Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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Expression of a mutated cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to enhance proliferation within CF airways, and cells expressing a mutated CFTR have been shown to be less susceptible to apoptosis. Because the CFTR is expressed in the epithelial cells lining the gastrointestinal tract and all CF mouse models are characterized by gastrointestinal obstruction, we hypothesized that CFTR null mice would have increased epithelial cell proliferation and reduced apoptosis within the small intestine. The rate of intestinal epithelial cell migration from crypt to villus was increased in CFTR null mice relative to mice expressing the wild-type CFTR. This difference in migration could be explained by an increase in epithelial cell proliferation but not by a difference in apoptosis within the crypts of Lieberkühn. In addition, using two independent sets of CF cell lines, we found that epithelial cell susceptibility to apoptosis was unrelated to the presence of a functional CFTR. Thus increased proliferation but not alterations in apoptosis within epithelial cells might contribute to the pathophysiology of CF.
cystic fibrosis transmembrane conductance regulator; animal model; pathophysiology
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INTRODUCTION |
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THE CYSTIC FIBROSIS TRANSMEMBRANE conductance regulator (CFTR) is a chloride channel that is mutated in CF. Mutation of the CFTR results in a decrease in epithelial cell chloride permeability. Although the defect affects all exocrine tissues, in humans it manifests itself primarily in the airways and intestine. CF is characterized by reduced chloride and fluid secretion, which results in accumulation of mucus within the bronchioles and in meconium ileus in CF newborns and intestinal obstructions in older CF patients. In all mouse models of CF in which the CFTR has been deleted by insertional or deletional mutagenesis, intestinal pathophysiology is characterized by mucus accumulation in the crypts of Lieberkühn, goblet cell hyperplasia, and intestinal obstruction. In addition, the CFTR is expressed in the epithelial cells lining the gastrointestinal tract with a reduction in expression along the crypt-to-villus axis (7, 26).
Previously, Gottlieb and Dosanjh (9) showed that cultured
C127 mouse mammary epithelial cells stably transfected with the F508-mutated CFTR remained viable and were less susceptible to apoptosis induced by cycloheximide or etoposide than cells
expressing the wild-type CFTR. Programmed cell death, or
apoptosis, is a physiologically relevant process that occurs in
regenerating tissues such as the lung and intestine. Cell turnover is a
balance between proliferation and elimination, and cell proliferation
has been shown to be greater in airway epithelia and nasal polyps in CF patients than in tissue from non-CF patients (12, 15).
Although this phenomenon has traditionally been attributed to
inflammation, the possibility that the proliferative differences might
be due to differential expression of the CFTR has not been addressed.
Because all mouse models of CF (CFTR /
) develop intestinal
obstructions (10) and the CFTR is expressed in epithelial
cells lining the gastrointestinal tract, we hypothesized that the CFTR
/
mouse intestine would show evidence of altered apoptosis
and/or enhanced proliferation relative to mice expressing the wild-type CFTR. The hypothesis was tested in vivo using a knockout mouse model of
CF. Mice expressing a mutant CFTR were found to have a greater rate of
migration of epithelial cells up the crypt-villus axis within the small
intestine. This increased rate could be attributed to an increase in
the proliferative capacity of cells within the crypts of
Lieberkühn but not to differences in apoptosis. Because
these current results contradict the previous findings (9)
of differences in apoptosis between cells transfected with the
F508 CFTR and the wild-type CFTR, studies were extended to further
examine apoptosis in two independent cell culture models of CF.
