. The folic acid-induced inhibition
of EGFR tyrosine kinase activity in colon cancer cell lines was also
associated with a concomitant reduction in the relative concentration
of the 14-kDa membrane-bound precursor form of TGF-
. In conclusion,
our data suggest that supplemental folic acid is effective in reducing
proliferation in two unrelated colon cancer cell lines and that EGFR
tyrosine kinase appears to be involved in regulating this process.
chemoprevention; epidermal growth factor receptor tyrosine kinase; transforming growth factor-
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INTRODUCTION |
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ALTHOUGH THE EPIDEMIOLOGY of colorectal cancer is related to genetic susceptibility, dietary factors such as vitamins and micronutrients are thought to influence tumorigenic processes, which include proliferation, differentiation, and apoptosis (13, 46). There is increasing evidence to suggest that the water-soluble vitamin folic acid may have a chemopreventive role in colon tumorigenesis. Diets deficient in folic acid have been associated with a higher incidence of adenomas, the most frequent premalignant colorectal lesion (4, 13, 37). Folate deficiency also enhances carcinogen-induced colonic neoplasia in rodents (10). In addition, several large case-controlled studies have noted an inverse relationship between dietary folic acid and the development of colorectal cancer (5, 12, 21, 34). Furthermore, in ulcerative colitis, red blood cell folate levels are greatly reduced and inadequate dietary supplementation of folic acid results in the development of colonic dysplasia or cancer, whereas supplementation of the vitamin may be chemopreventive (21, 23, 41).
Although the regulatory mechanisms for folic acid-induced suppression of colorectal neoplasia are poorly understood, we hypothesize that supplemental folic acid may inhibit mucosal cell proliferation, an event thought to be central to the initiation of carcinogenesis in the gastrointestinal tract (25). In support of this, we have observed that stimulation of ornithine decarboxylase (ODC, the rate-limiting enzyme in polyamine biosynthesis pathway) and tyrosine kinase activities in rat colonic mucosal explants in response to methylazoxymethanol (the active metabolite of the colonic carcinogen azoxymethane) is greatly attenuated by supplemental folic acid (35). Although neither ODC nor tyrosine kinases are directly involved in cell proliferation, they show a positive relationship with cell proliferation (26, 29, 36). Moreover, the activity of these enzymes is also increased in conditions predisposed to dysplasia and neoplasia (2). However, to the best of our knowledge, no information is available as to whether supplemental folic acid will inhibit proliferation of colon cancer cells. Therefore, the primary objectives of this investigation were to determine the effect of supplemental folic acid on proliferation of colon cancer cell lines Caco-2 and HCT-116 and the mechanisms that regulate this process.
Accumulating evidence suggests that tyrosine kinases, which are
associated with a number of growth factor receptors and products of
many protooncogenes, play a critical role in regulating proliferation of normal, preneoplasic, and neoplastic cells (16, 47). With respect to
the development and progression of colonic neoplasia, several lines of
evidence suggest a role for epidermal growth factor receptor (EGFR) in
regulating these processes. For example, colonic neoplasia is shown to
be associated with overexpression of EGFR (3, 19). In addition, we have
observed an increased tyrosine kinase activity of EGFR in the colonic
mucosa of patients with ulcerative colitis, adenomatous polyps, and
colon cancer (29). Moreover, in rats, administration of azoxymethane,
which augments colonic mucosal proliferative activity, also stimulates EGFR tyrosine kinase activity, and both processes can be attenuated by
tyrphostin (28, 40), an inhibitor of tyrosine kinases with a greater
specificity for EGFR than other protein kinases (24). It has also been
demonstrated that monoclonal antibody to EGFR inhibits proliferation in
certain colon cancer cell lines (18). Together, these results suggest a
role for EGFR in the development and progression of colonic neoplasia.
To determine, therefore, whether EGFR may also be involved in
regulating the folic acid-induced inhibition of proliferation of colon
cancer cells, tyrosine kinase activity of EGFR was measured in Caco-2
and HCT-116 cells following exposure to this vitamin. Furthermore,
because ligand-induced activation of EGFR is one of the primary causes
of induction of its intrinsic tyrosine kinase (26), we have also
evaluated the role of endogenous transforming growth factor-
(TGF-
) in regulating the enzyme activity in colon cancer cells.
