§
* Sealy Center for Oncology and Hematology, Department of Human Biological Chemistry & Genetics, and § Department of
Pharmacology, University of Texas Medical Branch, Galveston, Texas 77555-1048; and
Faculty of Nutrition, Molecular and Cell
Biology Section, Texas A&M University, College Station, Texas 77843-2471
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Abstract |
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Protein kinase C II (PKC
II) has been implicated in proliferation of the intestinal epithelium. To
investigate PKC
II function in vivo, we generated
transgenic mice that overexpress PKC
II in the intestinal epithelium. Transgenic PKC
II mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt
foci, preneoplastic lesions in the colon. Furthermore,
transgenic PKC
II mice exhibit elevated colonic
-catenin levels and decreased glycogen synthase kinase 3
activity, indicating that PKC
II stimulates the
Wnt/adenomatous polyposis coli (APC)/
-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC
II in colonic epithelial cell
proliferation and colon carcinogenesis, possibly through
activation of the APC/
-catenin signaling pathway.
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Introduction |
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COLON carcinogenesis is a complex multistep process
involving progressive disruption of homeostatic
mechanisms controlling intestinal epithelial cell
proliferation, differentiation, and programmed cell death.
This disruption appears to be mediated by dietary and environmental factors that modulate intestinal epithelial cell
signaling pathways, as well as genetic mutation of transforming oncogenes and deletion or mutation of DNA repair enzymes and tumor suppressor genes (Bertagnolli et
al., 1997). Recent studies have demonstrated the primary
importance of the Wnt/APC/
-catenin signaling pathway in
colon carcinogenesis (Pennisi, 1998
). Mutations in either
APC or
-catenin that lead to activation of this pathway
are present in the vast majority of colon cancers and colonic carcinoma cell lines (Pennisi, 1998
).
Accumulating evidence implicates protein kinase C
(PKC)1 in intestinal epithelial cell proliferation and colon
carcinogenesis both in rodents and humans (Weinstein,
1990; Chapkin et al., 1993
). PKC activity is higher in actively proliferating colonic epithelial cells than in their
quiescent counterparts (Craven and DeRubertis, 1987
),
suggesting a role for PKC activation in epithelial cell proliferation. A link between PKC and colon carcinogenesis
comes from the observation that components of cancer-promoting high fat diets lead to an increase in both colonic
epithelial cell PKC activity and cellular proliferation (Craven and DeRubertis, 1988
; Reddy et al., 1996
). High fat
diet-induced hyperproliferation is thought to predispose
the colonic epithelium to further genetic and biochemical
changes associated with progression along the carcinogenic pathway. PKC has also been shown to play a requisite role in the Wnt/APC/
-catenin proliferative signaling
pathway, suggesting a plausible molecular mechanism by
which PKC could stimulate colonic epithelial cell proliferation and colon carcinogenesis (Cook et al., 1996
).
Several lines of evidence indicate that the PKC II
isozyme (PKC
II) is selectively involved in colonic epithelial cell proliferation and colon carcinogenesis. First, PKC
II is the most responsive of the PKC isozymes expressed
in the colonic epithelium to activation by secondary bile
acids (Pongracz et al., 1995
). Secondary bile acid levels are
elevated in rodents fed a cancer-promotive high fat diet
and this increase has been implicated in early carcinogenic events (for review see Reddy, 1975
). Second, expression of
most colonic PKC isozymes (e.g., PKC
,
, and
) is reduced in the presence of chronically elevated diacylglycerol (DAG), such as is present in preneoplastic colonic epithelial cells (Wali et al., 1991
; Jiang et al., 1996
; Chapkin
et al., 1997
; Jiang et al., 1997
). However, intestinal PKC
II is largely resistant to such activator-mediated downregulation (Saxon et al., 1994
, Sauma et al., 1996
). Third,
the levels of PKC
II are dramatically elevated both during
the initial stages of tumorigenesis and in colonic carcinomas when compared with normal colonic tissue (Craven
and DeRubertis, 1992
; Davidson et al., 1994
, 1998
). Finally, PKC
II is directly involved in colon carcinoma cell
proliferation in vitro (Lee et al., 1993
; Sauma et al., 1996
).
These studies provide compelling but indirect evidence
that PKC II plays an important role in intestinal epithelial
cell proliferation and colon carcinogenesis, and are consistent with our studies demonstrating that PKC
II is
required for leukemia cell proliferation (Murray et al.,
1993
). Therefore, we hypothesized that PKC
II is directly
involved in intestinal epithelial cell proliferation in vivo
and that elevated colonic PKC
II expression and activity
would enhance colon carcinogenesis. To directly test this
hypothesis, we generated transgenic mice that express elevated levels of PKC
II in the intestinal epithelium. These
animals exhibit both hyperproliferation of the colonic epithelium and an increased susceptibility to colon carcinogenesis. Furthermore, our data indicate that the
-catenin/
APC proliferative signaling pathway is stimulated by PKC
II in these animals.
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Materials and Methods |
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Production and Maintenance of Transgenic Mice
A transgene construct consisting of the rat liver fatty acid binding protein
(FABP) promoter (596 to +21; kindly provided by Dr. Jeffrey Gordon,
Washington University, St. Louis, MO), the full-length human PKC
II
cDNA, and the SV40 large T antigen polyadenylation signal sequence was
produced by conventional cloning methods. The resulting PKC
II transgene construct was confirmed by direct microsequencing before microinjection. The pFABP/PKC
II transgene construct was propagated in the
mammalian expression vector pREP4 and the transgene insert was excised using NheI (5') and XbaI (3'), purified, and microinjected into
C57BL/6J × C3H/HeJ F2 mouse oocytes as previously described (Hogan
et al., 1994
). The microinjections and generation of transgenic founder
mice were conducted at the University of Texas Medical Branch Transgenic Mouse Facility. Transgenic founder mice were identified by Southern blot analysis. In brief, genomic tail DNA (5 µg) was digested to completion with Taq I (Roche), resolved by agarose gel electrophoresis, transferred to nylon membrane (Amersham), and transgenic DNA detected with a radiolabeled probe corresponding to the SV40 polyadenylation sequence. Three transgenic founder animals were identified from a
screen of 120 live births. Transgene copy number was determined for each
transgenic line by quantitative Southern blot analysis as previously described (Hogan et al., 1994
). Genotype was confirmed by slot blot analysis
using a radiolabeled probe corresponding to the polyadenylation sequences within the transgene (Sambrook et al., 1989
).
Founder mice were mated with C57BL/6J mice (The Jackson Laboratory) to establish the transgene on a stable genetic background. Transgenic PKC II mice and progeny were bred and housed in microisolator cages maintained at constant temperature and humidity on a 12-h on/12-h
off light cycle in a pathogen-free barrier facility. Mice were provided a
standard autoclavable chow (Purina 7012, 5% fat) and autoclaved water
ad libitum.
Detection of Transgenic PKC II RNA
Total RNA was extracted from tissue samples using a Totally RNA kit
(Ambion). Reverse transcription was carried out using 6 µg RNA, 1 µg
oligo(dT) primer, 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 U SuperScript II reverse transcriptase (GIBCO BRL). Amplification of the transgenic RNA was carried out using 20 ng of the following primers, which
amplify human PKC II but not endogenous mouse PKC
II: forward, 5'
CGTCCTCATTGTCCTC 3'; reverse 5' GACCTTGGTTCCCTGACTG
3'. An optimized amplification program of denaturation (94°C, 15 s), annealing (56°C, 15 s), and extension (74°C, 45 s) for 40 cycles using PCR Supermix (GIBCO BRL) was used. Human brain RNA was used as a positive control; mouse brain RNA and samples incubated without reverse
transcriptase served as negative controls.
