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Tumor Biology |
Department of Surgery II, Osaka University Medical School, Osaka 565-0871, Japan [I. T., H. Y., M. S., M. O., S. N., Y. M., T. M., Y. T., I. S., H. S., M. M.]; Department of Surgery, Osaka National Hospital, Osaka 540-0006, Japan [N. K.]; Department of Pathology, School of Allied Health Science, Faculty of Medicine, Osaka University, Osaka 565-0871, Japan [N. M.]; and Department of Surgery, Kansai Rosai Hospital, Hyogo 660-0064, Japan [T. A., N. T.]
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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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The cell cycle is a complex process in which many molecules are involved. Central to this process are the CDKs3 and their catalytic partners, cyclins. They are negatively regulated by CDK inhibitors (e.g., p16, p21, and p27) and positively activated by CDK-activating kinase (3 , 4) . CDC25 phosphatase is a novel class of CDK activator. In mammalian cells, CDC25A, CDC25B, and CDC25C are three CDK-activating phosphatases that remove the inhibitory phosphates of threonine and tyrosine residues in ATP-binding sites of CDKs at different points of the cell cycle (5, 6, 7) . In the CDC25 family, CDC25A and CDC25B types appear to be potential oncogenes because they have been found to transform primary murine fibroblasts in cooperation with either mutated Ha-ras or loss of Rb1 (8) . In fact, overexpression of CDC25A and CDC25B has been demonstrated in non-Hodgkin’s lymphoma, human carcinomas of the breast and lung, and head and neck tumors (8, 9, 10, 11) . Dysregulation of cell cycle progression is one evident alteration in human malignancies (12 , 13) . Colorectal carcinomatous tissues overexpress CDK1 and CDK2, possibly overexpress CDK4, and overexpress cyclins D1 and E (14, 15, 16, 17) . The CDK inhibitor p21waf1/cip1 is reduced, methylation of the p16INK4 gene occurs in the promoter region, and p27Kip1 appears to be decreased in a subset of colorectal carcinomas (18, 19, 20, 21) .
Recent studies have demonstrated overexpression of CDC25A phosphatase in azoxymethane-induced murine colon cancer (22) , but the expression and biological significance of CDC25A and CDC25B in human colorectal carcinoma have not yet been elucidated. Of considerable interest is that CDC25 phosphatase, especially the CDC25B type, is associated with the malignant properties of some human carcinomas. For example, expression of CDC25B is a poor prognostic factor for breast cancer when assessed by in situ hybridization and CDC25B overexpression was associated with aggressive non-Hodgkin’s lymphoma (8 , 9) . Furthermore, transgenic mice that overexpress the CDC25B gene display enhanced sensitivity to the carcinogen 9,10-dimethyl-1,2-benzanthracene (23) or develop mammary gland hyperplasia (24) .
To investigate the role of CDC25B phosphatase in the progression of colorectal carcinoma, we examined its expression using immunohistochemistry in 181 primary human colorectal carcinomas and analyzed the correlation between prognosis and the level of CDC25B protein. Western blot analysis and RT-PCR were used to quantitate the expression levels of CDC25B protein and mRNA in 10 paired samples of colonic normal mucosa and carcinomas. In addition, we compared the expression of CDC25A and proliferation marker Ki-67 with that of CDC25B in a subset of specimens. The present findings indicate that CDC25B is a novel, independent prognostic marker for colorectal carcinoma.
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MATERIALS AND METHODS |
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Clinical Features.
The selected patients included 79 (44%) males and 102 (56%) females,
with a mean age at surgery of 60 ± 10 years (range,
40–86 years). The primary tumors were evenly distributed in the colon
and rectum and ranged in size from 0.7–13.0 cm (mean size,
4.8 ± 1.9 cm). The majority of tumors were
well-differentiated carcinomas (56%), followed by moderately
differentiated carcinomas (40%) and poorly differentiated carcinomas
(4%). Dukes’ staging included 35 (19.3%) Dukes’ stage A
patients, 61 (33.8%) Dukes’ stage B patients, 69 (38.2%)
Dukes’ stage C patients, and 16 (8.7%) Dukes’ stage D
patients.
Antibodies.