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METHODS |
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Animal Model
A mouse model of CF generated by Dr. Beverly Koller (University of North Carolina) was used for all studies (3). In this model (cftrtm1Unc), the CFTR gene has been disrupted by gene targeting, and as with all mouse models of CF, the animals develop intestinal obstructions and typically die within weeks of birth. However, by replacing the drinking water with Colyte (Schwarz Pharma, Milwaukee, WI), a commercially available electrolyte solution containing 6% polyethylene glycol, the mice remain alive past 18 mo of age (10). Animals were maintained within the custom breeding colony at The Scripps Research Institute with all animals receiving Colyte in place of drinking water. At the time of weaning, the genotypes of all mice were determined by isolation of tail DNA and PCR amplification of the CFTR alleles as described previously by Koller et al. (14). All animal studies adhered to approved institutional animal protocols and American Association for Accreditation of Laboratory Animal Care guidelines.Epithelial Cell Migration
Bromodeoxyuridine (BrdU; Sigma Chemical, St. Louis, MO) was suspended in PBS at 20 mg/ml and administered via intraperitoneal injection at 150 µg/g body wt. The mice were killed by halothane overdose 2-48 h after BrdU injection. The small intestine was isolated and excised, rinsed in PBS, splayed open along its length, and rinsed twice to remove contents. The splayed intestine was fixed in Z-Fix (10% buffered formalin containing zinc; Anatech, Battle Creek, MI), coiled from proximal to distal end to form a "Swiss roll," dehydrated, and embedded in paraffin for tissue sectioning. Tissue sections of Swiss rolls allow for examination of the entire length of the intestine in a single section. BrdU incorporation on Swiss rolls was analyzed by immunohistochemistry. Briefly, 3-µm-thick tissue sections were cleared of paraffin and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in methanol for 15 min at room temperature. After pepsin digestion (0.4 mg/ml in 0.1 N HCl, 15 min at 37°C), tissue was treated with 2 N HCl for 30 min at 37°C and rinsed in 0.1 M sodium borate (pH 8.5). Tissue sections were incubated with anti-BrdU (Sigma Chemical) at 1:500 in 20% normal goat serum (90 min at 37°C). Incubation with biotinylated goat anti-mouse IgG (1:2,000 in PBS containing 1 mg/ml IgG-free BSA; Caltag, Burlingame, CA) for 60 min at room temperature was followed by enzymatic labeling with horseradish peroxidase-conjugated streptavidin (1:1,000 in PBS/BSA, 30 min at room temperature; Caltag). Diaminobenzidine (0.04%, Sigma Chemical) in 0.05 M Tris, pH 7.4, and 0.01% H2O2 were used for color development. Tissues were counterstained with Gill's hematoxylin (Fisher Scientific, Pittsburgh, PA), dehydrated through graded alcohols, and placed on a coverslip with Permount (Fisher Scientific).Epithelial cell migration was evaluated in CFTR +/ and CFTR
/
mice by measuring the distance between the base of the intestinal crypts at the most distal Paneth cell along the epithelial cells to
each BrdU-positive nuclei within the crypt-villus structure. The
investigator was blinded to animal identification and time after BrdU
injection. Through the use of a ×20 objective on a Nikon TE300
microscope and a Spot Diagnostics Instruments digital camera (Sterling
Heights, MI), 5 to 24 crypt-villus structures were captured for each
animal and analyzed using MetaMorph imaging software (Universal
Imaging, West Chester, PA). Three individual time courses for BrdU
migration were performed for a total of 20 animals (3 animals/genotype
at 2 and 24 h, 4 mice/genotype at 48 h). ANOVA was used to
assess whether there was a genotypic difference in migration rate, and
a two-tailed t-test was utilized to determine if there was a
difference in distance of migration at 48 h after BrdU injection.