PKC Immunoblot and Immunohistochemical Analysis
Immunoblot analysis for PKC II expression in mouse colonic epithelium
was performed essentially as previously described (Davidson et al., 1994
).
In brief, mice were killed by CO2 asphyxiation, the colons were isolated
and slit open longitudinally and rinsed well with PBS, and the colonic epithelium was scraped using a plastic coverslip. Total cell extracts were prepared in RIPA buffer [50 mM Tris, pH 7.2, 150 mM NaCl, 2 mM EDTA,
0.4 mM EGTA, 20 µM NaF, 0.5% deoxycholate, 1% NP-40, 0.1% SDS,
0.1 mM Na3VO4, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 25 µg/ml pepstatin, 1 µg/ml soybean trypsin inhibitor, and 34.5 µg/ml 4-(2-aminoethyl)
benzene sulfonyl fluoride]. Equal amounts (30 µg) of protein were subjected to immunoblot analysis using an isotype-specific antibody for PKC
II (Santa Cruz Biotechnology).
Immunohistochemistry was performed using an enhanced biotinyl tyramide system (New England Nuclear) on sections from the proximal and
distal colon fixed in ethanol, embedded in paraffin, and sectioned (5 µm)
as previously described (Jiang et al., 1995), with the following modifications. After deparaffinization and rehydration of tissues, sections were
treated with 3% hydrogen peroxide in methanol to inhibit endogenous
peroxidase, blocked with TNB reagent (Dupont New England Nuclear),
and incubated with polyclonal antibody to PKC
II (Hocevar and Fields,
1991
). Specificity was confirmed using antibody preincubated with excess
antigen peptide as previously described (Jiang et al., 1995
). Sections were
incubated with biotinylated secondary antibody followed by addition of
streptavidin-conjugated peroxidase. Biotinyl tyramide amplification reagent was then added followed by a second streptavidin-peroxidase incubation. Visualization was with DAB chromagen.
Measurement of Colonic Epithelial Cell Cytokinetics
12-wk-old mice were killed and their colons were dissected and measured
for overall length. The distal colon (1 cm from rectal end) was fixed in 4%
paraformaldehyde and processed for histology as described previously
(Jiang et al., 1995). Tissues were embedded in paraffin, sectioned (5-µm
thickness) and stained with hematoxylin and eosin. 25 full-length, longitudinally cut crypts from each animal were analyzed for crypt height (micrometer) and number of cells per crypt height. 25 crypts cut on the cross-section at random height were counted to determine the average crypt
circumference (in number of cells). These data were used to calculate cell
size (crypt height in micrometer/crypt height in cell number) and estimate the total cells per crypt (mean cells per crypt column × mean crypt circumference).
Proliferation.
Cell proliferation was determined by immunohistochemical detection for proliferating cell nuclear antigen (PCNA) in distal colon
sections. Primary antibody against PCNA (PC10 clone; DAKO) was diluted 1:50 in PBS and preincubated with 1:200 biotinylated anti-mouse
IgG (Santa Cruz Biotechnology) overnight at 4°C. After deparaffinization, sections were processed for antigen retrieval as described by the
manufacturer (DAKO), treated with 1% hydrogen peroxide for 10 min to
inactivate endogenous peroxidases, and blocked with normal goat serum.
The slides were then incubated with the PCNA/anti-mouse IgG antibody
conjugate for 60 min at room temperature. Antigen-antibody complexes
were detected with avidin and peroxidase-labeled biotin (ABC staining
system; Santa Cruz Biotechnology) and visualized with DAB. Slides were
counterstained with hematoxylin to provide contrast. 20 full-length, longitudinally cut crypts were divided into thirds and scored visually for cells staining darkly for PCNA (Lin et al., 1996). The labeling index (percent of
labeled cells) and proliferative zone (highest cell from the bottom of the
crypt staining for PCNA divided by the total cells per crypt height) were
calculated for each set of animals.
Differentiation. The differentiation status of colonic epithelial cells was measured by detection of the specific binding of three different lectins. After deparaffinization, sections were incubated for 60 min at room temperature in normal goat serum. Three different biotinylated lectins (dolichos biflorus agglutinin [DBA], peanut agglutinin [PNA], and Ulex europaeus-I [UEAI]; Vector Labs.) were diluted to 10 µg/ml in PBS. Sections were incubated with one of the three lectin solutions for 60 min at room temperature. Sections were then washed in three changes of PBS and incubated with 5 µg/ml of rhodamine red-X-conjugated Streptavidin (Jackson Immunoresearch Labs.) in PBS for 30 min at room temperature. After three 5-min washes in PBS, sections were mounted in aqueous media containing 95% glycerol in PBS and analyzed by fluorescence microscopy. Sections were also analyzed histologically by Alcian blue/periodic acid Schiff (PAS) staining for detection of mature, mucin-producing goblet cells.
Apoptosis.
The percentage of cells undergoing apoptosis (apoptotic index) was determined in paraformaldehyde-fixed distal colon tissue by the
TdT-mediated dUTP-biotin nick end labeling of fragmented DNA
(TUNEL) assay (Gavrieli et al., 1992) using the apoTACS kit from Trevigen. The tissue sections were counterstained with methyl green. 100 longitudinally cut, full-length crypts were scored for apoptotic cells based on a
combination of positive staining and morphological criteria as previously
described (Kerr et al., 1995
).
Carcinogen Treatment and Aberrant Crypt Foci Analysis
40 (20 transgenic PKC II mice, 20 nontransgenic littermates) 6-7-wk-old
female mice were injected intraperitoneally with azoxymethane (10 mg/kg
body wt) or saline weekly for 2 wk as previously described (Chang et al.,
1997
). At 5 and 20 wk after the second injection, five animals per group
were killed by CO2 asphyxiation and the colons were removed. The colons
were flushed with PBS to remove fecal pellets, slit open longitudinally,
and fixed flat between two pieces of filter paper under a glass plate in 70% ethanol for 24 h. Fixed colons were stained with 0.2% methylene blue in
PBS for 5 min before being mounted on a glass slide for observation at
low magnification (×40) on a light microscope. Aberrant crypt foci (ACF)
were scored blindly by a single observer (A.P. Fields) for total number
and multiplicity (number of crypts/focus) using previously defined criteria
(McLellan et al., 1991
).