Mouse antihuman CDC25B mAb and its blocking peptide, which was used as
an immunogen (NH2-terminal of human CDC25B; amino
acids 109 -122), were obtained from Transduction Laboratories
(Lexington, KY). The positive control lysate from HeLa cells was also
obtained from Transduction Laboratories. Rabbit polyclonal antibodies
for CDC25B and CDC25A and their blocking peptides were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). The CDC25B polyclonal
antibody was raised against the COOH-terminal of murine CDC25B. The
mouse antihuman Ki-67 mAb was purchased from DAKO (Carpinteria, CA;
Ref. 21
).
Specificity of Antibodies in Immunohistochemistry.
Specificity of staining obtained with CDC25B antibodies and CDC25A
antibody was assessed first by an absorption test in which immunogens
were used to generate the antibodies. This test resulted in the
disappearance of staining. For negative control, nonimmunized mouse or
rabbit IgG (Vector Laboratories, Burlingame, CA) or PBS alone was used
as a substitute for the primary antibody to exclude possible false
positive responses from secondary antibody or from nonspecific binding
of IgG. These control samples showed no cell staining. Staining of
Ki-67 was performed as described previously (21)
, using
tonsil samples as a positive control.
H&E Staining and Immunohistochemistry.
Tissue sections (4-µm thick) were deparaffinized in xylene,
rehydrated, and stained with H&E. The specimens were histologically
diagnosed by two pathologists from the Department of Pathology, Osaka
University Medical School. For immunostaining, sections were mounted on
charged glass slides, boiled for antigen retrieval (21)
,
and then processed for immunohistochemistry on the TeckMate Horizon
automated staining system (DAKO, Glostrup, Denmark) using the
Vectastain ABC peroxidase kit (Vector Laboratories) as described
previously (25)
. In the primary antibody reaction, the
slides were incubated with appropriate antibodies for 1 h at room
temperature. The dilution of each antibody was as follows:
(a) CDC25A polyclonal antibody, 1:50; (b) CDC25B
mAb, 1:200; (c) CDC25B polyclonal antibody, 1:50; and
(d) Ki-67 mAb, 1:50.
Immunohistochemical Assessment.
All immunostained tissue sections were evaluated in a coded manner
without knowledge of the clinical and pathological parameters. For
assessment of CDC25B, both cytoplasmic and nuclear staining were
evaluated. For each section, five high-power fields were selected at
random, and at least 700 cells were evaluated. The results were
expressed as a percentage of positively stained cells. In addition, the
cytoplasmic staining intensity for CDC25B was evaluated as follows:
(a) weak, 1; (b) moderate, 2; or (c)
strong, 3. Carcinoma samples containing >75% immunoreactive cells
with strong staining intensity (intensity = 3) were
classified as high expressors of CDC25B, and the remaining samples were
classified as low expressors of CDC25B. Agreement in the
above-mentioned tissue evaluation between the two investigators (H. Y.
and I. T.) was 98%. In cases of disagreement, the two investigators
reached the final evaluation by consensus after reexamining the tissue
using a multihead microscope. Staining was repeated in 50% of cases to
check for possible technical errors, but similar results were obtained.
For assessment of CDC25A and Ki-67, cells with positive nuclear
staining were counted as described above.
Western Blot Analysis for CDC25B.
Approximately 100 mg of each sample were homogenized in 1 ml of lysis
buffer [50 mM Tris (pH 8.0), 150 mM NaCl, and
0.5% NP40] with protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin). The homogenate was centrifuged at 14,000 rpm for 20 min at
4°C. The resulting supernatant was collected, and total protein
concentration was determined using the Bradford protein assay (Bio-Rad,
Hercules, CA). Western blotting was performed as described
previously (26)
. Briefly, 100 µg of the total protein
were subjected to 10% PAGE, followed by electroblotting onto a
polyvinylidene difluoride membrane. After blocking in 5% skim milk,
the membrane was incubated with 1 µg/ml CDC25B antibody, followed by
incubation with the secondary antibody at a dilution of 1:3000. For
detection of the immunocomplex, the enhanced chemiluminescence Western
blot detection system (Amersham, Aylesbury, United Kingdom) was used.
RNA Extraction and RT-PCR Analysis.
Total RNA was extracted with a single-step method using Trizol reagent
(Life Technologies, Inc., Gaithersburg, MD), and cDNA was generated
using avian myeloblastosis virus reverse transcriptase (Promega,
Madison, WI). Briefly, 1 µg of RNA was incubated at 70°C for 5 min
and then placed on ice before the addition of reverse transcription
reaction reagents with Oligo(dT)15 Primer. Reverse
transcription was performed at 42°C for 90 min, followed by heating
at 95°C for 5 min.