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MATERIALS AND METHODS |
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Cell culture and cell proliferation. Caco-2 and HCT-116 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), penicillin (10,000 U/ml), streptomycin (10,000 U/ml), and amphotericin (25 µg/ml) at 37°C in an atmosphere of 95% air and 5% CO2.
For determination of folic acid-induced changes in proliferation, 1-ml aliquots of cell suspension containing 7.5 × 104 cells in RPMI 1640-10% FBS were plated in 24-well culture dishes. At ~50% confluency, which occurs ~24 h after plating, cells were incubated in the absence (control) or presence of folic acid. In some experiments, aliquots of both cell lines, maintained in RPMI 1640 containing 1 µg/ml folic acid and 10% FBS, were preincubated in the absence (control) or presence of supplemental folic acid (1.25 µg/ml) for 48 h. After preincubation, the control and folic acid-preincubated cells were serum starved (0.1% FBS) for 24 h and then exposed to TGF-EGFR tyrosine kinase activity. The enzyme activity was determined as described previously (26). Briefly, cells were homogenized in RIPA buffer [20 mM sodium phosphate, pH 7.4, containing 5 mM sodium pyrophosphate, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 1 mM Na3VO4, 1 µg/ml aprotonin, and 1 mM 1,10-phenanthroline]. The homogenate was stirred for 30 min at 4°C and subsequently centrifuged at 10,000 g for 10 min. The supernatant, after it was diluted with an equal volume of homogenizing buffer (10 mM HEPES, pH 7.2, containing 150 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4, 10 µg/ml leupeptin, and 1 mM 1,10-phenanthroline), was used as the source for the enzyme. In all immunoprecipitation studies, protein concentrations were standardized among the samples. Protein content in all samples was measured using the bicinchoninic acid protein assay kit from Pierce (Rockford, IL) according to the manufacturer's instruction.
For determination of EGFR tyrosine kinase activity, aliquots of cell lysate containing 350 µg protein were incubated overnight at 4°C with 1 µg polyclonal antibody to EGFR (UBI, Lake Placid, NY). The immune complexes were precipitated with Sepharose-bound protein G (Sigma-Aldrich, Steinheim, Germany), washed several times with 1:1 RIPA-homogenizing buffer, and finally resuspended in 25 µl of assay buffer (100 mM HEPES, pH 7.5, 10 mM MnCl2, 80 mM KCl, and 40 mM 2-mercaptoethanol). The reaction at 30°C for 15 min was initiated by adding 20 µl of the reaction mix [2 µl of acid-denatured enolase (enolase was denatured by adding an equal volume of 0.1 M acetic acid and incubating the mixture for 8 min at 30°C), 5 µl of 20 µM ATP, 5 µl of [Western blot analysis of EGFR and TGF-.
EGFR was immunoprecipitated from detergent-solubilized cells containing
1 mg protein with EGFR antibodies as stated above. In the case of
TGF-
, cells were homogenized in homogenizing buffer and the
homogenate was centrifuged at 30,000 g
at 4°C for 30 min to obtain the crude membrane fraction. The
membrane fraction was suspended in RIPA buffer. Aliquots of the
membrane fraction containing 3 mg of protein were immunoprecipitated
with monoclonal antibodies against TGF-
(Santa Cruz Biotechnology,
Santa Cruz, CA). EGFR and TGF-
immunoprecipitates were subjected to
7.5% and 15% SDS-PAGE, respectively (27). After electrophoresis,
proteins were transferred electrophoretically onto polyvinylidene
difluoride membrane (Millipore, Bedford, MA) using a transfer cell
kit (International, Mount Prospect, IL). Membranes were
incubated overnight in 5% nonfat dry milk (Sanalac) and 0.1% Tween 20 in 1× PBS. After membranes were washed three times with 1×
PBS and 0.1% Tween 20, they were incubated at room temperature for 1 h
with respective antibodies at a final concentration between 1:2,500 and
1:1,000 in PBS containing 0.25% nonfat dry milk (Sanalac)-0.1% Tween
20. The membranes were washed and subsequently incubated with
horseradish peroxidase linked to anti-mouse/anti-rabbit antibody
conjugates (ICN) in PBS containing 5% nonfat dry milk and 0.1% Tween
20. After washing, the protein bands were visualized by an enhanced
chemiluminescence detection system (Amersham, Piscataway, NJ). The
membranes containing the electrophoresed proteins were exposed to
X-Omat film, and the density of the protein bands were analyzed by a
digital image analysis system (Molecular Dynamics Strom 860).