-Catenin Immunoblot Analysis and Glycogen Synthase
Kinase 3
Kinase Assay
Colonic epithelia from transgenic and nontransgenic mice were scraped
and equal amounts of protein from total tissue lysates were subjected to
immunoblot analysis using a specific -catenin polyclonal antibody (Santa
Cruz Biotechnology Inc.) or a specific GSK-3
monoclonal antibody
(Transduction Laboratories). For glycogen synthase kinase (GSK)-3
kinase assay, colonic epithelium scrapings were solubilized in lysis buffer [10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 25 µg/
ml pepstatin, 1 µg/ml soybean trypsin inhibitor, 34.5 µg/ml 4-(2-aminoethyl) benzene sulfonyl fluoride, 20 µM NaF, and 0.1 mM Na3VO4]. Lysates containing 300 µg of protein were precleared with 75 µl of protein A
agarose and then added to 75 µl of protein A agarose beads that had been preincubated with 5 µg of anti-GSK-3
monoclonal antibody (Transduction Labs.). Samples were incubated for 1 h at 4°C, and beads were pelleted and washed once with lysis buffer and once with kinase assay buffer
(8 mM MOPS, pH 7.4, 0.2 mM EDTA, 10 mM Mg acetate, and 0.1 mM
ATP). The washed and pelleted beads were then resuspended in 40 µl of
kinase assay buffer containing 10 µCi [
32P]ATP and 250 µmol of GSK-3
-specific substrate peptide (Upstate Biotechnology, Inc.). Reactions
were incubated for 20 min at 25°C and stopped by pelleting the beads and
adding the supernatant to 20 µl of 40% trichloroacetic acid. Reactions
were spotted on P-81 filters and washed three times in 0.75% phosphoric
acid and once with acetone. Incorporated radioactive phosphate was
quantitated by Cerenkov counting. Nonspecific and background counts
were calculated by performing parallel assays with a nonphosphorylatable
GSK-3
substrate peptide.
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Results |
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Generation and Identification of PKC II
Transgenic Mice
To investigate the role of PKC II in colonic epithelial cell
biology, we generated transgenic mice overexpressing
PKC
II in the intestinal epithelium. For this purpose, we
used the rat liver FABP promoter, which has been well
characterized to target transgene expression to the intestinal epithelium (Cohn et al., 1991
; Simon et al., 1993
). A
schematic diagram of the transgene construct is presented
in Fig. 1 A. The FABP promoter (
596 to +21) was fused
to the cDNA for human PKC
II and the SV40 poly A signal sequence by conventional cloning. Southern blot analysis of tail DNA identified four potential transgenic
founders (designated Nos. 54, 61, 78, and 92) from 120 live
births (Fig. 1 B). Animal 92 gave a reactive band of higher
mol wt than the expected 453 bp Taq 1 fragment generated from the intact transgene (Fig. 1, asterisk). Further
analysis using overlapping PCR primer sets demonstrated
that this animal contained a truncated transgene, whereas animals 54, 61, and 78 contained multiple copies of the intact
transgene construct. All three of the founder animals were fertile and subsequent analysis of progeny by quantitative
Southern blot analysis demonstrated that they carried 6, 15, and 31 copies of the transgene, respectively. Furthermore, all
three transgenic lines exhibit germline transmission of the
transgene to subsequent progeny (data not shown).
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Characterization of PKC II Transgene Expression
PKC II transgenic RNA expression was detected by reverse transcriptase (RT)-PCR using primers specific for
the human PKC
II transgene. A representative RT-PCR
analysis of a litter of mice from the 54 transgenic line is
shown in Fig. 1 C. As can be seen, transgenic PKC
II
mRNA is detected in the colonic epithelium of all three
transgenic mice, but not in nontransgenic littermates. Further analysis demonstrated that PKC
II transgene expression is fully penetrant, being detected in the colonic epithelium of all transgenic mice tested. Furthermore, no
false positive RT-PCR products have been detected in
nontransgenic animals, demonstrating the specificity of
the RT-PCR primers for the human PKC
II transgene construct. Similar results were obtained in the 61 and 78 transgenic lines (data not shown).
We next determined the level of PKC II protein expression in the colonic epithelium of transgenic mice. Colonic
epithelial cell lysates from transgenic and nontransgenic
animals from the 54 transgenic line were prepared and
subjected to immunoblot analysis using a PKC
II isozyme-specific antibody (Hocevar and Fields, 1991
). Consistent with the presence of transgenic PKC
II mRNA, PKC
II protein levels in the colonic epithelium of transgenic mice are elevated relative to their nontransgenic littermates (Fig. 2 A). Transgenic PKC
II exhibits a relative
molecular mass of ~85 kD, comigrating with mouse brain
PKC
II used as a positive control. Similar results were obtained in the small intestine of these animals and from animals in the 61 and 78 transgenic lines (data not shown).
Quantitation of PKC
II expression by densitometric analysis of the immunoblots indicated that 54 line transgenic
mice express an average of fivefold more PKC
II protein than do nontransgenic littermates. Immunoprecipitation
kinase assays showed an approximately fivefold increase
in calcium- and phospholipid-dependent PKC
II activity
in the colonic epithelium of transgenic mice, demonstrating that transgenic PKC
II is catalytically active and exhibits the same cofactor dependence of endogenous PKC/
II (data not shown). Animals in the 54 transgenic line
gave a consistently high level of transgene expression and
therefore this line was selected for further analysis.
|
We next assessed the pattern of transgenic PKC II protein expression within the colonic epithelium by immunohistochemistry (Fig. 2, B and C). For this purpose, tissue
from the proximal colon of transgenic and nontransgenic
mice was immunostained for PKC
II. Consistent with our
RT-PCR and immunoblot results, the colonic epithelium
from transgenic animals (Fig. 2 C) exhibits increased immunostaining for PKC
II when compared with nontransgenic littermates (Fig. 2 B). In nontransgenic mice, PKC
II staining is observed in the mid-crypt regions and on the
luminal surface of the epithelium. In transgenic animals,
PKC
II staining is greatest in the mid-crypt region but is
detectable throughout the entire crypt axis. Previous characterization of the transgene promoter demonstrated that
the rat liver FABP promoter is active in both proliferating
and postmitotic cells in the colonic epithelium of transgenic mice (Hansbrough et al., 1991
). The distribution of
endogenous PKC
II overlaps that of the stem cell population, which is located in the mid-crypt region in the proximal colon (Sato and Ahnen, 1992
). In the proximal colon,
maturing colonic epithelial cells migrate from the proliferative mid-crypt region toward the base and the luminal
surface of the crypt (Sato and Ahnen, 1992
). The fact that
endogenous PKC
II expression colocalizes with the stem
cell population is consistent with the hypothesis that PKC
II plays a functional role in colonic epithelial cell proliferation. Transgenic PKC
II expression was detected in both the proximal and distal colon, indicating transgene expression throughout the colonic epithelium.
Effect of Transgene Expression on Colonic Crypt Morphometry
To investigate the biological effects of overexpression of
PKC II in the colonic epithelium, we analyzed the following colonic morphometric parameters: colon length, colonic crypt height (in micrometer and cell number), crypt
circumference (in cell number), and cell size (crypt height
in micrometer/crypt height in cell number) (Table I). This
analysis revealed no statistical difference in the length of
the colon, cell size, crypt height in micrometers, or crypt
circumference between transgenic and nontransgenic littermates. However, colonic crypts from transgenic mice
tended to be longer and have a larger circumference than
those from nontransgenic mice. In addition, a highly significant increase in the number of cells per crypt height,
and in the total number of cells per crypt, was observed in
transgenic mice (Table I). Similar results were obtained in
a second transgenic mouse line (line 78; 22.2 cells per crypt
height in transgenic versus 20.7 in nontransgenic mice, P = 0.009; and 356.4 total cells per crypt in transgenic versus
321.1 in nontransgenic mice, P = 0.007), indicating that this effect is due to the presence of the PKC
II transgene
rather than an insertional mutagenic event. Both of these
cytokinetic parameters are highly regulated and are determined by the balance among cell proliferation, differentiation, and apoptosis. These results demonstrate that increased expression of PKC
II disrupts one or more of the
homeostatic mechanisms regulating cell number in the colonic epithelium.