Semiquantitative analysis for expression of CDC25B or CDC25A mRNA was performed by the multiplex RT-PCR technique, using PBGD (27 , 28) as the internal standard. To minimize the inter-PCR difference, PCR was performed with PBGD and CDC25A or CDC25B primers in identical tubes under unsaturated conditions, as described previously (25) . PCRs were performed in a total volume of 25 µl of reaction mixture containing 1 µl of cDNA template, 1x Perkin-Elmer PCR buffer, 1.5 mM MgCl2, 0.8 mM deoxynucleotide triphosphates, 20 pmol of each primer for CDC25A or CDC25B, 4 pmol of each primer for PBGD, and 1 unit of Taq DNA Polymerase (AmpliTaq Gold; Roche Molecular Systems, Inc., Belleville, NJ). The primer sets of CDC25A and CDC25B were designed to flank at least one intron and tested to ensure amplification of only cDNAs so that amplification of possibly contaminated genomic DNA could be avoided. The sequences of these PCR primers were as follows: (a) CDC25A sense primer, 5'-GAGGAGTCTCACCTGGAAGTACA-3' (nucleotides 1297–1569); (b) CDC25A antisense primer, 5'-GCCATTCAAAACCAGATGCCATAA-3'; (c) CDC25B sense primer, 5'-CACGCCCGTGCAGAATAAGC-3' (nucleotides 1059–1475); and (d) CDC25B antisense primer, 5'-ATGACTCTCTTGTCCAGGCTACAGG-3'. The primers for PBGD were synthesized as described previously (28) . The sizes of the amplicons for CDC25A, CDC25B, and PBGD were 272, 416, and 127 bp, respectively. The PCR conditions were as follows: (a) initial denaturing at 95°C for 12 min; (b) 35–40 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 1 min; and (c) a final extension at 72°C for 10 min. In the next step, 10 µl of each PCR product were electrophoresed on 2% agarose gels and stained with ethidium bromide. The PCR products were scanned by densitometry.
Statistical Analysis.
Statistical analysis was performed using the Statview J-5.0 program
(Abacus Concepts, Inc., Berkeley, CA). The postoperative period was
measured from the date of surgery to the date of the last follow-up or
death. The Kaplan-Meier method was used to estimate death from
colorectal cancer, and the log-rank test was used to examine
statistical significance. A Cox proportional hazards model was used to
assess the risk ratio under simultaneous contributions from several
covariates. The associations between the discrete variables were
assessed using Fisher’s exact test. Mean values were compared using
the Mann-Whitney test. P < 0.05 was accepted
as statistically significant.
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RESULTS |
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, Lane 1). Normal mucosa generally expressed low levels of
CDC25B (Lanes 2, 4, 6, and 8), whereas colorectal
carcinoma tissues showed wide variability in the expression of CDC25B
(strong expression, Lane 3; moderate expression, Lanes
5 and 7; weak expression, Lane 9). When 10
paired samples were examined, 8 of 10 (80%) carcinomas showed
overexpression of the CDC25B protein, which displayed a over 2-fold
band density, compared with their corresponding normal mucosa. When the
same series of carcinoma samples was immunostained with the CDC25B
antibody, the level of CDC25B in each sample paralleled the results
obtained by Western blot analysis (data not shown).
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). The CDC25B protein was randomly distributed from the
bottom to the top of the normal epithelium, and the intensity of
staining was generally weak, except in occasional cases. The germinal
center of the lymph follicle exclusively expressed CDC25B with weak
intensity. In carcinoma tissues, CDC25B protein was localized mainly in
the cytoplasm (Fig. 2B
). CDC25B expression was noted in 175
of 181 carcinoma cases (97%), with a wide variability in the
expression level, which ranged from 15% to 100%. Approximately
half of the samples displayed strong staining (Fig. 2B
),
whereas a subset of carcinomas showed weak staining for the protein
(Fig. 2C
). All carcinoma samples were evaluated with respect
to intensity and positivity as described in "Materials and
Methods," and the results are summarized in Table 1
. We then immunostained 117 duplicate slides from 181 specimens using
anti-CDC25B polyclonal antibody. The results of staining in the normal
colonic mucosa were identical to those obtained with CDC25B mAb
throughout the series. In carcinoma tissue, 113 of 117 samples (97%)
showed identical results, whereas 4 specimens (3%) showed discrepant
results. In the latter group, the use of the polyclonal antibody led to
a higher expression of CDC25B. For statistical analysis, we used the
data obtained with CDC25B mAb because the peptide used to generate it
was of human origin, whereas the immunogen for the polyclonal antibody
was of mouse origin, with a 1-amino acid difference from the human
CDC25B amino acid sequences.
