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Molecular Oncology, Markers, Clinical Correlates |
The 3rd Department of Surgery [M. K., T. E., Y. I., M. F.] and Department of Pharmacology [Y. T., T. N., S. A., K. I.], Nihon University School of Medicine, Tokyo 173-8610, Japan
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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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Recently, DeGregori et al. (11) have shown the genes encoding S phase-acting proteins, including TS, to be induced by the E2F1 transcription factor. Moreover, cells overexpressing E2F1, in a study of human fibrosarcoma cell lines, were reportedly resistant to 5-FU, with an up-regulation of TS expression (12) . Thus, the possibility that high TS expression in tumors may be the result of E2F1 overexpression has been suggested. To date, however, the relationship between TS and E2F1 expressions has not actually been studied in surgical specimens of primary colon cancer.
In this study, we investigated the intratumoral expression of both the TS and E2F1 genes in 23 colon cancers using the TaqMan RT-PCR assay and compared the results obtained. We found that TS expression correlates closely with E2F1 expression in colon cancer specimens. Moreover, this correlation was observed in all tumors, regardless of clinical stage. We discuss herein the significance of these observations from the clinical perspective.
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MATERIALS AND METHODS |
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TaqMan RT-PCR Assay.
The TaqMan 5' nuclease fluorigenic quantitative PCR assay that we used
is a well-established method of analyzing gene expression in a wide
range of samples (14
, 15)
. Fifty microliters of reaction
mixture for RT-PCR were prepared in a single tube: 30 ng of the
extracted total RNA; 1x TaqMan EZ buffer [50 mM Bicine,
115 mM potassium acetate, 0.01 mM EDTA, 60
nM Passive Reference 1, 8% glycerol (pH 8.2)]; 3
mM MgCl2; 300 µM dATP,
dGTP, and dCTP; 600 µM dUTP; 0.2 µM forward
primer; 0.2 µM reverse primer; 0.1 µM
TaqMan probe; 5 units of rTth DNA Polymerase; and 0.5 unit of AmpErase
UNG (the enzymes and the buffer containing the passive reference were
from Perkin-Elmer Corp.). The conditions of one-step RT-PCR were: 2 min
at 50°C, 30 min at 60°C, 5 min at 95°C, and then 40 cycles of
amplification for 20 s at 95°C and 1 min at 62°C. Triplicate
PCR amplifications were carried out for each sample.
Primers and TaqMan Probes.
Primers and the TaqMan probe for TS and E2F1 were designed in
accordance with Perkin-Elmer Corp. guidelines. Primers and the TaqMan
probe for GAPDH (TaqMan GAPDH control reagent kit) were also purchased.
The probes were labeled with a reporter dye [FAM or JOE
(2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein)], situated at the
5' end of the oligonucleotide, and a quencher dye (TAMRA), located at
the 3' end. The sequences of primers and probes used were: TS-forward:
5'-CCAGAGATCGGGAGACATGG-3' (bases 744–763 of the TS coding sequence;
Ref. 16
); TS-reverse: 5'-TACGTGAGCAGGGCGTAGCT-3' (bases
789–809 of the TS coding sequence; Ref. 16
); TS probe:
5'-FAM-CCTCGGTGTGCCTTTCAACATCGC-TAMRA-3' (bases 765–788 of the
TS coding sequence; Ref. 16
); E2F1-forward:
5'-GAGGTGCTGAAGGTGCAGAAG-3' (bases 607–627 of the E2F1 coding
sequence; Ref. 17
); E2F1-reverse:
5'-TTGGCAATGAGCTGGATGC-3' (bases 662–680 of the E2F1 coding sequence;
Ref. 17
); E2F1 probe:
5'-FAM-CGCATCTATGACATCACCAACGTCCTTG-TAMRA-3' (bases 631–658 of
the E2F1 coding sequence; Ref. 17
); GAPDH-forward:
5'-GAAGGTGAAGGTCGGAGT-3' (bases 1457–1474of the GAPDH genomic
sequence; Ref. 18
); GAPDH-reverse:
5'-GAAGATGGTGATGGGATTTC-3' (bases 3403–3412 of the GAPDH genomic
sequence; Ref. 18
); GAPDH probe:
5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3' (bases 3374–3393 of the GAPDH
genomic sequence; Ref. 18
). AmpliTaq DNA polymerase
extended the primer and displaced the TaqMan probe through its 5'-3'
exonuclease activity. When the probe is intact the emission spectrum of
the reporter is suppressed by the quencher. The nuclease degradation of
the hybridization probe releases the reporter, resulting in increased
fluorescence emission. The use of a sequence detector (ABI Prism 7700)
allows measurement of the amplified product in direct proportion to the
increase in fluorescence emission, continuously, during the PCR
amplification. The amplification plot is examined early in the
reaction, at a point that represents the logarithmic phase of product
accumulation. The point representing the detection threshold of the
increase in the fluorescent signal associated with the exponential
growth of the PCR product for the sequence detector is defined as the
cycle threshold (CT). CT
values are predictive of the quantity of input target (Refs.