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Increased PKC II Expression Induces
Hyperproliferation of the Colonic Epithelium
Elevated PKC II could increase the number of colonic epithelial cells by increasing the level of proliferation, or by
decreasing differentiation and/or apoptosis, in the colonic
crypt. To distinguish between these possibilities, each of
these cytokinetic parameters was measured. Immunohistochemical staining for PCNA revealed that the colonic
epithelium from transgenic mice contain significantly more PCNA-positive cells than those from nontransgenic
mice (Fig. 3). Quantitation of PCNA-positive nuclear
staining (indicative of cells in S-phase; Lin et al., 1996
;
Shpitz et al., 1997
) gave a labeling index of 28.3 ± 0.2% for
transgenic mice compared with 21.4 ± 0.9% for nontransgenic mice (Table II). This difference is highly significant
(P = 0.0001) and clearly contributes to the increase in
crypt cell number observed in transgenic mice. The difference in labeling index was most pronounced in the bottom third of the crypts, the region containing the stem cell population in the distal colon. The size of the proliferative
zone (calculated as the highest labeled cell in the crypt
column) was also larger in transgenic colons; however,
this difference was not statistically significant (Table II).
Taken together, these data demonstrate that elevated
PKC
II expression stimulates hyperproliferation of the
stem cell population residing within the base of the crypt, rather than stimulating postmitotic cells higher in the crypt to reenter the cell cycle.
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The differentiation state of the colonic epithelium was
examined by staining with a panel of lectins and histochemical markers to identify the major differentiated
colonic epithelial cell lineages. Fig. 4, A and B, shows distal colonic epithelium from transgenic and nontransgenic
mice stained with the two histochemical stains, Alcian blue
and PAS, that detect goblet cells. The staining pattern seen in transgenic and nontransgenic animals is indistinguishable. Mucin production was detected by staining with
several fluorescently labeled lectins (Fig. 4, C-H). DBA
binds fairly uniformly to mucin-producing cells in normal
distal colonic epithelium (Fig. 4, C and D; Campo et al.,
1988; Caldero et al., 1989
; Chang et al., 1997
; Hong et al.,
1997
). PNA gives a golgi (supranuclear) staining pattern on a subset of mucin-producing enterocytes (Fig. 4, E and
F; Freeman, 1983
; Campo et al., 1988
; Caldero et al., 1989
;
Boland and Ahnen, 1995
) and UEAI gives low level staining in normal mucosa of the distal colon (Fig. 4, G and H;
Caldero et al., 1989
). Analysis of the number and location
of cells staining with the various lectins revealed no significant changes in the number of goblet cells or in the intensity or pattern of lectin labeling in transgenic PKC
II versus nontransgenic mice. These data indicate that increased expression of PKC
II has no demonstrable effect on the
differentiation status of the major colonic enterocytic cell
lineages.
|
The level of apoptosis in the colonic epithelium was
measured using an in situ TUNEL assay (Fig. 5, A and B).
An example of TUNEL staining of an apoptotic cell,
which typically occurs near the top of the crypt, is shown in
Fig. 5 A. As expected, we detected a very low level of apoptosis in the colon of transgenic PKC II and nontransgenic
mice. The apoptotic index in the distal colon of nontransgenic mice was not significantly different from that in
transgenic PKC
II mice (Fig. 5 B). Apoptosis is thought to contribute to the loss of cells required to maintain a balance with cell proliferation within the colonic epithelium
(Chang et al., 1997
; Potten et al., 1997
). However, apoptotic cells are quickly eliminated in the colonic crypt, so
that apoptosis is detected at a very low level (Hall et al.,
1994
; Merritt et al., 1994
; Risio et al., 1996
). Our results are
similar to the level of apoptosis in mouse colon reported
by others (Merritt et al., 1994
; Risio et al., 1996
), and demonstrate that increased expression of PKC
II has no significant effect on the level of apoptosis in the colonic epithelium.
|
Transgenic PKC II Mice Are More Susceptible to
Formation of Carcinogen-induced Aberrant Crypt Foci
Increased cellular proliferation is a significant risk factor
for development of colon cancer (Chang et al., 1997; Einspahr et al., 1997
). Therefore, we assessed whether transgenic PKC
II mice exhibit an increased susceptibility to
colon carcinogenesis. 1,2-dimethylhydrazine and its metabolite, azoxymethane (AOM), are organ-specific carcinogens that have been extensively characterized for their
ability to induce colon cancer in rodents (Deschner and
Long, 1977
; Deschner et al., 1979
). AOM reproducibly induces colon tumors that exhibit many of the same genetic
and signal transduction defects identified in human colon
carcinomas (Deschner et al., 1977; Deschner et al., 1979
;
Erdman et al., 1997
; Maltzman et al., 1997
; DeFilippo et
al., 1998
; Sheng et al., 1998
). AOM also induces ACF,
which represent well-established preneoplastic colonic lesions in both rodents and humans (McLellan and Bird, 1988
; McLellan et al., 1991
; Takayama et al., 1998
). Both
the number and multiplicity (i.e., number of crypts per focus) of ACF are highly predictive of subsequent tumor development (Magnuson et al., 1993
; Bird, 1995
; Shivapurkar
et al., 1997
). Therefore, AOM-induced colon carcinogenesis is a highly relevant model for human colon cancer.
To determine whether transgenic PKC II mice differ
from nontransgenic mice in their sensitivity to AOM-induced colon carcinogenesis, 6-7-wk-old transgenic PKC
II mice and nontransgenic littermates (five mice/group)
received either AOM (10 mg/kg body wt) or saline by intraperitoneal injection once a week for 2 wk. At 5 and 20 wk after the second AOM injection, mice were killed and
their colons were analyzed for the presence of ACF. In
agreement with the literature (Bird, 1987
; McLellan and
Bird, 1988
; McLellan et al., 1991
), we observed no ACF in
saline-injected animals, confirming that ACF arise as a result of AOM exposure. Colons from both transgenic and
nontransgenic animals treated with AOM contained ACF
exhibiting the distinguishing characteristics described by
Bird and colleagues (Bird, 1987
; McLellan and Bird, 1988
;
McLellan et al., 1991
). Specifically, ACF appeared as enlarged crypts, often three or four times the size of adjacent
crypts, that were raised above the surface of the surrounding mucosa. ACF characteristically stained darker than
surrounding crypts, had thicker than normal intercryptal
spaces, and exhibited thickening of the crypt wall, suggestive of epithelial stratification. The crypt lumens in ACF were elongated and often serrated, in contrast to the
round, smooth lumens of normal crypts. ACF contained
either a single aberrant crypt or involved two or more adjacent crypts. Fig. 6 A shows the morphology of a typical
ACF consisting of three crypts from an AOM-treated animal. The total number of ACF/colon and the multiplicity of ACF was determined at 5 and 20 wk after the last AOM
injection (Fig. 6, B-D). AOM-treated transgenic mice had
a statistically significant increase in the total number of
ACF/colon and in the number of ACF of higher multiplicity at both 5 and 20 wk (Fig. 6, B and C). At 20 wk, the total number of ACF did not increase significantly from that
measured at 5 wk; however, the number of ACF of higher
multiplicity did increase in transgenic PKC
II mice (Fig. 6
D). Interestingly, at 5 wk, although the total number of
ACF and the number of ACF of higher multiplicity were
greater in transgenic mice, the average multiplicity of ACF
in these two groups did not differ (Fig. 6 D). However, by
20 wk, transgenic mice exhibited an increase not only in
the number of ACF but also in the average crypt multiplicity (Fig. 6 D). Since the number of ACF, particularly those
of higher multiplicity, are highly predictive of subsequent
colon tumor incidence, these data demonstrate that transgenic PKC
II mice are more susceptible to AOM-induced colon carcinogenesis than nontransgenic littermates. Furthermore, these data suggest that elevated PKC
II is involved not only in the early promotive phase of ACF development but also in their progression to lesions of higher
multiplicity and malignant potential.