Statistical analysis. When applicable, results were statistically evaluated with Student's t-test for unpaired values, with P < 0.05 designated as the level of significance.
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RESULTS |
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In the present investigation, all experiments were performed using two colon cancer cell lines, Caco-2 and HCT-116. Reasons for using these two colon cancer cell lines were to determine whether supplemental folic acid will be effective in inhibiting proliferation in both colon cancer cell lines. Although both cell lines are derived from colonic carcinomas, there are significant morphological and phenotypic differences between them. For example, Caco-2 cells lose their proliferative capacity on reaching confluence, resulting in a marked rise in activities of sucrase-isomaltase and other brush-border enzymes, which are considered equivalent to the crypt-to-villus differentiation occurring in vivo (14, 39). However, these changes are not apparent in HCT-116 cells, which are less differentiated compared with Caco-2 cells, and, unlike Caco-2 cells, they have lost the ability to spontaneously differentiate (7).
In the first set of experiments, Caco-2 and HCT-116 cells, which were
maintained in RPMI 1640 containing 1 µg/ml folic acid, were further
exposed to increasing concentrations (0-3.1 µg/ml) of folic acid
for 48 h. In both cell lines, supplemental folic acid inhibited
proliferation in a dose-dependent manner (Fig. 1).
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To further determine the antiproliferative effect of folic acid on
colon cancer cell lines, the next set of experiments was performed to
examine whether pretreatment of HCT-116 and Caco-2 cells for 48 h with
folic acid arrests TGF--induced proliferation of HCT-116 and Caco-2
cells. Aliquots of both cell lines were preincubated in the absence or
presence of folic acid (1.25 µg/ml), subsequently serum starved for
24 h, and then exposed to TGF-
(10
10 M) for another 24 h.
Preincubation with folic acid caused a 40-50% reduction in
proliferation when compared with the control (Fig. 2). Exposure of serum-starved control
Caco-2 or HCT-116 cells (those not preincubated with folic acid) to
10
10 M TGF-
for 24 h
resulted in a significant 70-80% increase in proliferation over
the corresponding basal levels (Fig. 2). However, pretreatment with
folic acid completely abrogated the stimulatory effect of the growth
factor by reducing proliferation by 59% and 81% in Caco-2 and HCT-116
cells, respectively, when compared with the corresponding basal levels
(Fig. 2).
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Although the regulatory mechanism(s) for folic acid-induced inhibition
of proliferation of colon cancer cell lines is unknown, we hypothesized
that certain tyrosine kinases, specifically the enzyme associated with
EGFR, may play a key role in this process. To test this hypothesis, we
studied the effect of supplemental folic acid on EGFR tyrosine kinase
activity in HCT-116 and Caco-2 cells. Exposure of Caco-2 and HCT-116
cells to supplemental folic acid at a dose of 0.625 µg/ml caused a
25% and 40% reduction, respectively, in EGFR tyrosine kinase activity
when compared with the respective basal levels (Fig.
3).
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To determine whether the suppressive effect of folic acid on EGFR
tyrosine kinase activity may be related in part to decreased levels of
the enzyme, Western immunoblot was performed to determine changes in
the relative concentration of EGFR in both Caco-2 and HCT-116 cells
following exposure to a supplemental dose of folic acid (0.625 µg/ml)
for 24 h. Folic acid caused a 75% and 80% reduction in EGFR
expression in HCT-116 and Caco-2 cells, respectively, when compared
with the corresponding basal levels (Fig.
4).
