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Tumor Biology |
Department of Microbiology and Immunology, Kimmel Cancer Institute [L. Y. Y. F., V. T. N., K. H., P. N. M.] and Department of Pathology, Anatomy & Cell Biology [J. L. F.], Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Major regulatory events leading to mammalian cell proliferation and differentiation occur in the G1 phase of the cell cycle (19) . Recent advances in cell cycle and cancer research have shown that tumor cells typically have acquired damage to genes that regulate G1-S progression pathways (20) . One such pathway, comprising p16ink4a, the product of the CDKN2 gene, cyclin D1, Cdk4 (catalytic partner of cyclin D1), and Rb, the product of the Rb gene, has emerged as a critical target in oncogenesis (21, 22, 23) . The assembly and catalytic activity of cyclin D1-Cdk4/6 complexes are positively regulated by mitogenic growth factors and negatively regulated by Cdk inhibitors. The latter include four distinct members of the INK4 gene family: p16ink4a, p15ink4b, p18ink4c, and p19ink4d (24, 25, 26) . The role of cyclin D1-Cdk4/Cdk6 complexes is to trigger the phosphorylation of the Rb protein (27) , thereby canceling its growth-suppressive function and enabling cells to enter the S phase (22) . Abnormal regulation of these steps can lead to uncontrolled cell proliferation and tumorigenesis (19) .
Indeed, many human cancers, including ESCC, display abnormalities in this pathway. Cyclin D1 overexpression attributable to gene amplification has been reported in ESCC (28 , 29) , and antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cancer cells (30) and the proliferation of lung cancer cells (31) . These studies provide evidence that overexpression of cyclin D1 in certain tumor cells contributes to their abnormal growth and tumorigenicity. Loss of heterozygosity of the Rb (a tumor suppressor gene) locus was observed in about 40% of ESCC, but mutations or deletions of Rb have not been found (32) . Mutations in the CDKN2 tumor suppressor gene have been detected in up to 50% of primary ESCC (33, 34, 35, 36) , and inhibition of esophageal cancer proliferation has been demonstrated by adenoviral-mediated delivery of CDKN2 (37) . Finally, inactivation of p16ink4a in ESCC, observed by immunohistochemistry, was reported to be associated with frequent aberrant methylation of the CDKN2 gene (38) . On the other hand, a sequential increase in expression of cyclin D1 (39 , 40) was reported in progression from normal epithelia to preneoplastic lesions to papillomas, in esophagi collected at the end point from nutritionally complete rats exposed to multiple doses of NMBA. In addition, Jenkins et al. (41) showed that in cyclin D1 overexpressing transgenic mice (42) , NMBA treatment increased the severity of dysplasia in esophageal epithelia, a prominent precursor to cancer development. In summary, alterations to genes regulating the G1-S transition, leading to overexpression of cyclin D1 or underexpression of p16ink4a, are frequently observed in esophageal cancers.
The present study was designed to determine the timing of expression and the interrelationship among key proteins at the G1-S checkpoint, relative to cell proliferation, at the very early stages of esophageal tumor development in ZD rats exposed to a dose of NMBA. At 14 weeks, 88% of ZD rats developed esophageal tumors compared with 0% in ZS rats. In addition, esophageal tumors were consistently found at very early time points after carcinogen treatment. By 24 h after NMBA treatment, FHLs in suprabasal layers showed cell proliferation, as revealed by PCNA immunohistochemistry. Concurrently, increased expression of cyclin D1, Cdk4, and Rb was observed in corresponding FHLs in adjacent esophageal sections, whereas p16ink4a expression was reduced or absent. These data demonstrate a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and initiation of esophageal tumors.
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MATERIALS AND METHODS |
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Experimental Design.
