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Regular Articles |
From the Experimental Chemotherapy Laboratory,*
the
Pathology Department,
and the Surgical
Department,
Regina Elena Cancer Institute,
Rome, Italy; the Peter MacCallum Cancer
Institute,§
Melbourne, Australia; the
Department of Microbiology and Immunology,¶
Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia,
Pennsylvania; and the Department of Biomedical Science,||
University of Modena, Modena, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of CRCs and have been associated with a poor
prognosis.5-8
However, available data are again
controversial.9,10
Thus, recent efforts have focused on
identifying new prognostic factors that accurately predict the clinical
outcome of CRC patients with the goal of providing a rational approach
for planning specific therapies. c-Myb is a 75- to 80-kd transcription factor11,12 expressed in immature and transformed hematopoietic cells. Hematopoiesis depends on c-Myb expression for the expansion of most cell lineages,13 whereas a decline in c-Myb expression can be a prelude to differentiation.12 c-Myb is also expressed in nonhematopoietic tissues of chickens, mice, and humans, with high levels of transcript and protein in the gastrointestinal tract.14-17 c-Myb expression increases from colonic normal mucosa through premalignant polyps up to colonic tumors.18 Cell lines derived from colon tumors also express c-Myb and seem to depend on c-Myb expression for growth.15,19,20
Deregulated expression of c-Myb inhibits growth arrest and accelerates apoptosis of myeloid cells, consistent with the involvement of c-Myb in the regulation of apoptotic process.21 Indeed, overexpression of c-Myb protects T lymphocytes from apoptosis induced by growth factor deprivation or dexamethasone treatment, and it is accompanied by enhanced Bcl-2 expression, dependent on activation of the Bcl-2 promoter.22 In colon cells, decreased c-Myb expression during the commitment to differentiation and apoptosis is accompanied by a decrease in Bcl-2 levels.23 Moreover, Bcl-2 expression is reduced and apoptosis is increased in colonic epithelium of embryos with a disrupted c-myb gene.24 The role of the anti-apoptotic Bcl-2 in CRC patients remains unclear. Several authors have found a lack of Bcl-2 expression in CRC25 and we recently found that only 30% of CRC patients studied were positive for Bcl-2 and that Bcl-2 expression predicted a better clinical outcome.26 Other authors found no prognostic significance of Bcl-2 expression in CRC.27,28 On the other hand, the anti-apoptotic protein Bcl-xL seems to play a major role in colorectal tumorigenesis and progression. A shift from expression of Bcl-2 to Bcl-xL has been demonstrated during progression of colorectal tumors,29 and significant Bcl-xL overexpression has been found in the majority of CRC patients when compared with the corresponding normal colonic tissue.30
Here we show that the expression of c-Myb correlates with that of Bcl-x and that the levels of these two proteins provide a reliable predictor of survival in CRC patients. Moreover, analysis of a human colon carcinoma cell line, transfected with the human c-myb cDNA, demonstrated that overexpression of c-Myb up-regulates Bcl-xL and increases tumorigenesis of colon carcinoma cells by inhibiting the apoptotic process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ninety-one patients surgically treated for CRC (70 colon and 21 rectum cancers) at the Regina Elena Cancer Institute between 1990 and 1998 have been included in this study. Multiple representative samples were collected from the tumor.
Tumor tissues were pathologically staged according to the Dukes’ classification as follows: 5 stage A (5.5%), 40 stage B (43.9%), 19 stage C (20.9%), and 27 stage D (29.7%). Tumors were categorized according to the World Health Organization classification, as well differentiated (G1; four cases, 4.4%), moderately-differentiated (G2; 65 cases, 71.4%), and poorly-differentiated (G3; 22 cases, 24.2%).
Flow Cytometry (FCM)
Frozen tissue specimens were mechanically desegregated and cell
suspensions were fixed at 4°C with a solution of acetone-methanol
(1:4, v/v). Indirect immunofluorescence was performed using the
anti-c-Myb primary monoclonal antibody (mAb) 1.1 (diluted
1:20).31
A fluorescein-isothiocyanate rabbit anti-mouse
(DAKO, Glostrup, Denmark) was used as secondary antibody and the
negative control was obtained by omitting the primary antibody.