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). There was no correlation between high expression of CDC25B and age,
gender, site of tumor, lymph node metastasis, depth of invasion, degree
of differentiation, and Dukes’ stage.
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). In the normal epithelium, CDC25A protein was weakly
expressed at the top and upper half of the gland, but a relatively
intense nuclear expression was noted in the lower parts of the gland.
The germinal center of the lymph follicle exclusively expressed marked
CDC25A expression and served as a positive control within the sections.
Furthermore, infiltrating lymphocytes frequently showed intense
staining. In colorectal carcinoma tissues, the CDC25A protein was
detected in 108 of 111 cases (97%), with a wide range of nuclear
expression (range, 30–100%; median, 75%). The carcinoma specimens
were divided into two groups, 52 high expressors and 59 low expressors,
according to the percentage of nuclear CDC25A-positive cells, using a
cutoff level of 75%. When we compared the results of CDC25A
immunostaining with those of CDC25B immunostaining with respect to the
level of expression, there was an appreciable mismatch, with 22 low
CDC25B expressors among 52 high CDC25A expressors and 21 high CDC25B
expressors among 59 low CDC25A expressors. Further analysis of CDC25A
expression indicated no significant association with the
clinicopathological parameters listed in Table 2
.
RT-PCR Analysis.
RT-PCR analysis for CDC25B and CDC25A mRNAs was performed using paired
normal-carcinoma mRNA extracts. The relative value of the CDC25A or
CDC25B band to the PBGD band was calculated for each sample, and the
T:N ratio was determined in each case. In five representative cases,
the T:N ratio was 7.0, 12.9, 3.0, 3.4, and 2.2 for CDC25A and 6.3, 4.1,
1.3, 0.7, and 1.2 for CDC25B (Fig. 3
). When the T:N ratio of >2.0 was defined as overexpression, CDC25A was
overexpressed in 9 of 10 cases tested, whereas CDC25B was overexpressed
in 6 of 10 cases.
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Analysis of Survival Rates.
In the next step, we analyzed the survival rates according to CDC25B
expression in colorectal carcinoma. Univariate analysis showed that
high expression of CDC25B, lymph node metastasis, depth of invasion,
and degree of differentiation were significant predictors of a poor
prognosis (P < 0.0001, 0.0004, 0.026, and
0.027, respectively). Other parameters, such as gender, age, tumor
size, tumor site, expression of CDC25A, and Ki-67 index, were not
significant predictors of a poor prognosis (Fig. 4A
). Furthermore, in the entire group, as well as in Dukes’ B
and C stage tumors, a high level of CDC25B expression was significantly
associated with poor prognosis (Fig. 4B
). The 5-year
survival rates of patients with tumors expressing high and low levels
of CDC25B were as follows: (a) entire group
(n = 181), 59% versus 82%
(P < 0.0001); (b) Dukes’ stage B
(n = 61), 77% versus 89%
(P < 0.05); and (c) Dukes’ stage
C (n = 69), 55% versus 77%
(P < 0.01). In contrast, there was no
significant difference in survival rates of patients with Dukes’ stage
A and Dukes’ stage D disease stratified by CDC25B level (data not
shown).