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Because ligand binding to the extracellular domain of EGFR is one of
the primary causes for activation of the receptor's intrinsic tyrosine
kinase activity (38), any event that affects the ligand-receptor interaction is likely to have a profound effect on EGFR activation. TGF-, one of the primary ligands of EGFR, is a membrane-bound peptide that also activates EGFR (6, 30, 36). To determine whether
folic acid-induced inhibition of EGFR tyrosine kinase activity in colon
cancer cell lines may be related to parallel changes in membrane-bound
TGF-
, we examined the effect of this vitamin on membrane expression
of TGF-
in both cell lines. Western blot analysis of the membrane
fraction revealed several molecular forms of TGF-
with relative
molecular masses of between 14 and 18 kDa in HCT-116 and Caco-2 cells,
whereas those exposed to folic acid (0.625 µg/ml) for 24 h showed
only one prominent molecular form of the peptide with a relative
molecular mass of 14 kDa in these cells (Fig.
5). The relative concentration
of the 14-kDa TGF-
in folic acid-treated cells was found to be
substantially lower compared with the corresponding band from control
cells (Fig. 5). Because the density of the 14-kDa band in folic
acid-treated Caco-2 cells could not be detected by the digital image
analysis system, quantitative analysis was not performed.
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DISCUSSION |
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There is an accumulating body of evidence suggesting that vitamins and micronutrients may have a key role in reducing the susceptibility to colorectal cancer (13, 46). It has been demonstrated that a diet deficient in folic acid may be associated with an increased risk of colorectal neoplasia (21, 37), whereas dietary supplementation of this nutrient may be chemopreventive (22, 23). Several case-control studies have noted an inverse relationship between folic acid intake and the risk for developing colorectal cancer (5, 12, 13, 21, 34). The more profound beneficial effect of supplemental folate may relate to the relatively higher concentration of this nutrient in typical supplements as well as its increased bioavailability (13).
Although the number of studies performed is relatively small, murine and human studies suggest a role for folic acid in reducing colon carcinogenesis (10, 33). Cravo et al. (10) demonstrated that modest folate deficiency in rats is associated with enhanced development of colon tumors in dimethylhydrazine-treated rats. Alternatively, folate supplementation in this model protected against the development of colonic neoplasms in a dose-dependent manner (20). Moreover, recent human studies noted significantly lower colonic mucosal concentrations of folate in subjects with adenomatous polyps compared with those with hyperplastic polyps despite insignificant differences in serum folate levels (15). Recently, Meenan et al. (33) found folate levels to be lower in adenoma and carcinoma than in normal-appearing adjacent mucosa, suggesting that folic acid may indeed play a key role in the development of colorectal neoplasia.
The mechanisms responsible for the reduction of colon carcinogenesis by folic acid are speculative but may be related in part to the requirement of folic acid in DNA methylation and cellular homeostasis. Folate deficiency may, therefore, result in a variety of cellular consequences, including misincorporation of uracil for thymidine during DNA synthesis (45), resulting in increased spontaneous mutation (44), as well as chromosomal abnormalities and errors in DNA synthesis (11, 17). The restoration of DNA methylation status in patients with colorectal neoplasms treated with supraphysiological doses of folic acid (9) lends further support to this hypothesis.
Our current data suggest that supplemental folic acid may have a role
in modulating cellular hyperproliferation, an event that is considered
central to the initiation of carcinogenesis in the gastrointestinal
tract (25). This interpretation comes from the observation that
exposure of colon cancer cell lines, Caco-2 and HCT-116, to
supplemental folic acid results in inhibition of proliferation in a
dose-dependent manner. Furthermore, pretreatment of both cell lines
with supplemental folic acid completely abrogated the TGF--induced
stimulation of cell proliferation. These observations are in agreement
with our earlier findings that supplemental folic acid also suppressed
carcinogen-induced ODC and tyrosine kinase activities in rats (35).
Furthermore, our observation that folic acid is equally effective in
inhibiting proliferation of Caco-2 and HCT-116 cells, which are
morphologically and phenotypically different, suggests that
supplemental folic acid can inhibit proliferation in different colon
cancer cells. However, this observation is contrary to what has been
noted for normal renal tubular cells, whose proliferation has been
shown to be accelerated following a bolus injection of folic acid in
rats (31). Although the reasons for this discrepancy is not fully
understood, it is plausible that the responsiveness of highly
proliferative cancer cells, especially colon cancer cells, to
supplemental folic acid is quite different from that of normal cells.