Specificity for the c-Myb protein of the mAb 1.1 in FCM was tested
using resting and phytohemagglutinin-activated (Sigma Aldrich, Milano,
Italy) lymphocytes. Resting lymphocytes were negative for c-Myb protein
expression, whereas 50% of lymphocytes exposed to 2 µg/ml of
phytohemagglutinin for 72 hours were c-Myb-positive. Suspended cells
were incubated with antibodies, stained for DNA content determination
in a solution containing 75 kU/ml RNase A and 50 µg/ml propidium
iodide, and then analyzed using a FACScan cytofluorimeter (Becton
Dickinson, San Jose, CA). Linear propidium-iodide red fluorescence was
monitored through a LP 620 filter and logarithmic green fluorescence
from fluorescein isothiocyanate-labeled anti-c-Myb antibody was
measured through a BP 530/15 filter. Debris, damaged cells, and
doublets were excluded by gating on a forward and side-scatter dot
plot. At least 30,000 events per sample were stored as list mode data
and the monodimensional or bidimensional FCM distributions were
analyzed using Lysis II C32 Becton Dickinson software. Each tumor was
analyzed in parallel with normal mucosa of the same patients. In each
sample the percentage of c-Myb-positive cells was calculated by
subtracting a background of 2 to 5% from the positive cell
distribution. The cut-off of c-Myb and percent S-phase, calculated on
the median value of tumor specimens, were 58 and 20.1, respectively.
Primary Antibodies and Immunohistochemistry
Immunoreactivity for Bcl-x protein was detected by using affinity-purified polyclonal antibody S-18 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:100, raised against a peptide mapping at the N terminus of human bcl-x gene (identical to corresponding mouse sequence). Bcl-2 protein expression was evaluated using mAb 124 (Dakopatts, Copenhagen, Denmark), diluted 1:20. Immunohistochemical staining was performed on 5-µm thick sections on silane-treated (APES; Sigma, St. Louis, MO) slides from routinely fixed paraffin-embedded blocks. The deparaffinized and rehydrated sections were pretreated twice in a microwave oven at 750 W for 5 minutes in citrate buffer and incubated for 60 minutes at room temperature with primary antibodies. The reaction was visualized using a streptavidin-biotin immunoperoxidase system (LSAB 2 kit; DAKO, Milan, Italy) and 3-amino-9-ethyl carbazole solution (DAKO) as chromogenic substrate. Sections were then slightly counterstained with Mayer’s hematoxylin and mounted in aqueous mounting medium (Glycergel, DAKO). Sections without primary antibodies served as negative controls.
Bcl-x and Bcl-2 expression was determined semiquantitatively by
assessing the whole tumor section by two investigators (MM and SB), who
were blinded to the clinical data at the time of interpretation. Tumors
were classified, according to Sinicrope and colleagues32
with minor modifications, into four categories and assigned to one of
the four: 0, <5%; 1, 5 to 25%; 2, 25 to 50%; 3, >50%. The
staining intensity was scored as follows: 0, completely negative; 1+,
weak; 2+, moderate; and 3+, strong. For tumors showing heterogeneous
staining, the predominant pattern was taken into account for
scoring. The percentage of positive tumor cells and staining
intensity were multiplied to produce a weighted score for each case.
Cases with weighted scores 
1 were defined as negative; otherwise they
were defined as positive.
Cells and Transfection Experiments
Human colon carcinoma LoVo cells were grown in HAM’S-F12 (Bio-Whittaker, Walkersville, MD) supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, and antibiotics.