Spontaneous and Induced Epithelial Cell Apoptosis
In situ terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) has been found to be unreliable as a method of detecting apoptosis in the gastrointestinal tract (21). Therefore, a method previously described by Potten (21) in which apoptosis is quantified by calculating the percentage of cells with condensed and/or fragmented nuclei within the first 10 cells following the Paneth cells in the crypts of Lieberkühn was used to quantify apoptosis in the small intestine of CFTR mice. Crypts were scored by an investigator blinded to the identity of animals that met the following criteria: 1) Paneth cells could be viewed, 2) the crypt lumen could be seen, and 3) a minimum of 10 nonoverlapping epithelial cells in the crypt column could be distinguished. Levels of spontaneous apoptosis were evaluated in control animals. Whole body cesium gamma irradiation was used to induce apoptosis in the small intestine of CFTR mice in an attempt to uncover genotypic differences in susceptibility to apoptosis. An initial dose-response curve using CFTR +/Intestinal Epithelial Cell Proliferation
Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) was evaluated within the crypts of control (nonirradiated, no BrdU) CFTR mice by an investigator blinded to animal identity. Tissues were acquired and processed as described above, and paraffin-embedded Swiss rolls were dewaxed and rehydrated through graded alcohols. Tissues were incubated with Target retrieval solution (DAKO, Carpinteria, CA) at 95°C for 20 min and at room temperature for an additional 20 min. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in methanol before incubation with anti-PCNA (clone PC10, DAKO) for 90 min at 37°C in 20% normal goat serum. After rinsing with PBS, tissue was incubated with biotinylated goat anti-mouse IgG (Caltag) suspended 1:2,000 in PBS containing 1 mg/ml IgG-free BSA. After 30 min incubation with horseradish peroxidase-conjugated streptavidin (Caltag), color was developed with SigmaFast diaminobenzidine (0.7 mg/ml diaminobenzidine in 0.06 M Tris buffer containing 0.2 mg/ml urea-H2O2), counterstained with hematoxylin, dehydrated through alcohols, and placed on a coverslip with Permount. Proliferation was evaluated by determining the average number of PCNA-positive cells present within the first 10 cells following the Paneth cells in a minimum of 25 crypts/animal. Twelve animals (17) were used to determine if there was a genotypic difference in epithelial cell proliferation. A two-tailed t-test was used to determine statistical significance.Cell Culture Models
C127 cell lines.
C127 mouse mammary epithelial cells stably transfected with the
wild-type CFTR or the CFTR with the deletion of phenylalanine at
position 508 (C127 F508) were obtained from Dr. G. M. Denning (Howard Hughes Medical Institute, University of Iowa College of Medicine) (4). These cells were grown in DMEM
(GIBCO BRL, Rockville, MD) supplemented with 10% FBS (HyClone, Logan,
UT), penicillin, streptomycin, and Fungizone (Sigma Chemical) and
passaged 1:5 two times per week. The cell lines were grown in the
presence of Geneticin (500 µg/ml; GIBCO BRL).
CFT1 cell lines.
CFT1 cell lines, which were derived from tracheal epithelial cells
isolated from a 24-year-old CF patient homozygous for the F508
mutation, were obtained from J. Yankaskas (University of North
Carolina). The parental cell line (CFT1-C2) was used to establish the
other three cell lines and has been immortalized with human papilloma
virus. CFT1-LCFSN cells overexpress the wild-type CFTR introduced by
retroviral vector (20). CFT1-
F508 cells express an
additional copy of the CFTR with the
F508 mutation, and the CFT1-LC3
is a retroviral vector control cell line overexpressing the
-galactosidase gene. These cells were grown in Ham's F-12 (Fisher
Scientific) supplemented with insulin (10 µg/ml), hydrocortisone (1 µM), endothelial cell growth supplement (ECGS; 3.75 µg/ml), epidermal growth factor (25 ng/ml), T3 (30 nM), transferrin (5 µg/ml), and cholera toxin (10 ng/ml) (all hormones were obtained from
Sigma Chemical). All cells, except the CFT1-C2 cells, were grown in the presence of Geneticin (500 µg/ml) and were passaged 1:3
two times per week.
Analyses of death
Membrane destabilization detected by YOPRO-1 uptake. Cells were plated at 10,000 cells/well into 96-well tissue culture plates. After 24 h, the medium was aspirated and replaced with 100 µl sterile PBS. Cells were ultraviolet (UV) irradiated at 0-8,000 J/m2 using a Fisher Scientific UV crosslinker, the PBS was replaced with fresh medium, and the cells were incubated at 37°C with 5% CO2. After 16 h, 25 µM YOPRO-1 (10 µl) was added to each well and incubated at 37°C for 10 min. YOPRO-1 is taken up by cells with an unstable membrane and binds to double-strand DNA; therefore, apoptotic or necrotic cells will be labeled with YOPRO-1 (13). With the use of a fluorescence plate reader (Bio-Tek Instruments, Winooski, VT), YOPRO-1 was found to be excited at 530 nm and emissions were detected at 480 nm. Cells were lysed with 10 µl of lysis buffer (125 mM EDTA and 2.5% Nonidet P-40) and incubated at 37°C with 5% CO2 for 30 min after which fluorescence was evaluated to determine the total number of cells per well. The ratio of the first fluorescence (apoptotic and necrotic cells) to the second fluorescence (total cells) was taken to evaluate percent death and plotted against UV dose.