The current observation of the folic acid-induced inhibition of
proliferation of colon cancer cells could not be attributed to cellular
toxicity, since removal of folic acid from the medium after 24 h
reverses the inhibition with restoration of proliferative activity
(data not shown).
Although the regulatory mechanisms for folic acid-induced inhibition in proliferation of colon cancer cell lines are not fully known, our observation that supplementation of this vitamin also inhibits tyrosine kinase activity suggests a role for these enzymes in regulating the folic acid-induced inhibition of proliferation. Numerous studies suggest that tyrosine kinases play a crucial role in regulating proliferation, differentiation, and transformation of cells (16, 47). However, tyrosine kinases are associated with products of many protooncogenes and with receptors of a number of growth factors (47). Increased activity of several Src-related tyrosine kinases, including pp60c-src, pp56lck, and c-Yes has been reported in certain premalignant colon conditions and in colon carcinoma (8, 38, 43). Tyrosine kinase associated with EGFR also appears to be involved in the induction and progression of colorectal neoplasia. We have reported increased EGFR tyrosine kinase activity in the colonic mucosa from patients with ulcerative colitis, adenomatous polyps, and colon cancer (29). In rats, a single injection of the colonic carcinogen azoxymethane markedly stimulates EGFR tyrosine kinase activity and proliferative processes in the colonic mucosa, and both parameters are inhibited by tyrphostin (40). Others have noted that monoclonal antibody to EGFR inhibits proliferation in colon cancer cell lines (18). Considering that activation of EGFR is an important event in the development and progression of colonic carcinogenesis, our observation that folic acid-induced inhibition of proliferation is accompanied by a concomitant reduction in EGFR tyrosine kinase activity suggests that this vitamin may exert its antiproliferative role by modulating EGFR tyrosine kinase.
A number of factors may regulate the intrinsic tyrosine kinase activity
of EGFR. The fact that the relative concentrations of EGFR in both
Caco-2 and HCT-116 cells, as assessed by Western immunoblot, are
greatly decreased following folic acid exposure suggests that decreased
activation of the enzyme in response to folic acid may be partly the
result of reduced levels of the enzyme protein. Whether the latter is
the result of decreased synthesis of EGFR, however, remains to be
determined. TGF-, one of the primary ligands of EGFR, may also
modulate the intrinsic tyrosine kinase of EGFR. Because ligand binding
is one of the primary causes of activation of EGFR signaling pathways
(42), any event(s) that affects the ligand-receptor interaction is
likely to have a profound effect on EGFR function. In general,
expression of TGF-
is greatly increased in colon cancer cells and
may be partly responsible for increased activation of EGFR in colonic
neoplasia (1, 3, 18). However, TGF-
is a membrane-bound peptide and
the transmembrane precursor form(s) of the peptide is able to activate
EGFR through an autocrine/juxtacrine mechanism (22, 30, 36). Our
observation that folic acid-induced inhibition of EGFR tyrosine kinase
activity in colon cancer cell lines is associated with a concomitant
reduction in the relative concentration of the 14-kDa precursor form of
TGF-
in membranes suggests that the peptide might be partly
responsible for modulating EGFR tyrosine kinase through an
autocrine/juxtacrine mechanism. Although it remains plausible that
other tyrosine kinases may also be involved in regulating the folic
acid-induced attenuation of proliferation of colon cancer cells, the
fact that supplemental folic acid not only inhibits the expression and
activation of EGFR but also diminishes membrane accumulation of TGF-
in colon cancer cells strongly suggests a key role for EGFR tyrosine
kinase in modulating the antiproliferative effect of folic acid.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sasi Boppana, for technical help during the early part of this investigation.
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FOOTNOTES |
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This work was supported by grants from the Department of Veterans Affairs.
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. P. N. Majumdar, Research Service-151, VA Medical Center, 4646 John R., Detroit, MI 48201 (E-mail: a.majumdar{at}wayne.edu).