LoVo cells (1 x 106/200 µl) were transfected by electroporation (960 µF, 200 V, Gene Pulser; Bio-Rad, Milano, Italy) with the expression vector pcDNA3c-myb carrying the c-myb gene and the gene for the resistance to neomycin (G418). c-Myb full-length cDNA was obtained by digesting the plasmid LXSNc-myb22 with EcoRV, by end-blunting and by cloning it into pcDNA3 vector (CMV promoter). pcDNA3c-myb constructs were screened by sequencing and plasmids with c-myb gene in sense orientation were chosen for the experiments. Transfected LoVo cells were grown in G418-containing medium (0.8 mg/ml; Life Technologies, Inc., Gaithersburg, MD) to obtain individual clones. Two weeks later, clones were expanded and screened for c-Myb expression by Western blot analysis.
Western Blotting
Western blot and detection were performed as previously reported33 using 40 µg of total proteins loaded from each sample on denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anti-c-Myb mAb 1.131 was used at 1:1,000 dilution; anti-Bcl-x (S-18, Santa Cruz) polyclonal antibody and nonspecific purified IgG were used at 1:500 dilution. To check the amount of proteins transferred to nitrocellulose membrane, heat shock protein (HSP) was used as control and detected by an anti-human HSP 72/73 mAb (Ab-1, clone W27; Calbiochem, Cambridge, MA). The relative amounts of the transferred proteins were quantified by scanning the autoradiographic films with a gel densitometer scanner (Bio-Rad) and then normalized to the related HSP 72/73 amounts.
Northern Blotting
Total RNA was isolated by Trizol (Life Technologies, Inc.) following a standard protocol, and 30 µg was size-fractionated on denaturing formaldehyde agarose gel, blotted onto nylon filter, and hybridized with a 920-bp fragment cut from a human bcl-xL plasmid. The filter was exposed to an autoradiographic film for 3 days. After stripping (0.1% sodium dodecyl sulfate for 5 minutes at 100°C) the filter was reprobed with the glyceraldehyde-3-phosphate dehydrogenase probe to assess equivalence of RNA loading and transfer.
In Vitro and In Vivo Cell Growth
The growth of LoVo parental line, one neo clone (LoVo/neo) and three c-Myb-overexpressing clones (M11, M18, and M30) was assessed by seeding 8 x 104 cells in 60-mm Petri dishes (Nunc, Mascia Brunelli, Milano, Italy). Cell counts (Coulter Counter model ZM, Kontron) and viability (trypan blue dye exclusion) were determined daily, from day 1 to day 15 of culture.
CD-1 male nude (nu/nu) mice, 6 to 8 weeks old and 22 to 24 g in body weight, were purchased from Charles River Laboratories, Calco, Italy. All procedures involving animals and their care have been previously described and were in accordance with institutional guidelines in compliance with national and international laws and policies.34 Mice were injected intramuscularly with parental LoVo cells, LoVo/neo clone or each of c-Myb-overexpressing clones at 2 x 106 of tumor cells/mouse. Eight mice for each group were used and tumor mass was monitored daily.34
Terminal Deoxynucleotide Transferase-Mediated dUTP Nick-End Labeling (TUNEL)
Apoptotic activity on sections of the murine tumors was analyzed by TUNEL with a commercial kit (ApopDetek in situ apoptosis detection kit; Enzo Diagnostic, New York, NY), following the manufacturer’s instructions. Briefly, formalin-fixed, paraffin-embedded tissue sections (4 µm) were deparaffinized with xylene and stripped of proteins by incubation with 25 µg/ml proteinase K for 1 hour at room temperature before the TUNEL reaction. The incorporated Bio-16-dUTP was then stained with streptavidin-biotinylated horseradish peroxidase complex, AEC (3-amino-9-ethyl carbazole) and hematoxylin. Apoptotic cells had red nuclear staining. Omission of the terminal deoxynucleotide transferase enzyme in the TUNEL reaction was used as negative control and resulted in no staining.
Three mice for each group were sacrificed at day 50 after cell injection. The percentage of apoptotic cells was determined by light microscopic examination of sections at x400. For each slide, five high-power fields of nonnecrotic tumor areas were examined and 300 cells in each field were counted. For each tumor, four different sections were evaluated. Results were expressed as mean ± SD of the samples analyzed. The apoptotic index was calculated as number of apoptotic cells per 100 cells.