This study used the cell proliferation-driven esophageal cancer model
(9)
to investigate mechanism(s) of esophageal
tumorigenesis in very early stages. Using immunohistochemical detection
of protein expression on adjacent esophageal sections, the spatial and
topographical distribution and localization of PCNA-positive cells was
correlated with the expression of G1-S
cell-cycle-related genes. Weanling male Sprague Dawley rats
(48.8 ± 3.9 g) were randomized into two dietary
groups. ZD rats were fed a deficient diet ad libitum, and
control animals were pair-fed a ZS diet to match the food consumption
of rats on the ZD diet. ZS rats were thus calorie-restricted
(5)
. All animals were given deionized water and weighed
weekly. After 5 weeks, each animal received an intragastric dose of
NMBA at 2 mg/kg body weight. At 0 h (before NMBA dosing), 24 h, 48 h, 1 week, 3 weeks, 7 weeks, and 10 weeks after NMBA
treatment, 5–8 animals/group were sacrificed. At 14 weeks, end point
tumor incidence analysis was performed on the remaining 14 ZS and 26 ZD
animals.
At each time point, whole esophagus was excised from each rat and opened longitudinally. A small portion of the uppermost esophagus was cut, fixed in buffered formalin for a few hours, and embedded in paraffin. Serial cross sections (4 µm) were cut, mounted on Superfrost/Plus glass slides (Fisher Scientific, Pittsburgh, PA), and air-dried overnight. The sections were stained with H&E or reserved for immunohistochemical studies. Esophageal epithelium was prepared from the remaining esophagus using a blade to strip off the connective tissue layer. Samples containing only the esophageal epithelia were snap-frozen in liquid nitrogen and stored at -80°C until protein preparation for Western blotting. Expression of cyclin D1, Cdk4, p16ink4a, and Rb in relation to that of PCNA was characterized in consecutive esophageal sections from ZD and ZS rats by immunohistochemistry and in some cases, by Western blotting.
Immunohistochemical Detection of p16ink4a, Rb, Cyclin
D1, and Cdk4.
After deparaffinization and rehydration in graded alcohols, the
sections were heated in citrate buffer [0.01 M (pH 6.0)]
for Rb and Cdk4 detection and in 1 mM EDTA (pH 8.0) for
cyclin D1 in a microwave oven (90°C-95°C; 3 x 5
min) before nonspecific binding sites were blocked with goat serum. The
antigen retrieval procedure was not done on sections for
p16ink4a staining (43)
. Sections
were incubated overnight at 37°C in a humidified chamber with
respective primary antibodies: mouse
anti-p16ink4a monoclonal antibody (Santa Cruz,
CA) at 1:300 dilution; mouse anti-Rb monoclonal antibody (PharMingen,
San Diego, CA) at 1:20 dilution; mouse anticyclin D1 monoclonal
antibody (Santa Cruz, CA) at 1:60 dilution; and goat anti-Cdk4
polyclonal antibody (Santa Cruz, CA) at 1:200 dilution. Incubation with
appropriate biotinylated secondary antibodies followed. Slides were
then incubated with streptavidin horseradish peroxidase; expression of
p16ink4a, Rb, cyclin D1, and Cdk4 was localized
by a final incubation with 3,3'-diaminobenzidine tetrahydrochloride and
a light hematoxylin counterstain. The cyclin D1 monoclonal antibody
used has no cross-reactivity with cyclin D2 or D3, and Cdk4 polyclonal
antibody has no cross-reactivity with Cdk6 or any other Cdks. These
antibodies recognize the rat versions of
p16ink4a, cyclin D1, Cdk4, and Rb faithfully.
Cell Proliferation Determination by PCNA Immunohistochemistry.
Monoclonal mouse anti-PCNA (Santa Cruz, CA) was used at 1:250 dilution,
followed by incubations with biotinylated goat antimouse antibody and
streptavidin horseradish peroxidase, as described above. PCNA was
localized by a final incubation with
3-amino-9-ethylcarbazole-substrate-chromogen system (Dako Corp.,
Carpinteria, CA) and a light hematoxylin counterstain. Cells with red
reaction product in the nucleus were considered positive for the
presence of PCNA. PCNA analysis has the potential to identify cell
cycle subpopulations (G1, S,
G2, M; Ref. 44
): dark-staining
nuclei represent S-phase cells, light-staining nuclei represent
G1-S and G2 cells, cells with cytoplasmic
staining usually depict mitoses, and nonstaining nuclei represent
quiescent (G0) cells. Preliminary analysis showed
a good correlation between S-phase cells measured with PCNA or
5-bromo-2'-deoxyuridine (data not shown).