|
The APC/-Catenin Signaling Pathway Is Activated in
the Colonic Epithelium of Transgenic PKC
II Mice
Colonic epithelial cell proliferation is under the control of
the Wnt/APC/-catenin proliferative signaling pathway
(Pennisi, 1998
). PKC has recently been demonstrated to
play a key role in Wnt/wingless signaling in tissue culture
cells (Cook et al., 1996
). Selective PKC inhibitors can
block Wnt-mediated inhibition of GSK-3
activity, whereas
activation of PKC with PMA leads to inactivation of
GSK-3
kinase activity in the absence of Wnt (Cook et al., 1996
). GSK-3
is a constitutively active serine/threonine
kinase that is a critical downstream target in the Wnt signaling pathway. GSK-3
-mediated phosphorylation of
APC facilitates binding of
-catenin to APC, which targets
-catenin for degradation. The ability of PKC to inhibit
GSK-3
activity is probably due to its direct phosphorylation of GSK-3
, since PKC has been shown to directly
phosphorylate GSK-3
and inhibit its activity in vitro (Goode et al., 1992
). To determine whether PKC
II activates the Wnt/APC/
-catenin pathway in vivo, we assessed GSK-3
levels and activity in the colonic epithelium of transgenic PKC
II mice (Fig. 7, A and B).
Immunoblot analysis reveals that GSK-3
protein levels
are similar in transgenic and nontransgenic mice (Fig. 7
A). However, immunoprecipitation kinase assays demonstrate that GSK-3
activity in transgenic mice is 50% of
that observed in nontransgenic littermates (Fig. 7 B). The
observed decrease in GSK-3
activity is due to a decrease
in the specific kinase activity of the enzyme since GSK-3
expression was unchanged in transgenic PKC
II mice
(Fig. 7 A). The extent of GSK-3
inhibition is similar to
that observed in response to optimal concentrations of either soluble Wnt or PMA in fibroblasts in vitro (Cook et
al., 1996
). As another measure of Wnt pathway activation,
-catenin protein levels were assessed by immunoblot
analysis (Fig. 7, C and D).
-catenin levels are elevated in
transgenic PKC
II mice when compared with nontransgenic littermates (Fig. 7 C). Densitometric analysis of the
immunoblot data indicate that on average
-catenin levels
are ~40% higher in transgenic PKC
II mice. These data indicate that the Wnt/APC/
-catenin signaling pathway
can be stimulated by
II and provide a plausible molecular
mechanism by which PKC
II causes hyperproliferation
and increased susceptibility to colon carcinogenesis in
these animals.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Colonic Crypt Homeostasis/Role of (Hyper)proliferation in Susceptibility to Colon Carcinogenesis
Colon carcinogenesis is a multistep process involving the
progressive loss of growth control mechanisms and accumulation of genetic mutations that result in an increasing
level of neoplasia (Bertagnolli et al., 1997). The process
of multistage carcinogenesis has been described as "a
progressive disorder in signal transduction" (Weinstein,
1990
). According to this model, nongenetic changes in
normal signal transduction pathways which increase the
susceptibility to further genetic "hits" and therefore play a
critical role in the pathogenesis of colon cancer occur early
in the carcinogenic process. However, the nature of these
early cancer-promotive changes is not well understood.
Members of the PKC family of enzymes have been implicated in the regulation of colonic cell proliferation, differentiation, and apoptosis. PKC
II plays a direct role in cellular proliferation in both human leukemia cells and colon cancer cell lines (Murray et al., 1993
; Sauma et al., 1996
),
and increases in PKC
II expression are early events in colon carcinogenesis in vivo (Davidson et al., 1998
). Our
present data demonstrate that this increase in PKC
II expression plays a promotive role in colon carcinogenesis.
To directly assess the role of PKC II in colonic epithelial cell proliferation and colon carcinogenesis, we developed a transgenic mouse model in which PKC
II is overexpressed in the intestinal epithelium. Transgenic PKC
II
mice exhibit hyperproliferation of the colonic epithelium
characterized by an increase in the labeling index and an
increase in the number of cells per colonic crypt. Interestingly, no significant changes were observed in colonocyte
differentiation status or apoptotic index, indicating a selective effect of PKC
II on the proliferative program of the
colonic epithelium. Although we cannot eliminate the possibility that subtle changes have occurred in the regulation
of differentiation or susceptibility to apoptosis, our data
clearly demonstrate that the change in proliferation is a
major contributing factor to the increased colonic crypt
cell number observed in transgenic PKC
II mice.
Increased proliferation is an important risk factor for induction of colon cancer and is a key biomarker of preneoplastic events (Chang et al., 1997; Einspahr et al., 1997
).
Our data indicate that PKC
II acts early in the carcinogenic pathway to increase the proliferation of the colonic
epithelium, perhaps making it more susceptible to further
genetic mutations and formation of preneoplastic lesions,
including ACF. The effect of increased PKC
II expression
on the susceptibility to induction of colon cancer was
tested using a well-characterized rodent carcinogenesis model (Deschner and Long, 1977
; Deschner et al., 1979
).
AOM-induced colon tumors are a good model for sporadic human colon cancer because they exhibit many of
the same properties as human colon tumors, including increased proliferation, development of tumors predominantly in the distal colon, and the presence of many of the
same genetic mutations found in human tumors. In addition, ACF, the earliest preneoplastic lesions observed in
this model, are also thought to be preneoplastic lesions in
humans (Pretlow et al., 1992
; Takayama et al., 1998
). ACF
exhibit many of the early phenotypic markers of colon
cancer including increased proliferation and frequent mutations in the APC and ras genes (Pretlow et al., 1993
;
Smith et al., 1994
; Shivapurkar et al., 1997
). We demonstrate that increased PKC
II expression makes transgenic
mice more susceptible to AOM-induced colon carcinogenesis as measured by an increase in the total number of
ACF and in the number of ACF of higher multiplicity than
nontransgenic mice. ACF are highly predictive of subsequent tumor formation and multiplicity in the rodent carcinogenesis model and of adenoma formation and colon
cancer risk in humans (Magnuson et al., 1993
; Bird, 1995
;
Roncucci et al., 1991
). Our data indicate that elevated
PKC
II expression not only promotes ACF formation,
but also stimulates progression of these lesions. These results suggest that PKC
II plays a critical role at multiple stages in the colon carcinogenic pathway.