Statistical Analyses
Association between variables was tested by the Pearson’s chi-square test. The TUNEL results were expressed as mean ± SD of the samples analyzed. Student’s t-test was used to evaluate statistical significance. Disease-free survival (DFS) and overall survival (OS) curves were estimated by the Kaplan-Meier product-limit method; the log-rank test was used to assess differences between subgroups. A P value < 0.05 was considered significant. The BMDP statistical package program (BMDP, Los Angeles, CA) was used for analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-Myb levels were monitored in 91 colorectal tumor samples using FCM. This methodology permits the simultaneous evaluation of more than one biological parameter and might allow to accurately calculate the percentage of c-Myb-positive cells in heterogeneous cell populations. Thus, c-Myb expression, DNA ploidy, and percent S-phase were determined in each tumor sample and compared to those of the corresponding normal mucosa.
Two-parameter FCM analysis (c-Myb expression and DNA content) in four
representative samples of normal mucosa and primary tumor demonstrates
that c-Myb is differentially expressed in the same neoplastic sample
containing diploid and aneuploid cell populations (Figure 1)
. The expression of c-Myb was
relatively low in cells with a diploid DNA content (window 1), whereas
an increase in c-Myb positivity was observed in cells with aneuploid
DNA content (window 2). Because these results suggested that there was
a relationship between aneuploidy and c-Myb overexpression, the
association between c-Myb expression, ploidy, and percent S-phase cells
was evaluated in each tumor sample.
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shows the statistical evaluation
of the relationship between clinicopathological and biological
variables. Tumor cell differentiation and Dukes’ stage did not
correlate with c-Myb overexpression, whereas a significant relationship
between c-Myb overexpression and aneuploidy (P =
0.0003) was observed. On the other hand, expression of c-Myb, a cell
cycle-related gene, did not correlate with the percentage of cells in S
phase (P = 0.4534). In fact, the number of cases
with more or less 20.1% S-phase cells was similar regardless the
degree of c-Myb expression.
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. Although the expression of
c-Myb did not correlate with that of Bcl-2 (P =
0.2549), it was directly related to that of Bcl-x
(P < 0.0001). In fact the majority of tumors
expressing high c-Myb levels were positive for Bcl-x (87%, Figure 2a
), whereas tumors expressing low c-Myb
levels were negative for Bcl-x (93%, Figure 2b
). In addition,
tumors positive for Bcl-x staining were associated with aneuploid CRC
(P = 0.0115) and Dukes’ stage. Pearson
chi-square test revealed a borderline correlation between Bcl-x
positivity and tumor grading.
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To evaluate the prognostic impact of c-Myb and Bcl-x, patients who died within 30 days after surgery or those who died from noncancer-related causes were excluded from DFS and OS Kaplan-Meier analysis. This selection permitted us to analyze 81 cases, with a median follow-up of 50 months (range, 24 to 77 months), for DFS and OS, respectively. In this series of patients, 33 relapses were recorded and 30 patients died from cancer-related causes.
Kaplan-Meier curves, stratified for c-Myb and Bcl-x expression, showed
a significantly longer DFS and OS in patients with low c-Myb expression
(Figure 3, a and b)
and in patients with
Bcl-x-negative tumors (Figure 3, c and d)
. To better investigate the
predictive value of c-Myb and Bcl-x in patients not presenting distant
metastases, DFS and OS Kaplan-Meier analysis was performed excluding
the Dukes’ D patients. As shown in Figure 4
c-Myb low-expressing and Bcl-x-negative
patients had a significant increased DFS and OS benefit compared to
those with high c-Myb expression and Bcl-x-positive.
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, the results obtained in the
entire series of patients (Dukes’ A to D) provide statistically
significant evidence that the concomitant expression of c-Myb and Bcl-x
identifies a group of patients with a higher probability of relapsing
and dying. Similar results were observed excluding Dukes’ D patients
(Figure 5, c and d)
.