Received 20 May 1999; accepted in final form 15 July 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anzane, M. A.,
D. Rieman,
W. Pritchelet,
D. F. Bowen Pope,
and
F. Greig.
Growth factor production by human colon carcinoma cell lines.
Cancer Res.
49:
2898-2904,
1989[Abstract].
2.
Arlow, F. L.,
S. M. Walczak,
G. D. Luk,
and
A. P. N. Majumdar.
Attenuation of zoxymethane-induced colonic mucosal ornithine decarboxylase and tyrosine kinase activity by calcium in rats.
Cancer Res.
49:
5884-5888,
1989[Abstract].
3.
Barnard, J. A.,
J. Beauchamp,
W. E. Russell,
R. N. Dubois,
and
R. J. Coffey.
Epidermal growth factor-related peptides and their relevances to gastrointestinal pathophysiology.
Gastroenterology
108:
564-580,
1995[Medline].
4.
Baron, J. A.,
R. S. Sandler,
R. W. Haile,
J. S. Mandel,
L. A. Mott,
and
E. R. Greenberg.
Folate intake, alcohol consumption, cigarette smoking, and risk of colorectal adenomas.
J. Natl. Cancer Inst.
90:
57-62,
1998
5.
Benito, E.,
A. Stiggelbout,
F. Bosch,
A. Obrador,
J. Kaldor,
M. Mulet,
and
N. Munoz.
Nutritional factors in colorectal cancer risk: a case-control study in Majorca.
Int. J. Cancer
49:
161-167,
1991[Medline].
6.
Brachmann, R.,
P. B. Lindquist,
M. Nagashima,
W. Kohr,
T. Lipari,
M. Napier,
and
R. Derynck.
Transmembrane TGF- precursors activates EGF/TGF-
receptors.
Cell
56:
691-700,
1989[Medline].
7.
Brattain, M. G.,
W. D. Fine,
F. M. Khaled,
J. Thompson,
and
D. E. Brattain.
Heterogeneity of malignant cells from a human colonic carcinoma.
Cancer Res.
41:
1751-1756,
1981[Abstract].
8.
Cartwright, C. A.,
C. A. Coed,
and
B. M. Egbert.
Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis.
J. Clin. Invest.
93:
509-515,
1994[Medline].
9.
Cravo, M.,
P. Fidalgo,
A. D. Pereira,
A. Gouvela-Oliveira,
C. Nobre Leitao,
and
F. Costa Mira.
Folate supplementation increases the degree of DNA methylation in patients with colonic neoplasms (Abstract).
Gastroenterology
104:
A394,
1993.
10.
Cravo, M. L.,
J. B. Mason,
Y. Dayal,
M. Hutchinson,
D. Smith,
J. Selhub,
and
I. H. Rosenberg.
Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats.
Cancer Res.
52:
5002-5006,
1992[Abstract].
11.
Fenech, M.,
and
J. Rinaldi.
The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12, and folic acid.
Carcinogenesis
15:
1405-1411,
1994[Abstract].
12.
Freudenheim, J.,
S. Graham,
J. Marshall,
B. Haughey,
S. Cholewinski,
and
G. Wilkinson.
Folate intake and carcinogenesis of the colon and rectum.
Int. J. Epidemiol.
20:
368-374,
1991[Abstract].
13.
Giovannucci, E.,
M. J. Stampfer,
G. A. Colditz,
E. B. Rimm,
D. Trichopoulos,
B. A. Rosner,
F. E. Speizer,
and
W. C. Willett.
Folate, methionine and alcohol intake and risk of colorectal adenoma.
J. Natl. Cancer Inst.
85:
846-848,
1993[Medline].
14.
Hauri, H. P.,
E. E. Sterchi,
D. Benise,
J. A. M. Fransen,
and
A. Marxer.
Expression and intracellular transport of microvillus membrane hydrolases in human intestinal endothelial cells.
J. Cell Biol.
10:
838-851,
1985.
15.
Hossain, M. Z.,
L. R. Wilkens,
and
P. P. Mehta.
Enhancement of gap junctional communication by retinoids correlates with their ability to inhibit neoplastic transformation.