Nuclear condensation and fragmentation. Cells were plated at 30,000 cells/chamber into four-well chamber slides. One cell type was plated per chamber slide with cells plated in the first and fourth chambers only. After allowing the cells to adhere and reach ~80% confluence, the medium was aspirated and replaced with 500 µl sterile PBS. One chamber (containing control cells) was covered with an opaque material to prevent irradiation while the chamber slide was irradiated at 2,000 J/m2. The position of the control and irradiated cells was randomized to ensure that the analysis was done under blinded conditions. The PBS was aspirated and replaced with fresh medium. Cells were incubated at 37°C with 5% CO2 for 6 h after which 37% formaldehyde was added to each chamber (final concentration: 3.75% formaldehyde). During fixation, the slides were centrifuged at 1,200 rpm for 15 min to ensure minimal loss of cells during the fixation procedure. The cells were fixed for an additional 15 min, rinsed with PBS, and allowed to air dry. At the time of analysis, 30 µM Hoechst no. 33342 in PBS were added to each chamber, slides were coverslipped, and a minimum of 300 cells/chamber were analyzed for nuclear condensation and fragmentation by fluorescence microscopy. The percentage of fragmented and condensed nuclei from three separate experiments was obtained with the experimenter blinded to cell type and irradiation dose.
CFTR Adenovirus Infection
To confirm that the mutated CFTR was responsible for the resistance to apoptosis, C127 ![]() |
RESULTS |
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Mouse Model of CF
Mouse models of CF are consistent in their susceptibility to gastrointestinal obstruction; therefore, studies were designed to determine whether a nonfunctional CFTR alters the kinetics of epithelial cell migration within the gastrointestinal tract and ultimately might underlie the pathophysiology within the intestine.The rate of migration of BrdU-labeled cells up the crypt-villus axis is
faster in CFTR /
mice than in their CFTR +/
littermates (P = 0.048). By 48 h, the distance that the
epithelial cells from the CFTR
/
mice have migrated is
significantly greater than the distance of migration in the CFTR +/
mouse intestine (P = 0.018) (Fig.
1). Because epithelial cell migration
from the crypts of Lieberkühn up the intestinal villi is the net
result of proliferation and apoptosis within the crypts, both
components were evaluated separately to see if either or both are
altered as a result of the expression of the mutant CFTR. An increase
in epithelial cell migration might occur if apoptosis is
reduced and/or proliferation within the crypts of Lieberkühn is
increased.
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Levels of spontaneous apoptosis are not different between CFTR
genotypes, with CFTR /
mice exhibiting 5.4 ± 4.6%
apoptosis within the crypts and CFTR +/
and CFTR +/+ mice
presenting 6 ± 2.5% and 2.1 ± 2.1% apoptosis,
respectively (Fig. 2). To determine whether the low levels of spontaneous apoptosis seen within the crypts masks a difference in susceptibility to apoptosis,
intestinal apoptosis was induced with whole body cesium gamma
irradiation. Irradiation significantly increased apoptosis in
all genotypes (P < 0.001) but did not uncover any
differences in susceptibility to apoptosis between genotypes
(Fig. 2). Epithelial cells at the villus tips do not show nuclear
fragmentation and condensation and, additionally, detection of
apoptosis in the gastrointestinal tract by TUNEL is highly
variable (19). Therefore, apoptosis at the villus
tips could not be examined in this study.
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To determine whether the difference in epithelial cell migration is the
result of differences in crypt cell proliferation, epithelial cells
within the crypts of Lieberkühn were evaluated for expression of
PCNA. A significantly greater percentage of epithelial cells from CFTR
/
mice (49.4 ± 6.6%) express PCNA than those from animals
expressing at least one copy of the functional gene (CFTR +/
,
36.8 ± 2.7%; P = 0.003) (Fig.
3).
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The results of the animal studies contradict the previous cell culture findings presented by Gottlieb and Dosanjh (9), in which cells expressing the mutated CFTR were more resistant to apoptosis than cells expressing the wild-type CFTR. Therefore experiments were designed to determine whether the cell culture results were misleading.