14
and 15
; i.e., when the PCR
conditions are the same, the larger the initial template concentration,
the lower the CT).
Statistical Analysis.
The standard curve was created automatically by the ABI Prism
7700 detection system by plotting the
CT against each input amount
(containing 100, 50, 10, 5, 1, or 0.1 ng) of control total RNA (total
starting RNA) supplied in TaqMan EZ RT-PCR Kit (Perkin-Elmer Corp.).
The coefficient of linear regression (r) for each standard
curve was calculated. When the CT
value of a sample was substituted into the formula for each standard
curve, the relative concentration of TS, E2F1, or
GAPDH could be calculated. To normalize for differences in the amount
of total RNA added to each reaction, GAPDH was selected as an
endogenous RNA control. The normalized concentration of TS
or E2F1, an arbitrary number that can be used to compare the
relative amounts of TS or E2F1 in different
samples, was determined by dividing the concentration of TS
or E2F1 by the concentration of GAPDH.
Data are presented as mean ± SD. Statistical comparisons between groups of samples were made by ANOVA with the Mann-Whitney test. Differences were considered to be statistically significant when the P was <0.05. Pearson’s correlation coefficient analysis was used to evaluate the relation between TS and E2F1 expression levels.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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presents the
CT values plotted versus
the input amount (total starting RNA) to produce standard curves for
TS, E2F1, and GAPDH quantitation. The
standard curves that were generated to span three logs showed
linearity over the entire quantitation range, and provided accurate
measurements over a very large range of starting target quantities.
Equations were derived from the lines of the standard curves. When the
CT value of a sample was substituted
into the formula for each standard curve, the relative concentration of
TS, E2F1,or GAPDH could be calculated.
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shows the clinicopathological factors
correlating with TS and E2F1 expression. The mean
levels of TS and E2F1 mRNAs in the colon tumors
of all 23 patients were 0.98 and 1.52, respectively. The levels of
TS or E2F1 expression for men tended to be higher
than those for women; the mean respective levels of TS and
E2F1 for 13 men were 1.32 and 2.11, versus 0.54
and 0.76 for the 10 women. The sex difference was significant
(P < 0.05) for TS expression, but not for
E2F1 expression. The median age of the 23 patients in this
study was 65 years. The mean TS mRNA level for those younger
than the median age was 1.04 versus 0.94 for those older
than 65 years, the mean E2F1 mRNA levels 2.30
versus 1.10. TS and E2F1 expressions
did not differ among stages; the mean TS mRNA levels for
stage II, III, and IV patients were 0.94, 0.87, and 1.16, respectively,
whereas the corresponding E2F1 mRNA levels were 1.88, 1.36,
and 1.30. Fig. 2
shows the significant
relationship between TS and E2F1 expressions
(r2
= 0.598, P <
0.001) in the 23 tumor samples. Interestingly, at each stage, the level
of TS expression correlated closely with that of
E2F1 expression: stages II
(r2
= 0.846, P <
0.001), III (r2
= 0.868, P = 0.002), and IV (r2
= 0.988,
P < 0.001).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As to clinicopathological variables, no significant relation was found
in the current study between the expression of E2F1 and
three important clinicopathological variables: age, sex, and staging
(Table 1)
. With the exception of sex, similar results were obtained for
TS expression. Previous studies found TS
expression to be associated with clinical stage in colon (5
, 6)
and gastric cancer (19)
. However, there were no
differences in TS expression among stages in this study.
These results may be explained as follows. First, the sample size was
small, and levels of TS and E2F1 expression were
low in most (Fig. 2)
. Second, we evaluated TS expression
using a new detection system, the TaqMan RT-PCR assay. There is a
possibility that results using this new system may not be totally
consistent with those using previous methods. Many samples will need to
be analyzed using the TaqMan RT-PCR assay, to allow comparison of the
results with those of previous studies (2
, 5
, 6)
.
This study is the first to compare TS and
E2F1 expressions in the same primary colon cancer specimen.