Carcinogenesis
10:
1743-1748,
1989[Abstract].
16.
Hunter, T.,
and
J. A. Cooper.
Protein tyrosine kinases.
Annu. Rev. Biochem.
54:
897-930,
1985[Medline].
17.
James, S. J.,
A. G. Basnakian,
and
B. J. Miller.
In vitro folate deficiency induces deoxynucleotide pool imbalance, apoptosis, and mutagenesis in Chinese hamster ovary cells.
Cancer Res.
94:
5075-5080,
1994.
18.
Karnes, W. A., Jr.,
J. H. Walsh,
V. Wu,
R. S. Kim,
M. G. Martin,
H. C. Wong,
J. Mendelsohn,
J.-G. Park,
and
F. Cuttita.
Autonomous proliferation of colon cancer cell lines that co-express transforming growth factor and its receptor. Variable effects of receptor-blocking antibody.
Gastroenterology
102:
474-487,
1992[Medline].
19.
Khasharyasha, K.,
V. Schirrmacher,
and
R. B. Lichtner.
EGF receptor in neoplasia.
Cancer Metastasis Rev.
12:
255-274,
1993[Medline].
20.
Kim, Y. I.,
S. W. Choi,
R. N. Solomon,
D. Graeme-Cook,
D. Smith,
M. Nadeau,
G. Dallal,
and
J. B. Mason.
Dietary folate protects against the development of macroscopic colonic neoplasms in a dose-responsive manner in the dimethylhydrazine rat model (Abstract).
Gastroenterology
106:
A402,
1994.
21.
Lashner, B. A.
Red blood cell folate is associated with the development of dysplasia and cancer in ulcerative colitis.
J. Cancer Res. Clin. Oncol.
119:
549-554,
1993[Medline].
22.
Lashner, B. A.,
P. A. Heidenreich,
G. L. Su,
S. V. Kane,
and
S. B. Hanauer.
Effect of folate supplementation on the incidence of dysplasia and cancer in chronic ulcerative colitis. A case-control study.
Gastroenterology
97:
255-259,
1989[Medline].
23.
Lashner, B. A.,
K. S. Provencher,
D. L. Seidner,
A. Knesebeck,
and
A. Brzezinski.
The effect of folic acid supplementation on the risk for cancer or dysplasia in ulcerative colitis.
Gastroenterology
112:
29-32,
1997[Medline].
24.
Levitzki, A.
Tyrphostins: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction.
FASEB J.
6:
3275-3283,
1992
25.
Lipkin, M.
Biomarkers of increased susceptibility to gastrointestinal cancer, their development and application to studies of cancer prevention.
Gastroenterology
92:
1083-1086,
1987[Medline].
26.
Majumdar, A. P. N.,
and
J. R. Goldenring.
Localization and significance of pp55, a gastric mucosal membrane protein with tyrosine kinase activity.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G863-G870,
1998
27.
Majumdar, A. P. N.,
J. A. Moshier,
F. L. Arlow,
and
G. D. Luk.
Biochemical changes in the gastric mucosa after injury in young and aged rats.
Biochim. Biophys. Acta
992:
35-40,
1989[Medline].
28.
Malecka-Panas, E.,
S. E. G. Fligiel,
N. K. Relan,
S. Dutta,
and
A. P. N. Majumdar.
Azoxymethane enhances ligand-induced activation of EGF receptor tyrosine kinase in the colonic mucosa of rats.
Carcinogenesis
17:
233-237,
1996[Abstract].
29.
Malecka-Panas, E.,
K. Radzislaw,
W. Biernat,
J. Tureaud,
P. P. Liberski,
and
A. P. N. Majumdar.
Differential activation of total and EGF receptor (EGF-R) tyrosine kinase (Tyr-k) in the rectal mucosa in patients with adenomatous polyps, ulcerative colitis and colon cancer.
Hepatogastroenterology
44:
435-440,
1997[Medline].
30.
Massague, J.
Epidermal growth factor-like transforming growth factor. II. Interactions with epidermal growth factor receptors in human placenta membranes and A431 cells.
J. Biol. Chem.
258:
13614-13620,
1983
31.