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To examine the potential relationship between c-Myb and
Bcl-xL in tumor cells, the human colon cancer
cell line LoVo was transfected with a full-length c-myb cDNA
driven by a constitutively active promoter. LoVo cells were selected as
low c-Myb-expressing cells from a panel of five colon carcinoma lines
(Colo205, HT29, LoVo, SW, DLD-1) (data not shown). Figure 6
shows different c-Myb protein levels in
the parental LoVo cells, LoVo/neo clone (the vector control) and in
some of the transfectants. Clones M11, M18, and M30, which show a 5- to
10-fold increase in c-Myb expression compared to the LoVo/neo clone,
were used for further experiments.
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shows Western blot analysis of Bcl-xL protein in
LoVo/neo control clone and three clones overexpressing c-Myb (M11, M18,
and M30). Compared to vector-transfected LoVo cells, c-Myb-transfected
cells show a twofold to threefold increase in
Bcl-xL protein. Bcl-xL
protein corresponds to the lower band (29 kd) in the top panel of the
figure because the top prominent 35 kd is a nonspecific band recognized
by a nonspecific rabbit IgG (middle panel). A twofold to threefold
increase in bcl-xL mRNA was also observed in
M11, M18, and M30 clones (Figure 7B)
. The LoVo parental cells showed
the same level of Bcl-xL expression observed in
the LoVo/neo clone (data not shown).
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c-Myb-overexpressing LoVo cells were analyzed for in
vitro and in vivo growth characteristics. The in
vitro growth curves of the c-Myb overexpressing M11, M18, and M30
transfectants were similar to those of the parental cells and the
LoVo/neo clone (Figure 8)
. Similar
results were obtained by performing the growth curves in medium
supplemented with 1% FCS (data not shown). Thus, overexpression of
c-Myb protein did not affect proliferation of LoVo cells. By contrast,
in vivo growth of c-Myb-overexpressing LoVo cells was
different (Figure 9A)
. The tumor mass of the c-Myb-overexpressing
clones was larger than control tumors, even at a late stage of tumor
growth; ie, 60 days after cell injection the weight of tumors
originating from LoVo and LoVo/neo cells was 1,200 mg compared to
the 3,000 mg from c-Myb-overexpressing clones. In addition, 75% of
mice injected with parental cells or with the LoVo/neo control clone
developed tumors, whereas tumors formed in all mice injected with the
M11, M18, or M30 c-Myb transfectants.
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shows TUNEL staining
in tumor section of LoVo/neo control clone (a) and one representative
c-Myb-overexpressing clone (b). Apoptosis was detected in tumors from
LoVo/neo cells but not in tumors from the M30 c-Myb-overexpressing
clone. In the LoVo/neo clone, the mean apoptotic index was 10.1 ±
2.02, whereas in the M30 c-Myb-overexpressing clone it was 2.36 ±
0.98 and this difference was significant (P <
0.0001). Similar results have been obtained with M11 and M18 clones
(data not shown). These results are consistent with the hypothesis that
the increased tumorigenicity observed in c-Myb-overexpressing clones is
closely correlated to decreased apoptosis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of colorectal malignancies is known to involve the interplay between genes that drive cell division and regulate cell cycle checkpoints.35 c-Myb protein is required for entry into the cell cycle and its persistent expression is required for cells to remain in the cell cycle.16 In CRC cells, this continual cell cycling might favor the accumulation of additional genetic alterations28 that, in turn, may enhance survival of tumor cells.36 On the other hand, in our model, the high level of c-Myb expression in tumor cells is not directly proportional to the fraction of S-phase cells. The results using tumor specimens are in agreement with the findings that no changes occur in the proliferation of c-Myb-overexpressing LoVo colon carcinoma cells. Thus, c-myb oncogene seems to be involved in the regulation of other aspects of cell behavior underlying colorectal tumorigenicity.