Mouse mammary epithelial cells expressing a mutated CFTR (C127 F508)
are more resistant to death induced by 1,000 or 2,000 J/m2
UV irradiation than cells expressing the wild-type CFTR (Fig. 4). With UV irradiation doses at or above
4,000 J/m2, a similar percentage of cell death was seen in
both cell types. This is consistent with the previous findings
(9) that C127
F508 cells are more resistant than C127
wild-type cells to induction of apoptosis by etoposide and
cycloheximide.
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To determine whether the difference in susceptibility to UV irradiation
seen in the C127 cell lines is a result of differences in expression of
a functional CFTR, C127 F508 cells were infected by an adenovirus
expressing the wild-type CFTR before induction of apoptosis.
Although cells overexpressing the mutant CFTR may have a low level of
CFTR activity (2), expression of the wild-type CFTR in the
C127
F508 cells did not increase the susceptibility of these cells
to death relative to cells infected with the
-galactosidase adenovirus or uninfected cells (Fig. 5).
These data suggest that expression of the functional CFTR does not make
C127 cells more susceptible to apoptosis than cells expressing
a mutated CFTR. Instead, the data suggest that the observed difference
in apoptosis is unrelated to the CFTR but instead may have been
a property acquired during the clonal selection process.
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To further assess whether the original data were misleading, a second
set of cell lines was tested to determine susceptibility to
apoptosis based on expression of a functional or mutated CFTR. In the CFT1 cell lines, at 16 h after UV irradiation, cell death increased in a dose-dependent manner to a similar extent across cell
lines (Fig. 6). Similar data were
obtained 6 h after UV irradiation using nuclear condensation and
fragmentation as markers of apoptosis (Fig.
7). In addition, UV irradiation of CFT1
cell lines resulted in a similar time course of DNA degradation (data
not shown) with DNA laddering present at 3 h after UV irradiation
in all four cell lines. Furthermore, cleavage of caspase 3 (PharMingen,
La Jolla, CA) and caspase 9 (New England Biolabs, Beverly, MA) after UV
irradiation was not different across cell lines (data not shown). Thus
the current data from the C127 and CFT1 cell lines suggest that the
CFTR does not play a role in apoptosis.
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DISCUSSION |
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Many CF patients and all mouse models of CF have a common
pathology in their susceptibility to gastrointestinal obstruction. In
the normal intestine, epithelial cells line the villi of the absorptive
surfaces. These cells are continuously regenerated by proliferation in
the crypts of Lieberkühn, differentiation, and migration up the
villi. At the tips of the villi, the cells undergo apoptosis
and are shed into the lumen (11, 24). In addition, Martin
et al. (18) have shown that both proliferation and
apoptosis occur in the stem cells within the crypts and that apoptosis within the crypts is likely to be an important part of the homeostatic process for tissue maintenance. Under normal conditions, stem cell proliferation and epithelial sloughing are in
equilibrium. As we have shown in the current study, epithelial cells in
CFTR /
mice migrate up the crypt-villus axis at a greater rate than
in animals expressing a functional CFTR. The epithelial cells from CFTR
/
mice migrated 27% farther in 48 h than those from the CFTR
+/
mice. This difference in rate is due to differences in
proliferation and not apoptosis within the crypts of
Lieberkühn with ~12% more cells with proliferative capacity in
the CFTR
/
mice than the CFTR +/
mice. Because villus length is
not different between the genotypes (data not shown), the increased
number of cells proliferating within the crypt and migrating up the
villi would lead to an increase in the mass of cells that are sloughed into the gastrointestinal tract.
The increased proliferative capacity within the CFTR-null mice may be
attributed to a cellular defect within the cells as a result of a
nonfunctional CFTR, although the mechanism by which this might happen
is unknown. Potentially, differences in intracellular pH might account
for the enhanced proliferation in CFTR /
cells. Cytosolic pH is
thought to be one of the factors that control the rate of proliferation
(8) with cytoplasmic alkalinization constituting a signal
for mitogenesis (1, 22, 27). In addition, cells with
higher intracellular pH are more likely to be in the S, G2,
or M phases of the cell cycle, whereas cells with lower pH are more
likely to be in G1 (19). Cells expressing a
mutated CFTR have been reported (5, 6) to have an elevated
cytosolic pH, and overexpression of the wild-type CFTR but not a mutant CFTR results in growth arrest (23). Thus a mutation in the
CFTR may result in an elevated intracellular pH, which stimulates the cell to undergo proliferation more readily and thus increase the rate
at which epithelial cells of the crypts of Lieberkühn proliferate and migrate up the intestinal villi.