In this study, the spatial and temporal distribution of PCNA-stained nuclei (S phase, G1-S/G2) in esophageal epithelia were assessed. In the scoring of labeled cells in the S phase and G1-S/G2, dark-staining and light-staining nuclei of the cross section of an entire esophagus were counted by light microscopy. LI was calculated by dividing the number of respective labeled cells by the total number of cells, and the result was expressed as a percentage.
Protein Extraction and Western Blotting.
Esophageal epithelia were homogenized in a buffer containing 10
mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 100
µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM benzamidine,
7 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl
fluoride. Debris was removed by centrifugation at 16,000 x g for 20 min. The protein concentration in the
lysates was measured using a Bradford protein assay kit (Bio-Rad,
Hercules, CA). Proteins (100 µg) were separated by 7.5% (for
Rb), 12% (for cyclin D1 and Cdk4), and 14% (for
p16ink4a) SDS-PAGE and transferred onto
Immobilon-P membranes (Millipore, Bedford, MA). After transfer,
membranes were stained with Ponceau S (Sigma, St. Louis, MO) to test
for equal loading of the samples and washed three times with 1x
PBS-0.1% Tween 20. Membranes were individually probed with goat
polyclonal antibody against Cdk4 or mouse monoclonal antibodies
against cyclin D1, p16ink4a, or Rb after they
were treated with blocking solution of 2% BSA. Molt-4 cell lysate,
which overexpresses Rb, was used as a positive control for the
detection of Rb. After antibody binding, membranes were incubated with
appropriate horseradish peroxidase conjugate (Pierce, Rockford, IL).
All incubations and washes were performed in PBS. Immunodetection was
performed using the enhanced chemiluminescence method for Western
blotting detection (Pierce).
Tumor Analysis and Zinc Determination.
At sacrifice, the animals were anesthetized with isoflurane (Ohmeda
Inc., Madison, WI), blood was collected from the retro-orbital
venous plexus of each animal, and serum was prepared for zinc analysis
by atomic absorption spectroscopy (5)
. Esophageal tumors
>1 mm in diameter were mapped and counted.
Statistical Analysis.
Data on cell proliferation were analyzed by one-way ANOVA using the SAS
statistical computer program (45)
. Tumor incidence
differences were analyzed by Fisher’s exact test, two-tailed
(46)
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RESULTS |
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Esophageal Cell Proliferation Determined by Quantitative PCNA
Immunohistochemistry
The three separate indicators of epithelial cell proliferation in
the esophagus, as measured by PCNA immunohistochemistry, are:
(a) the number of labeled cells (S phase;
G1-S/G2 cells) per cross
section of an esophagus averaged statistically for the group of rats at
the same time point; (b) the total number of cells, both
labeled and unlabeled, for the group; and (c) LI, the
percentage of labeled cells for the group. Fig. 1
shows that at all time points, ZD rats had considerably higher LIs for
S-phase, and S-phase and
G1-S/G2 cells than ZS
animals. In addition, ZD esophagi had substantially higher numbers of
labeled cells for both the S phase and
G1-S/G2 (results not
shown), and total numbers of cells than ZS esophagi (Fig. 1
, legend).
These results affirm that dietary zinc deficiency induces sustained
increased esophageal cell proliferation in rodents (5
, 7
, 9)
. With the already high level of cell proliferation induced by
dietary zinc deficiency (0 h), stimulus by NMBA had no effect on
esophageal cell proliferation in ZD rats, whereas in ZS animals, NMBA
transiently increased the LI of both S-phase (P < 0.004), S-phase and
G1-S/G2 cells
(P < 0.001) at 48 h; these levels
returned to threshold levels (0 h) after 1 week (Fig. 1
).