A Model for the Role of PKC II in Sporadic
Colon Cancer
Accumulating evidence suggests that PKC II plays a direct role in intestinal epithelial cell proliferation and colon
carcinogenesis in both rodents and humans. PKC
II levels
and activity are elevated in preneoplastic and neoplastic
colons, demonstrating that these changes precede colon
carcinoma development (Craven and DeRubertis, 1992
;
Wali et al., 1995
; Davidson et al., 1998
). Here, we demonstrate that overexpression of PKC
II in the colonic epithelium leads to hyperproliferation and increased susceptibility to colon carcinogenesis. Furthermore, we demonstrate
that elevated PKC
II leads to inhibition of GSK-3
activity and an increase in
-catenin levels. These observations
are consistent with in vitro data demonstrating a requisite
role for PKC in the Wnt proliferative signaling pathway (Cook et al., 1995), and suggest that PKC
II may play
such a role in vivo. Further studies will be required to determine whether PKC
II-mediated activation of this
pathway is required for its ability to stimulate proliferation
and cancer susceptibility in the transgenic mouse setting.
Taken together, the data lead us to propose a molecular
mechanism by which PKC II stimulates colonic epithelial
cell hyperproliferation and increased colon carcinogenesis
in transgenic mice (Fig. 8). In this model, PKC
II either
directly or indirectly leads to GSK-3
inactivation. PKC
has been shown to phosphorylate GSK-3
and inactivate
the enzyme in vitro (Goode et al., 1992
), suggesting that
PKC
II can inhibit GSK-3
by direct phosphorylation and inactivation. Inhibition of GSK-3
leads to an accumulation of
-catenin by decreasing the interaction of
-catenin with APC, which targets
-catenin for degradation.
Accumulation of
-catenin causes Tcf-dependent transcriptional activation of growth-related genes to stimulate
colonocyte proliferation (Pennisi, 1998
). The APC/
-catenin pathway is a major site for mutation during colon carcinogenesis (Pennisi, 1998
). Mutations in either APC or
-catenin that disrupt
-catenin degradation are present in
the vast majority of colon cancers, providing strong evidence that elevated
-catenin levels are important in colon
carcinogenesis (Ilyas et al., 1997
; Pennisi, 1998
). Furthermore, overexpression of a proteolytically-stable NH2-terminal truncated
-catenin in the intestinal epithelium of
transgenic mice leads to hyperproliferation (Wong et al.,
1998
). Our data suggest that accumulation of
-catenin
through PKC
II-mediated inhibition of GSK-3
may play
an important promotive role in colon carcinogenesis before the acquisition of mutations in members of this critical signaling pathway.
|
A major question is how PKC II activity is modulated
during the early stages of colon carcinogenesis. One attractive hypothesis arises from the finding that colonocyte
PKC activity can be stimulated by cancer-promotive components of a high fat diet. Diets high in certain fatty acids
have been shown to increase the proliferative activity of
the colonic epithelium, stimulate colonocyte PKC activity, and increase susceptibility to carcinogen-induced ACF
(Craven and DeRubertis, 1988
; Risio et al., 1996
, Morotomi et al., 1997
). This finding is of significance since increased colonic proliferation is a well-established risk factor and biomarker for colon cancer in individuals with
familial adenomatous polyposis and ulcerative colitis, as
well as in carcinogen-treated rodents (Einspahr et al.,
1997
).
Cancer-promotive dietary fats function to increase the
level of secondary bile acid and fatty acids in the intestinal
lumen. Secondary bile acids can in turn activate colonic
PKC by a number of mechanisms. First, secondary bile acids and fatty acids can directly activate PKC II activity
and stimulate cellular proliferation in the colonic epithelium (DeRubertis et al., 1984
; Fitzer et al., 1987
; Ward and
O'Brian, 1988
; Pongracz et al., 1995
). Second, bile acids
can promote DAG production by intestinal bacteria, which in turn stimulate colonocyte PKC activity (Morotomi et al., 1990
; Morotomi et al., 1991
). Third, bile acids
can stimulate phospholipid breakdown and DAG generation in colonic epithelial cells (DeRubertis and Craven,
1987
), leading to PKC activation. Therefore, we hypothesize that these dietary risk factors increase PKC
II activity
in intestinal epithelial cells by multiple mechanisms, resulting in increased epithelial cell proliferation through activation of the APC/
-catenin signaling pathway in a Wnt-independent fashion (Fig. 8 C). This model provides a
plausible link between a critical intracellular signaling
pathway that is known to be important in colon cancer,
and known dietary risk factors for colon carcinogenesis. Our transgenic PKC
II mice will provide a valuable
model to test the hypothesis that PKC
II is a relevant target for these cancer-promotive dietary risk factors, and to
explore the mechanism by which these factors may impinge on the APC/
-catenin signaling pathway.
![]() |
Footnotes |
---|
Address correspondence to Alan P. Fields, Sealy Center for Oncology & Hematology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1048. Tel.: 409-747-1940. Fax: 409-747-1938. E-mail: afields{at}utmb.edu
Received for publication 10 March 1999.
We thank Dr. Jeffrey Gordon for the generous gift of the rat liver FABP promoter; Dr. Jeffrey Ceci for generation of the founder transgenic mice and expert technical advice on genotypic analysis; Jenny Cui, Dr. Wenchi Chang, and Lisa Nash for excellent technical assistance; Dr. Leonard H. Augenlicht for advice on in situ apoptosis measurements; and Dr. Joanne Lupton, and Marco Velasco for helpful discussions.
This work was supported in part by National Institutes of Health grants CA59034 and CA81436. A.P. Fields is a Leukemia Society of America Scholar.
![]() |
Abbreviations used in this paper |
---|
ACF, aberrant crypt foci;
AOM, azoxymethane;
APC, adenomatous polyposis coli;
DAG, diacylglycerol;
DBA, dolichos biflorus agglutinin;
FABP, fatty acid binding protein;
GSK-3, glycogen synthase kinase 3
;
PAS, periodic acid Schiff;
PKC, protein kinase C;
PKC
II, protein kinase C
II isozyme;
PNA, peanut agglutinin;
RT-PCR, reverse transcriptase PCR;
TUNEL, TdT-mediated
dUTP-biotin nick end labeling;
UEAI, Ulex europaeus-I.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bertagnolli, M.M., C.J. McDougall, and H.L. Newmark. 1997. Colon cancer prevention: intervening in a multistage process. Proc. Soc. Exp. Biol. Med. 216: 266-274 [Abstract]. |
2. | Bird, R.P.. 1987. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37: 147-151 |
3. | Bird, R.P.. 1995. Role of aberrant crypt foci in understanding the pathogenesis of colon cancer. Cancer Lett. 93: 55-71 |
4. | Boland, C.R., and D.J. Ahnen. 1995. Binding of lectins to goblet cell mucin in malignant and premalignant colonic epithelium in the CF-1 mouse. Gastroenterology. 89: 127-137 |
5. | Caldero, J., E. Campo, J. Vinas, and A. Cardesa. 1989. Lectin-binding sites in neoplastic and non-neoplastic colonic mucosa of 1,2-dimethylhydrazine-treated rats. Lab. Invest. 61: 670-676 |
6. | Campo, E., E. Condom, A. Palacin, E. Qusada, and A. Cardesa. 1988. Lectin binding patterns in normal and neoplastic colonic mucosa. Dis. Colon Rectum. 31: 892-899 |
7. | Chang, W.C.L., R.S. Chapkin, and J.R. Lupton. 1997. Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis. 18: 721-730 [Abstract]. |
8. | Chapkin, R.S., J. Gao, D.Y. Lee, and J.R. Lupton. 1993. Dietary fibers and fats alter rat colon protein kinase C activity: correlation to cell proliferation. J. Nutr. 123: 649-655 |
9. | Chapkin, R.S., Y.H. Jiang, L.A. Davidson, and J.R. Lupton. 1997. Modulation of intracellular second messengers by dietary fat during tumor development. In Dietary Fat and Cancer. American Institute for Cancer Research, editor. Plenum Press, New York, NY. 85-96. |
10. | Cohn, S.M., K.A. Roth, E.H. Birkenmeier, and J.I. Gordon. 1991. Temporal and spatial patterns of transgene expression in aging adult mice provide insights about the origins, organization, and differentiation of the intestinal epithelium. Proc. Natl. Acad. Sci. USA. 88: 1034-1038 [Abstract]. |
11. | Cook, D., M. Fry, K. Hughes, R. Sumathipala, J. Woodgett, and T. Dale. 1996. Wingless inactivates glycogen synthase kinase-3 via an intracellular signaling pathway which involves a protein kinase C. EMBO (Eur. Mol. Biol. Organ.) J. 15: 4526-4536 [Abstract]. |
12. | Craven, P.A., and F.R. DeRubertis. 1987. Subcellular distribution of protein kinase C in rat colonic epithelial cells with different proliferative activities. Cancer Res 47: 3434-3438 [Abstract]. |
13. | Craven, P.A., and F.R. DeRubertis. 1988. Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation by unsaturated fatty acids. Gastroenterology. 95: 676-685 |
14. | Craven, P.A., and F.R. DeRubertis. 1992. Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis. Cancer Res. 52: 2216-2221 [Abstract]. |
15. | Davidson, L.A., Y.H. Jiang, J.N. Derr, H.M. Aukema, J.R. Lupton, and R.S. Chapkin. 1994. Protein kinase C isoforms in human and rat colonic mucosa. Arch. Biochem. Biophys 312: 547-553 |
16. |
Davidson, L.A.,
C.M. Aymoud,
Y.H. Jiang,
N.D. Turner,
J.R. Lupton, and
R.S. Chapkin.