Although the stage of disease usually determines the prognosis of CRC
patients, in the 91 CRCs tested no relationship was found between
Dukes’ stage and c-Myb overexpression. We also found a direct
correlation between high c-Myb expression and Bcl-x-positive tumor
cells in 90% of the patients, a finding consistent with previous
studies suggesting that the apoptosis inhibitor
Bcl-xL protein plays a major role in colorectal
tumorigenesis and progression.29
Overexpression of
Bcl-xL as well as a shift from expression of the
anti-apoptotic Bcl-2 to Bcl-xL protein have been
detected during progression of colorectal tumors.29,30
Kaplan-Meier curves show that c-Myb and Bcl-x are predictors of poor
clinical outcome. Of particular clinical interest is that the
significant prognostic value of c-Myb and Bcl-x is also retained in the
Dukes’ A to C patients both for DFS and OS. Moreover, the concomitant
high expression of c-Myb and Bcl-x positivity identifies a group of
patients at higher risk, probably requiring more aggressive therapies.
The relationship between c-Myb and Bcl-x observed in tumor specimens may reflect the modulation of Bcl-xL by c-Myb. Indeed, three c-Myb-overexpressing clones showed increased levels of Bcl-xL protein and mRNA expression. Because several studies have recently shown that genes involved in inhibition of apoptosis can confer growth advantage and increased tumorigenicity,37,38 we studied the growth characteristics of three clones of LoVo cells overexpressing c-Myb and with up-regulation of Bcl-xL. In vitro, parental, and c-Myb-overexpressing LoVo cells were indistinguishable. In vivo, the behavior of c-Myb-overexpressing LoVo cells was markedly different from the parental line as indicated by the high tumorigenicity of c-Myb-transfected cells. However the degree of c-Myb expression does not seem to directly correlate with the expression of Bcl-xL and tumorigenicity. In fact, in transfectants, c-Myb overexpression is always associated with increased Bcl-xL expression and tumorigenicity, but the enhancement of these two parameters does not reflect the different c-Myb overexpression levels. The apparent discrepancy could be explained by the mechanism of negative autoregulation of c-Myb activity by homodimer formation as reported by Nomura and colleagues.39
The increase in tumorigenicity of c-Myb-overexpressing clones was closely related to the inhibition of apoptotic process as determined by TUNEL assay. A positive relationship between enhanced cell survival and tumorigenic phenotype has recently been suggested by our and other studies showing that breast and melanoma cells overexpressing Bcl-2 were more tumorigenic than the nontransfected counterpart.40,41 Similar conclusions were drawn from models of experimental tumorigenesis where reduced apoptosis marked a late stage of tumor development.20 Thus, the enhanced tumorigenic potential of c-Myb-overexpressing LoVo cells might be because of a reduced susceptibility to apoptosis.
In conclusion, our results demonstrate that overexpression of c-Myb is associated with enhanced Bcl-x protein levels in primary CRC and the co-expression of these two markers is a predictor of poor prognosis. The increased levels of Bcl-xL are probably the direct consequence of c-Myb overexpression as observed in LoVo cells transfected with c-Myb. Overexpression of c-Myb in these cells is associated with a lower frequency of apoptotic cells in tumor mass that likely contributes to the enhanced tumorigenicity of c-Myb-overexpressing LoVo cells.
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Acknowledgements |
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Footnotes |
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Supported by grants from Italian Association for Cancer Research (AIRC), Italy-USA Project on Therapy of Tumors (to G. Z.), NIH (to B. C.), in part by a Research Fellowship from the National Health and Medical Council of Australia (to R. G. R.); and by a fellowship from Italian Foundation for Cancer Research (FIRC) (to B. B. and S. B.).
Igea D’Agnano’s present address: Institute of Biomedical Technology, CNR, Rome, Italy.
Accepted for publication January 9, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) was set
to the negative control of each sample. Windows 1 and 2 indicate the
diploid and aneuploid cell populations, respectively.
), and
M11 (
), M18
(
), M30
(
)
c-Myb-overexpressing clones. B: Detection of apoptosis
(TUNEL) in LoVo/neo
(a) and M30
(b) tumor
cells grown in nude mice. Original magnification, x200.