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. By 14 weeks, 23 of 26 (88%) ZD rats had esophageal tumors, with a
multiplicity of 2.8 ± 1.9 tumors/rat. Interestingly,
grossly visible tumors were consistently evident at early time points
after NMBA treatment in ZD rats, although the limited number of animals
precluded statistically significant differences from ZS animals. The
tumors were mostly exophytic papillomas and occurred more frequently in
the upper two-thirds of the esophagus. The small papillomas observed at
48 h after NMBA dosing are interpreted as the effects of the
previous 5 weeks of feeding the ZD diet alone because we (5
, 9)
, as well as others (6)
, have reported that rats
fed a ZD diet can develop esophageal papillomas in the absence NMBA
treatment. Nevertheless, our present findings underscore that in the
presence of sustained esophageal cell proliferation induced by zinc
deficiency, a single dose of NMBA substantially shortens the latency
period of esophageal tumor development in these animals.
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Histological examination of ZS esophagi collected at 0 h
typically showed a single layer of basal cells with an overlying
stratum two to four cells thick covered by a thin keratinous layer (not
shown). Twenty-four h after treatment with a single dose of NMBA, there
was a patchy and mild increase in hyperplasia, which became more
noticeable at 48 h (results not shown; Ref. 48
). At 1 week, the
esophageal basal cell layer still exhibited some mild folding and was
slightly thickened (Fig. 2
A). PCNA immunohistochemistry demonstrated that darkly
stained S-phase cells were found mostly in the basal cell layer (Fig. 2
B). Although there was variation among animals in the
extent of esophageal hyperplasia after NMBA dosing, ZS esophagi
displayed no abnormal microscopic pathology throughout the 14-week
experimental period. However, an increased rate of apoptosis was
observed in these pair-fed ZS
esophagi.4
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). Corresponding lesions in consecutive
esophageal sections contained clusters of darkly stained PCNA-positive
nuclei or S-phase cells (Fig. 2D
), an observation
that documents the proliferative activity of such cells in suprabasal
layers. FHLs are considered to be neoplastic precursors in rodent
esophageal and skin carcinogenesis (49
, 50)
.
At 24 h after NMBA dosing, there was an expansion in the size of
FHLs (Fig. 2E
, rat 56 and Fig. 3A
, rat 59). Again, corresponding FHLs in their respective
consecutive sections demonstrated the presence of PCNA-positive nuclei
(Fig. 2F
and Fig. 3B
), a result
denoting the proliferative activity in these expanded focal lesions.
More importantly, concurrent overexpression of cyclin D1 (Fig. 3C
) and Cdk4 (Fig. 3D
) were found in
corresponding expanded lesions in adjacent tissue sections. These
lesions showed very reduced or absent p16ink4a
expression, but an overexpression of Rb (results not shown). Together,
these findings revealed that deregulation of the
p16ink4a-cyclin D1/Cdk4-pRb pathway, a critical
target for oncogenesis, was a very early event in the cell
proliferation-driven, esophageal carcinogenesis in ZD rats, occurring
as early as 24 h after NMBA dosing.
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), which correlates well with the pattern of PCNA-stained
nuclei in a corresponding lesion of an adjacent esophageal section
(Fig. 2H
).
Expression and Localization of Cell Cycle Proteins in Esophageal
Tumor Progression
p16ink4a Expression.
In agreement with accepted criteria of p16ink4a
staining (43)
, only distinct nuclear immunoreactivity was
considered a sign of positivity, and positively
p16ink4a-stained cells in the submucosal layer
served as an internal control. At all time points, esophagi from ZS
rats showed strong nuclear staining for p16ink4a
predominantly in the basal and immediate suprabasal cell layers, with
the percentage of positively stained cells (per cross section of an
esophagus) ranging from 10 to 35%. Fig. 4 and C
depicts examples of
p16ink4a staining in ZS rat esophagus at 24 h and 14 weeks, respectively. In contrast, the highly proliferative ZD
esophageal epithelia typically showed absent or very reduced staining
for p16ink4a (Fig. 4E
) at all time
points. In addition, FHLs and dysplastic areas demonstrated mostly
negative p16ink4a immunostaining (data not
shown), as did esophageal papillomas (Fig. 4G
).