1998.
Non-invasive detection of fecal protein kinase C ![]() ![]() |
17. | DeFilippo, C., G. Caderni, M. Bazzicalupo, C. Briani, A. Giannini, M. Fazi, and P. Dolara. 1998. Mutations of the Apc gene in experimental colorectal carcinogenesis induced by azoxymethane in F344 rats. Brit. J. Cancer. 77: 2148-2151 |
18. | Deschner, E.E., and F.C. Long. 1977. Colonic neoplasms in mice produced with six injections of 1,2-dimethylhydrazine. Oncology. 34: 255-257 |
19. | Deschner, E.E., F.C. Long, and A.P. Maskens. 1979. Relationship between dose, time, and tumor yield in mouse dimethylhydrazine-induced colon tumorigenesis. Cancer Lett. 8: 23-28 |
20. | DeRubertis, F.R., and P.A. Craven. 1987. Relationship of bile salt stimulation of colonic epithelial phospholipid turnover and proliferative activity: role of protein kinase C. Prev. Med. 16: 572-579 |
21. | DeRubertis, F.R., P.A. Craven, and R. Saito. 1984. Bile acid stimulation of colonic epithelium proliferation. J. Clin. Invest. 74: 1614-1624 |
22. | Einspahr, J.G., D.S. Alberts, S.M. Gapstur, R.M. Bostick, S.S. Emerson, and E.W. Gerner. 1997. Surrogate end-point biomarkers as measures of colon cancer risk and their use in cancer prevention trials. Cancer Epidemiol. Biomark. Prev. 6: 37-48 [Abstract]. |
23. | Erdman, S.H., H.D. Wu, L.J. Hixson, D.J. Ahnen, and E.W. Gerner. 1997. Assessment of mutations in Ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats. Mol. Carcinogenesis. 19: 137-144 |
24. | Fitzer, C.J., C.A. O'Brian, J.G. Guillem, and I.B. Weinstein. 1987. The regulation of protein kinase C by chenodeoxycholate, deoxycholate and several structurally related bile acids. Carcinogenesis. 8: 217-220 [Abstract]. |
25. | Freeman, H.J.. 1983. Lectin histochemistry of 1,2-dimethyhydrazine-induced rat colon neoplasia. J. Histochem. Cytochem. 31: 1241-1245 [Abstract]. |
26. | Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in-situ via specific labeling of nuclear DNA fragments. J. Cell Biol. 119: 493-501 [Abstract]. |
27. |
Goode, N.,
K. Hughes,
J. Woodgett, and
P. Parker.
1992.
Differential regulation of glycogen synthase kinase-3![]() |
28. |
Hall, P.A.,
P.J. Coates,
B. Ansari, and
D. Hopwood.
1994.
Regulation of cell
number in the mammalian gastrointestinal tract: the importance of apotosis.
J. Cell Sci.
107:
3569-3577
|
29. |
Hansbrough, J.R.,
D.M. Lublin,
K.A. Roth,
E.A. Birkenmeier, and
J.I. Gordon.
1991.
Expression of a liver fatty acid binding protein/human decay-accelerating factor/HLA-B44 chimeric gene in transgenic mice.
Am. J. Physiol.
260:
G929-G939
|
30. |
Hocevar, B.A., and
A.P. Fields.
1991.
Selective translocation of ![]() |
31. | Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 497 pp. |
32. | Hong, M.Y., W.C.L. Chang, R.S. Chapkin, and J.R. Lupton. 1997. Relationship among colonocyte proliferation, differentiation, and apoptosis as a function of diet and carcinogen. Nutr. Cancer. 28: 20-29 |
33. |
Ilyas, M.,
I. Tomlinson,
A. Rowan,
M. Pignatelli, and
W. Bodmer.
1997.
![]() |
34. | Jiang, Y.H., H.M. Aukema, L.A. Davidson, J.R. Lupton, and R.S. Chapkin. 1995. Localization of protein kinase C isozymes in rat colon. Cell Growth Differ. 6: 1381-1386 [Abstract]. |
35. | Jiang, Y.H., J.R. Lupton, W.C. Chang, C.A. Jolly, H.M. Aukema, and R.S. Chapkin. 1996. Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon. Carcinogenesis. 17: 1227-1233 [Abstract]. |
36. | Jiang, Y.H., J.R. Lupton, and R.S. Chapkin. 1997. Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes. Carcinogenesis. 18: 351-357 [Abstract]. |
37. | Kerr, J.F.K., G.C. Gobé, C.M. Winterford, and B.V. Harmon. 1995. Anatomical methods in cell death. Methods Cell Biol. 46: 1-27 |
38. |
Lee, H.,
J. Ghose-Dastidar,
S. Winawer, and
E. Friedman.
1993.