All the mouse models of CF show evidence of inflammation and intestinal
obstructions (10); thus the possibility that the stimulus
to enhanced proliferation within the intestine of the CFTR /
mice
might be due to differences in inflammation cannot be ruled out.
Proliferation in nasal polyps (12, 16) and epithelium of
bronchial airways (15) has been shown to be increased in CF patients and has been attributed to airway inflammation. Chronic inflammation has been shown to be present in the CF intestine with
increased luminal content of immune cell constituents, secretory immunoglobulins, and cytokines (25). Therefore,
inflammation within the gastrointestinal tract potentially can be a
stimulus to enhanced proliferation.
Initial studies by Gottlieb and Dosanjh (9) showed
decreased susceptibility to apoptosis in cells expressing the
mutated CFTR relative to cells expressing the wild-type CFTR. In this initial study, cycloheximide and etoposide were used to induce apoptosis. The CFTR is an anion channel that when mutated does not allow for chloride to be transported across the plasma membrane. Because this is a transport problem, one concern with the use of any
drug or chemical to induce apoptosis is a difference in the
ability of the cells to transport the drug or chemical across the cell
membrane. To avoid this potential problem in the current studies, UV
irradiation was utilized as the apoptotic stimulus. Cells
expressing the mutated CFTR were more resistant to apoptosis induced by UV irradiation than cells expressing the wild-type CFTR,
which is in accordance with the findings of Gottlieb and Dosanjh
(9). However, one control that was lacking from the original set of experiments was the introduction of the wild-type CFTR
into these resistant cell lines to restore susceptibility to
apoptosis. In the current study, infection of C127 F08 cells with an adenovirus expressing the wild-type CFTR failed to restore susceptibility to apoptosis.
-galactosidase
adenovirus-infected cells were found to be more susceptible to death
than both the uninfected and infected CFTR cells. The reason
behind this increased propensity toward death is not known. The
-galactosidase construct used in these experiments was the
nuclear-targeted protein, which, because of its location of expression,
may make the cells more susceptible to the DNA-damaging effect of UV
irradiation. To confirm the findings from the C127 cells, an
independent set of cell lines was used to determine whether expression
of a mutated or wild-type CFTR results in differences in susceptibility
to apoptosis. Cells with a mutated CFTR were found to be no
different in their sensitivity to UV irradiation than cells expressing
the wild-type CFTR when evaluated for membrane destabilization detected
by the uptake of YOPRO-1, nuclear condensation and fragmentation, DNA
degradation, or caspase cleavage. Thus taken together the data suggest
that the CFTR does not play a role in a cell's susceptibility to apoptosis.
In the CFTR-null mice, epithelial cell kinetics within the gastrointestinal tract differ from mice expressing at least one copy of the functional CFTR. The altered proliferation within the crypts of Lieberkühn is not accompanied by changes in apoptosis but results in an increased rate of cell migration up the villus. The increased number of cells reaching the intestinal lumen might contribute to the viscosity of the luminal contents and increase the likelihood of intestinal blockage, one of the pathophysiological hallmarks of CF. If epithelial cell proliferation and turnover is increased in parallel in the CF lung and pancreas, this might contribute to the viscosity of the bronchial and pancreatic secretions and exacerbate the symptoms of CF.
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ACKNOWLEDGEMENTS |
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This research was supported by a postdoctoral fellowship from the Cystic Fibrosis Foundation (A. M. Gallagher), a grant from the Cystic Fibrosis Foundation (R. A. Gottlieb), and an award from the Pew Foundation Scholars Program in Biomedical Sciences (R. A. Gottlieb).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. Gottlieb, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MEM220, La Jolla, CA 92037 (E-mail: robbieg{at}scripps.edu).
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. Section 1734 solely to indicate this fact.
Received 5 January 2001; accepted in final form 4 May 2001.
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