Also, small areas of multiple or single cells positive for
p16ink4a were found within large areas lacking
staining (data not shown). In summary, 56% or 14 of 25 of the ZD
esophagi showed lack of p16ink4a staining at the
14-week end point. Immunoblotting analysis confirmed the strong
expression of p16ink4a in ZS esophagi (Fig. 5
A, Lanes 6–9) but absent or reduced expression in
ZD rats at various time points (Fig. 5A
, Lanes
1–5).
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) and 48 h, ZS esophagi showed increased Rb staining
in basal cells, an effect that reverted to mostly suprabasal cell
staining at later time points (Fig. 4
D). On the other hand,
there was a marked increase in the number of basal cells displaying
nuclear staining for Rb in ZD esophagi at 24 (Fig. 4
F) and
48 h, a change that was still evident at the end point (Fig. 4H
). FHLs and tumor areas in ZD esophagi also exhibited
increased staining for Rb (data not shown). Importantly, there was an
inverse relationship between Rb and p16ink4a
staining in adjacent esophageal sections, e.g.,
hyperplastic, FHLs, or tumor areas that showed lack of
p16ink4a expression always displayed strong
nuclear staining for Rb.
The Rb antibody recognizes the underphosphorylated (pRb),
phosphorylated, and hyperphosphorylated (ppRb) forms of Rb.
Thus, immunoblotting analysis was performed to examine Rb expression in
ZD and ZS esophagi. Based on equal loading of total cellular proteins,
the levels of Rb in ZD esophagi were increased (Fig. 5
B,
Lanes 2–9), as compared with ZS esophageal epithelium (Fig. 5
B, Lane 1). In addition, ZD esophagi showed
increased expression of the hyperphosphorylated forms of Rb.
Cyclin D1 and Cdk4 Expression.
ZS esophagi exhibited weak cyclin D1 and Cdk4 nuclear immunoreactivity
in 10–15% of the basal cells at 0 h. In response to NMBA dosing,
transient overexpression of both proteins was apparent at 24 and
48 h (data not shown). In contrast, ZD esophagi showed moderate to
strong nuclear staining in basal and immediate suprabasal layers at
0 h and in FHLs (data not shown). At 24 h, overexpression of
both proteins was detected in basal and immediate suprabasal layers, as
well as in hyperplastic areas and FHLs (Fig. 3 and D
for cyclin D1 and Cdk4, respectively). Importantly,
overexpression of both proteins in ZD esophagi persisted and was found
in the hyperplastic, dysplastic, and tumor areas at all later time
points. Also, analyses performed on adjacent esophageal sections
demonstrated that the staining patterns of cyclin D1, Cdk4, and PCNA
were correlated well with each other at all time points; an example is
provided in Fig. 3
. Immunoblotting results for cyclin D1 and Cdk4 (Fig. 5 and D
) analysis in ZD and ZS animals are
consistent with those obtained using immunohistochemistry.
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DISCUSSION |
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-difluoromethylornithine (47)
. Zinc-replenished and
-difluoromethylornithine-treated deficient animals exhibited
substantially reduced tumor incidence (51
, 47)
.
To the best of our knowledge, the present study is the first report
that investigates biological changes in very early stages of esophageal
tumorigenesis. Two significant findings emerge from this study.
Firstly, small pre-existing FHLs showing proliferation activity of
suprabasal cells were detected in the highly proliferative esophagi
after 5 weeks of zinc deficiency (Fig. 2A). These
small tumor precursors also overexpressed cyclin D1 and Cdk4 (data not
shown). Twenty-four h after NMBA dosing, the FHLs were larger with an
increased number of PCNA-positive S-phase cells (Fig. 3 and B
), and they exhibited overexpression of cyclin D1 and
Cdk4 in adjacent tissue sections (Fig. 3 and D
). Concurrent overexpression of these two proteins was
consistently detected in hyperplastic and dysplastic cells, in FHLs,
and in the papillomas present in ZD esophagi collected at later time
points. These results provide evidence that overexpression of cyclin D1
and Cdk4 is associated with esophageal tumor initiation.