Surprisingly, a significant relationship between TS and
E2F1 expressions (r2
=
0.598, P < 0.001), as shown in Fig. 2
, was found
despite the number of patients being relatively small. Our results
reinforce previous studies indicating that E2F1 is a
transcription factor regulating TS expression (11
, 12)
. We also made several novel observations. As to clinical
stage, surprisingly, a high correlation between TS mRNA and
E2F1 mRNA expressions was observed in stages II
(r2
= 0.846, P <
0.001) and III (r2
= 0.868,
P = 0.002), and even in stage IV
(r2
= 0.988, P <
0.001), colon cancer. It is noteworthy that this correlation was
observed regardless of clinical stage, even in advanced tumors from
stage IV colon cancer patients. These results suggest that the
gene-regulatory pathway from E2F1 to TS may be
highly conserved during malignant progression.
Only four (two stage II, one stage III, and one stage IV) of the
23 tumors showed TS overexpression (Fig. 2)
, with increased
E2F1 expression, although it was difficult to determine a
cutoff level between high and low TS expression. These
results suggest that tumors with high TS expression in these
cases may be secondary to E2F1 overexpression. Similarly,
DeGregori et al. (11)
and Banerjee et
al. (12)
have shown that
E2F1-overexpressing cells had increased TS
levels, which is consistent with our results. Because elevated
TS mRNA in colon cancer correlates with a poor response to
5-FU treatment (5)
, tumors with a high level of
TS expression would be predicted to be 5-FU resistant. On
the other hand, Banerjee et al. (12)
have also
shown cells overexpressing E2F1 to be more sensitive to
etoposide and doxorubicin (i.e., topo II inhibitors) and
SN38 (the active metabolite of irinotecan; i.e., a topo I
inhibitor), despite being resistant to 5-FU. Therefore, tumors with a
high level of E2F1 expression, as in this study, may be more
sensitive to topo I and topo II inhibitors. The levels of both
E2F1 and TS mRNAs in tumors are thus potential
indicators of which anticancer agents are likely to be effective for
colon cancer patients. The ability to predict response and outcome
based on E2F1 and TS expression in the primary
tumor would provide useful information for many clinicians in planning
chemotherapy.
We speculate that one mechanism by which tumor cells increase
TS expression may be overexpression of E2F1, even
in primary colon cancers. Because many of the genes encoding S
phase-acting proteins (including DNA polymerase 
, proliferating cell
nuclear antigen, ribonucleotide reductase, and TS) are reportedly
induced by E2F1 (11)
, tumors with high
E2F1 expression, as demonstrated herein, may be predictive
of the overexpression of not only TS but also many other
genes participating in the progression of cells from the
G1 to the S phase of the cell cycle. Although the
ability of a tumor to overexpress TS may represent an
important protective mechanism in response to 5-FU, as has previously
been discussed (1
, 4
, 5)
, the possibility that many genes
associated with E2F1 contribute to the mechanism of 5-FU
resistance in tumors with a high level of TS expression
cannot be ruled out. Additional studies are needed to define the
molecular mechanism underlying the development of resistance to
chemotherapy in colon cancer patients.
In conclusion, we have demonstrated that the level of TS mRNA expression correlates closely with the level of E2F1 mRNA expression; that is, E2F1 regulation of TS expression was demonstrated in colon cancer specimens. These results suggest that the ability of a tumor to overexpress TS may be due to enhanced expression of E2F1. Although the number of patients was relatively small, our study provides new insights into the molecular mechanisms underlying the regulation of TS expression in colon cancers.
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FOOTNOTES |
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1 Supported in part by a Grant-in Aid for the
High-Tech Research Center from the Ministry of Education, Science,
Sports and Culture, Japan, awarded to Nihon University. 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology and 3rd Department of Surgery,
Nihon University School of Medicine, 30-1 Oyaguchi Kamimachi,
Itabashi-ku, Tokyo 173-8610, Japan. Phone: 81-3-3972-8111, ext. 2246.
Fax: 81-03-5995-5914. E-mail: yasuot{at}med.nihon-u.ac.jp
3 The abbreviations used are: TS, thymidylate
synthase; 5-FU, 5fluorouracil; RT-PCR, reverse transcription-PCR;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TAMRA,
6-carboxy-tetramethylrhodamine; FAM, 6-carboxyfluorescein.
Received 10/ 6/99; revised 4/ 3/00; accepted 4/10/00.
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represents the results of PCR
amplification of samples containing unknown quantities of target RNAs.
The three formulas for log ng of TS, E2F1, and GAPDH are: TS,
y = -3.81x +28.74
(r = 1.000); E2F1, y =
-3.31x +28.18 (r = 0.998); and
GAPDH, y = -3.94x +24.12
(r = 0.999).
, stage II
tumor; gray circle, stage III tumor; •, stage IV
tumor.