Matousovic, K,
T. Yasuboi,
H. Walker,
J. P. Grande,
and
T. P. Dousa.
Inhibitors of cyclic nucleotide phosphodiesterase isozymes block renal tubular cell proliferation induced by folic acid.
J. Lab. Clin. Med.
130:
487-495,
1997[Medline].
32.
McCormack, S. A.,
and
L. R. Johnson.
Role of polyamines in gastrointestinal mucosal growth.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G795-G806,
1991
33.
Meenan, J.,
E. O'Hallinan,
J. Scott,
and
D. G. Weirt.
Epithelial cell folate depletion occurs in neoplastic but not adjacent normal colon mucosa.
Gastroenterology
112:
1163-1168,
1997[Medline].
34.
Meyer, F.,
and
E. White.
Alcohol and nutrients in relation to colon cancer in middle-aged adults.
Am. J. Epidemiol.
138:
225-236,
1993[Abstract].
35.
Nensey, Y. M.,
F. L. Arlow,
and
A. P. N. Majumdar.
Aging. Increased responsiveness of colorectal mucosa to carcinogen stimulation and protective role of folic acid.
Dig. Dis. Sci.
40:
396-401,
1995[Medline].
36.
Pandiella, A.,
and
J. Massague.
Cleavage of membrane precursor for transforming growth factor is a regulated process.
Proc. Natl. Acad. Sci. USA
88:
1726-1730,
1991[Abstract].
37.
Paspatis, G. A.,
E. Kalafatis,
L. Oros,
V. Xourgias,
P. Koutsioumpa,
and
D. G. Karamanolis.
Folate status and adenomatous colonic polyps. A colonoscopically controlled study.
Dis. Colon Rectum
38:
64-68,
1995[Medline].
38.
Pena, S. V.,
M. F. Melhem,
A. I. Meisler,
and
C. Cartwright.
Elevated c-Yes tyrosine kinase activity in premalignant lesions of the colon.
Gastroenterology
108:
117-124,
1995[Medline].
39.
Pinto, M.,
S. Robine-Leon,
M. D. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. V. Laccroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human carcinoma cell line CaCo-2 in culture.
Biol. Cell
47:
323-330,
1983.
40.
Relan, N. K.,
K. Ponduri,
S. E. G. Fligiel,
S. Dutta,
and
A. P. N. Majumdar.
Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats.
Biochim. Biophys. Acta
1244:
368-376,
1995[Medline].
41.
Riddell, R. H.,
H. Goldman,
D. F. Ransohoff,
H. D. Appelman,
C. M. Fenoglio,
R. C. Haggitt,
C. Ahren,
P. Correa,
S. R. Hamilton,
B. C. Morson,
S. C. Sommers,
and
J. H. Yardley.
Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications.
Hum. Pathol.
14:
931-968,
1983[Medline].
42.
Ullrich, A.,
and
J. Schlessinger.
Signal transduction by receptors with tyrosine kinase activity.
Cell
61:
203-212,
1990[Medline].
43.
Veillette, A.,
F. M. Foss,
E. A. Sausville,
J. B. Bolen,
and
N. Rosen.
Expression of the lck tyrosine kinase gene in human colon carcinoma and other non-lymphoid human tumor cell lines.
Oncol. Res.
1:
357-374,
1987.
44.
Weinberg, G.,
B. Ullman,
and
D. W. Martin, Jr.
Mutator phenotypes in mammalian cell mutants with distinct biochemical defects and abnormal deoxyribonucleoside triphosphate pools.
Proc. Natl. Acad. Sci. USA
78:
2447-2451,
1981[Abstract].
45.
Wickramasinghe, S.,
and
S. Fida.
Bone marrow cells from vitamin B12 and folate-deficient patients misincorporate uracil into DNA.
Blood
83:
1656-1661,
1994
46.
Willett, W. C.
Micronutrients and cancer risk.
Am. J. Clin. Nutr.
59:
1162s-1165s,
1994[Medline].
47.
Yarden, Y.,
and
A. Ullrich.
Growth factor receptor tyrosine kinases.
Annu. Rev. Biochem.
57:
443-487,
1988[Medline].