Secondly, markedly reduced or absent staining for
p16ink4a, an inhibitor of Cdk4-mediated
phosphorylation of Rb, was seen in the proliferative ZD esophagi at all
time points. Loss of functional p16ink4a leads to
deregulated activity of Cdk4 and Cdk6, an effect that causes the loss
of growth control owing to persistent Rb phosphorylation
(24, 25, 26)
. In this regard, ZD esophagus showed a
substantial increase in the number of basal cells immunoreactive for Rb
at 24 (Fig. 4F
) and 48 h after NMBA dosing as compared
with 0 h. Immunoblotting analysis showed increased expression of
the hyperphosphorylated form of Rb in ZD esophagi at various time
points after NMBA administration (Fig. 5B
), a result that
indicates the release of cells from Rb-mediated growth inhibition, and
the entry of quiescent cells into the S phase. In contrast, ZS
esophagi, which showed sustained reduced cell proliferation
(5)
, exhibited strong nuclear staining for
p16ink4a in basal and suprabasal layers at all
time points (Fig. 4 and C
; Fig. 5A
). In summary, our data demonstrate that
increased cell proliferation induced by zinc deficiency is associated
with deregulation of the p16ink4a-cyclin
D1/Cdk4-Rb regulatory pathway. This effect is enhanced on
administration of a single dose of NMBA, unleashing a cascade of
genetic events that lead to esophageal tumor development in ZD rats.
Overexpression of cyclin D1 has been reported in preneoplastic lesions
and tumors in esophagi collected at end point from nutritionally
complete rats treated with multiple doses of NMBA, a finding that
suggests such overexpression occurs relatively early in esophageal
carcinogenesis (39
, 40)
. More recently (41)
,
cyclin D1 overexpression in transgenic mice, in combination with NMBA,
was shown to increase the severity of esophageal squamous dysplasia, a
prominent precursor to cancer. In human ESCC, Shamma et al.
(52)
demonstrated a strong correlation between
overexpression of cyclin D1 and the PCNA-determined cell proliferation
index, suggesting that cyclin D1 plays a major role and is closely
related to abnormal cell proliferation in esophageal cancer. In
addition, concurrent overexpression of cyclin D1 and Cdk4 has been
reported in colon tumorigenesis of both humans and rodents (53
, 54)
, and it is associated with increased proliferative activity
in preneoplastic cells. These and other studies indicate that abnormal
increases in the level and/or activity of cyclin D1 and Cdk4 are common
events in tumorigenesis. Because increased cellular proliferation is a
hallmark of cancer cells, and PCNA expression (a cofactor of DNA
polymerase-
) shows a strong correlation with the proliferative
activity of the cell (44)
, our data, which show a
consistent correlation between the spatial and temporal distribution of
PCNA-positive cells and that of cyclin D1 and Cdk4 coexpression in
tumor precursors, thus provide new insights into the interaction of
these molecules in the very early stages of esophageal carcinogenesis
in ZD rats.
Direct evidence that p16ink4a can inhibit cell growth (55) was demonstrated in p16ink4a-deficient mice (56) , which developed spontaneous tumors at an early stage and were highly sensitive to carcinogens. In this study, ZD esophagus that exhibited unrestrained cell proliferation throughout the experimental period showed reduced/absent p16ink4a expression. Also, an inverse relationship was observed between the expression of p16ink4a and Rb, suggesting that the Rb expressed in ZD esophagus may be phosphorylated forms that are permissive for proliferation. Immunoblotting results were consistent with this interpretation. However, the genetic basis for the increased or absent expression of these cell cycle proteins in ZD esophagi awaits further investigation.