Signal transduction through extracellular signal-regulated kinase-like pp57 blocked in
differentiated cells having low protein kinase C![]() |
39. | Lin, H-C., A.V. Sotnikov, L. Fosdick, R.M. Rostnick, and W.C. Willett. 1996. Quantification of proliferating cell nuclear antigen in large intestinal crypt by computer-assisted image analysis. Cancer Epidemiol. Biomark. Prev. 5: 109-114 [Abstract]. |
40. | Magnuson, B.A., I. Carr, and R.P. Bird. 1993. Ability of aberrant crypt foci characteristics to predict colonic tumor incidence in rats fed cholic acid. Cancer Res. 53: 4499-4504 [Abstract]. |
41. | Maltzman, T., J. Wittington, L. Driggers, J. Stephens, and D. Ahnen. 1997. AOM-induced mouse colon tumors do not express full-length APC protein. Carcinogenesis. 18: 2435-2439 [Abstract]. |
42. | McLellan, E.A., and R.P. Bird. 1988. Aberrant crypts: potential preneoplastic lesions in the murine colon. Cancer Res. 48: 6187-6192 [Abstract]. |
43. | McLellan, E.A., A. Medline, and R.P. Bird. 1991. Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon. Carcinogenesis. 12: 2093-2098 [Abstract]. |
44. | Merritt, A.J., C.S. Potten, C.J. Kemp, J.A. Hickman, A. Balmain, D.P. Lane, and P.A. Hall. 1994. The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice. Cancer Res. 54: 614-617 [Abstract]. |
45. | Morotomi, M., J.G. Guillem, P. LoGerfo, and I.B. Weinstein. 1990. Production of diacylglycerol, an activator of protein kinase C, by human intestinal microflora. Cancer Res 50: 3595-3599 [Abstract]. |
46. | Morotomi, M., P. LoGerfo, and I.B. Weinstein. 1991. Fecal excretion, uptake and metabolism by colon mucosa of diacylglycerol in rats. Biochem. Biophys. Res. Commun. 181: 1028-1034 |
47. | Morotomi, M., Y. Sakaitani, M. Satou, T. Takahashi, A. Takagi, and M. Onoue. 1997. Effect of a high fat diet on AOM-induced aberrant crypt foci and fecal biochemistry and microbial activity in rats. Nutr. Cancer. 27: 84-91 |
48. |
Murray, N.R.,
G.P. Baumgardner,
D.J. Burns, and
A.P. Fields.
1993.
Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation.
J. Biol. Chem.
268:
15847-15853
|
49. |
Pennisi, E..
1998.
How a growth control path takes a wrong turn to cancer.
Science.
281:
1438-1441
|
50. | Pongracz, J., P. Clark, J.P. Neoptolemos, and J.M. Lord. 1995. Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids. Int. J. Cancer. 61: 35-39 |
51. |
Potten, C.S.,
J.W. Wilson, and
C. Booth.
1997.
Regulation and significance of
apoptosis in the stem cells of the gastrointestinal epithelium.
Stem Cells.
15:
82-93
|
52. | Pretlow, T.P., M.A. O'Riordan, T.G. Pretlow, and T.A. Stellato. 1992. Aberrant crypts in human colonic mucosa: putative preneoplastic lesions. Cell. Biochem. Suppl. 16G:55-62. |
53. | Pretlow, T.P., T.A. Brasitus, N.C. Fulton, C. Cheyer, and E.L. Kaplan. 1993. K-ras mutations in putative preneoplastic lesions in the human colon. J. Natl. Cancer Inst. 85: 2004-2007 [Abstract]. |
54. | Reddy, B.S.. 1975. Role of bile metabolites in colon carcinogenesis. Cancer. 36: 2401-2406 |
55. |
Reddy, B.S.,
B. Simi,
N. Patel,
C. Aliaga, and
C.V. Rao.
1996.
Effect of amount
and types of dietary fat on intestinal bacterial 7![]() |
56. | Risio, M., M. Lipkin, H. Newmark, K. Yang, F.P. Rossini, V.E. Steele, C.W. Boone, and G.J. Kelloff. 1996. Apoptosis, cell replication, and Western-style diet-induced tumorigenesis in mouse colon. Cancer Res 56: 4910-4916 [Abstract]. |
57. | Roncucci, I., D. Stamp, A. Medline, J.B. Cullen, and W.R. Bruce. 1991. Identification and quantification of aberrant crypt foci and microadenomas in the human colon. Hum. Pathol. 22: 287-294 |
58. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
59. | Sato, M., and D.J. Ahnen. 1992. Regional variability of coloncyte growth and differentiation in the rat. Anat. Rec. 233: 409-414 |
60. |
Sauma, S.,
Z. Yan,
S. Ohno, and
E. Friedman.
1996.
Protein kinase C![]() ![]() |
61. | Saxon, M.L., X. Zhao, and J.P. Black. 1994. Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. J. Cell Biol. 126: 747-763 [Abstract]. |
62. | Sheng, H., J. Shao, C.S. Williams, M.A. Pereira, M.M. Taketo, M. Oshima, A.B. Reynolds, M.K. Washington, R.N. DuBois, and R.D. Beauchamp. 1998. Nuclear translocation of beta-catenin in hereditary and carcinogen-induced intestinal adenomas. Carcinogenesis. 19: 543-549 [Abstract]. |
63. | Shivapurkar, N., L. Huang, B. Ruggeri, P.A. Swalsky, A. Bakker, S. Finkelstein, A. Frost, and S. Silverberg. 1997. K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients. Cancer Lett. 115: 39-46 |
64. | Shpitz, B., Y. Bornstein, Y. Mekori, R. Cohen, Z. Kaufman, M. Grankin, and J. Bernheim. 1997. Proliferating cell nuclear antigen as a marker of cell kinetics in aberrant crypt foci, hyperplastic polyps, adenomas, and adenocarcinomas of the human colon. Am. J. Surg. 174: 425-430 |
65. |
Simon, T.C.,
K.A. Roth, and
J.I. Gordon.
1993.
Use of transgenic mice to map
cis-acting elements in the liver fatty acid-binding protein gene (Fabpl) that
regulate its cell lineage-specific, differentiation-dependent, and spatial patterns of expression in the gut epithelium and in the liver acinus.
J. Biol.
Chem.
268:
18345-18358
|
66. | Smith, A.J., H.S. Stern, M. Penner, K. Hay, A. Mitri, B.V. Bapat, and A. Gallinger. 1994. Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons. Cancer Res. 54: 5527-5530 [Abstract]. |
67. |
Takayama, T.,
S. Katsuki,
Y. Takahashi,
M. Ohi,
S. Nojuri,
S. Sakamaki,
J. Kato,
K. Kogawa,
H. Miyake, and
Y. Niitsu.
1998.
Aberrant crypt foci of the
colon as precursors of adenoma and cancer.
N. Engl. J. Med.
339:
1277-1284
|
68. | Wali, R.K., C.L. Baum, M.J.G. Bolt, P.K. Dudeja, M.D. Sitrin, and T.A. Brasitus. 1991. Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine-induced rat colonic tumors. Biochim. Biophys. Acta. 1092: 119-123 |
69. |
Wali, R.K.,
B.P. Frawley,
S. Hartmann,
H.K. Roy,
S. Khare,
B.A. Scaglione-Sewell,
D.L. Earnest,
M.D. Sitrin,
T.A. Brasitus, and
M. Bissonnette.
1995.
Mechanism of action of chemoprotective ursodeoxycholate in the
azoxymethane model of rat colonic carcinogenesis: potential roles of protein
kinase C-![]() ![]() ![]() |
70. | Ward, N.E., and C.A. O'Brian. 1988. The bile acid analog fusidic can replace phosphatidylserine in the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate in vitro. Carcinogenesis. 8: 1451-1454 . |
71. | Weinstein, I.B.. 1990. The role of protein kinase C in growth control and the concept of carcinogenesis as a progressive disorder in signal transduction. Adv. Second Messenger Phosphoprotein Res. 24: 307-316 |
72. |
Wong, M.H.,
B. Rubinfeld, and
J.I. Gordon.
1998.
Effects of forced expression
of an NH2-terminal truncated ![]() |