In conclusion, the present study showed that in this in vivo model for esophageal cancer with a uniquely rapid tumor initiation, the appearance of the various proliferative and oncogenic changes can be precisely delineated in relation to time of application of the carcinogenic stimulus. In addition, we have observed that the highly proliferative esophagi from ZD rats had altered expression profiles for the genes that control G1-S progression. Most importantly, this study demonstrates an association between the deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and initiation of esophageal tumors by NMBA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants 97B115-REV and 99B045-REN
(to L. Y. Y. F.) from the American Institute for Cancer Research,
and by Cancer Center Grant 56336 from National Cancer Institute,
NIH. 
2 To whom requests for reprints should be
addressed, at Department of Microbiology and Immunology, Kimmel Cancer
Institute, Thomas Jefferson University, 1020 Locust Street,
Philadelphia, PA 19107. Phone: (215) 503-4763; Fax: (215) 923-7144;
E-mail: L_Fong{at}hendrix.JCI.TJU.EDU
3 The abbreviations used are: NMBA,
N-nitrosomethylbenzylamine; ZD, zinc-deficient; ZS,
zinc-sufficient; ESCC, esophageal squamous cell carcinoma; PCNA,
proliferating cell nuclear antigen; Cdk, cyclin-dependent
kinase; FHL, focal hyperplastic lesion; LI, labeling index; Rb,
retinoblastoma; pRb, under-phosphorylated form of Rb; ppRb,
hyperphosphorylated form of Rb.
Received 1/10/00. Accepted 6/19/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Difluoromethylornithine inhibits N-nitroso-methylbenzylamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on esophageal cell proliferation and apoptosis. Cancer Res., 58: 5380-5388, 1998.This article has been cited by other articles:
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  G. Jayachandran, J.-i. Sazaki, M. Nishizaki, K. Xu, L. Girard, J. D. Minna, J. A. Roth, and L. Ji Fragile Histidine Triad Mediated Tumor Suppression of Lung Cancer by Targeting Multiple Components of the Ras/Rho GTPase Molecular Switch Cancer Res., November 1, 2007; 67(21): 10379 - 10388. [Abstract] [Full Text] [PDF]
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C.-G. Liu, L. Zhang, Y. Jiang, D. Chatterjee, C. M. Croce, K. Huebner, and L. Y.Y. Fong Modulation of Gene Expression in Precancerous Rat Esophagus by Dietary Zinc Deficit and Replenishment Cancer Res., September 1, 2005; 65(17): 7790 - 7799. [Abstract] [Full Text] [PDF]
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L. Y. Y. Fong, R. Mancini, H. Nakagawa, A. K. Rustgi, and K. Huebner Combined Cyclin D1 Overexpression and Zinc Deficiency Disrupts Cell Cycle and Accelerates Mouse Forestomach Carcinogenesis Cancer Res., July 15, 2003; 63(14): 4244 - 4252. [Abstract] [Full Text] [PDF]
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L. Y. Y. Fong, V. T. Nguyen, and J. L. Farber Esophageal Cancer Prevention in Zinc-Deficient Rats: Rapid Induction of Apoptosis by Replenishing Zinc J Natl Cancer Inst, October 17, 2001; 93(20): 1525 - 1533. [Abstract] [Full Text] [PDF]
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F. Biramijamal, A. Allameh, P. Mirbod, H.-J. Groene, R. Koomagi, and M. Hollstein Unusual Profile and High Prevalence of p53 Mutations in Esophageal Squamous Cell Carcinomas from Northern Iran Cancer Res., April 1, 2001; 61(7): 3119 - 3123. [Abstract] [Full Text]
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 L. Y. Y. Fong, V. T. Nguyen, A. E. Pegg, and P. N. Magee {{alpha}}-Difluoromethylornithine Induction of Apoptosis: A Mechanism Which Reverses Pre-established Cell Proliferation and Cancer Initiation in Esophageal Carcinogenesis in Zinc-deficient Rats Cancer Epidemiol. Biomarkers Prev., March 1, 2001; 10(3): 191 - 199. [Abstract] [Full Text]
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