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. The relative risk of death in patients with carcinomas expressing
high levels of CDC25B was 3.7 that of patients with tumors expressing
low levels of CDC25B (P < 0.0001). The
effect of CDC25B was also clearly observed when patients with Dukes’ B
and C stage tumors were analyzed by multivariate analysis. In these two
groups, a high expression of CDC25B alone was associated with poor
prognosis and a relative risk of death due to colorectal cancer of 5.2
(P < 0.05) and 3.1 (P < 0.05), respectively; none of the other factors were associated
with prognosis and relative risk of death due to colorectal cancer
(data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our comparative study revealed several differences in the expression level and cellular localization of CDC25B and CDC25A. RT-PCR and immunohistochemical assays showed that the expression levels of CDC25A and CDC25B were not always similar in individual colorectal carcinoma specimens. Such a difference has also been described in head and neck tumors, non-Hodgkin’s lymphoma, and non-small lung cancer (9, 10, 11) . These findings suggest that although the two molecules share a high homology in the COOH-terminal domain, their expressions may be regulated separately. Immunohistochemical analysis showed that the CDC25B protein was localized mainly in the cytoplasm, whereas the CDC25A protein was found in the nucleus. These results are in agreement with those of previous studies showing abundant CDC25B protein in the cytoplasm and CDC25A in the nuclear fraction (22 , 29, 30, 31, 32) .
The major finding of this study is the striking correlation between
high CDC25B expression and poor prognosis in patients with colorectal
carcinoma (Fig. 4A
; P < 0.0001).
When the Cox proportional hazards model was constructed for the entire
series, high CDC25B expression remained an independent predictive
factor of death; surprisingly, it displayed a higher relative risk for
death than lymph node metastasis (relative risk, 3.7 versus
2.4), which is one of the strongest predictors of poor prognosis in
colorectal carcinoma, and was used for this purpose in Dukes’ staging
system. The mechanism of the negative effect of CDC25B
expression on the progression of colorectal carcinoma is not yet clear.
Although CDC25B is a positive regulator of the cell cycle, it is
unlikely that the prognostic value of CDC25B expression would be due to
the rapid growth of carcinoma cells because of a lack of association
between CDC25B expression and cell proliferation as assessed by the
Ki-67 index. One clue is that high CDC25B expression was frequently
noted in patients with distant metastasis, i.e., those with
Dukes’ stage D disease. Although the latter by itself is a high risk
factor for death, we postulate that CDC25B itself may enhance the
malignant nature, apart from distant metastasis, because notable
differences in survival rates were also identified in the presence of
different levels of CDC25B expression in Dukes’ B and C stage tumors
that had escaped from distant metastasis (Fig. 4B
). In
support of this hypothesis, mechanistic studies of in vitro
transformation of fibroblasts and CDC25B transgenic mice have shown
that CDC25B displays oncogenic properties under certain conditions
(8
, 23 , 24)
.
Recent studies indicated that CDC25B is involved in G2-M-phase transition through the activation of CDC2 kinase (29, 30, 31, 32) . Cyclin B is synthesized during S phase and G2 phase and immediately forms complexes with CDC2 in the cytoplasm. The complex is inactivated by phosphorylation of the threonine 14 and tyrosine 15 residues of CDC2 by Wee-1 or Mik1 until G2-M-phase transition (33 , 34) but is activated on dephosphorylation by CDC25B. Ectopic expression of the CDC25B gene shows that prophase microtubule nucleation on the centrosomes is a consequence of cytoplasmic CDC25B activity (29) . Because the activity of CDC2 kinase is increased in a subset of colon carcinoma (35) , overexpression of CDC25B might contribute to the constitutively active status of CDC2 kinase and accelerate the transition from G2 to M phase. Consequently, alteration of G2-M-phase transition may lead to inappropriate distribution of the chromosome and result in aneuploidy. Indeed, there is evidence that overexpression of Cdc25B causes S phase and G2 phase cells to rapidly enter mitosis, irrespective of the completion of DNA replication (31) . Moreover, it has been demonstrated that introduction of CDC25B cDNA into normal mouse embryo fibroblasts leads to aneuploidy (8) . Interestingly, aneuploidy is known to be associated with poor prognosis in colorectal carcinoma (36 , 37) . Introduction of CDC25B cDNA into colon carcinoma cell lines may offer some insight into the underlying mechanisms of ploidy status and other aspects of the malignant properties of colorectal cancers including invasiveness, neovascularization, and metastatic ability.
The present study clearly showed that CDC25B, but not CDC25A and Ki-67,
was a significant poor prognostic factor in colorectal carcinoma (Fig. 4A
). Previous studies indicated that CDC25A plays a crucial
role in G1-S-phase transition (6
, 38
, 39)
, whereas CDC25B is essential for the
G2-M-phase transition (29, 30, 31, 32)
.
Among the components engaged in G2-M-phase
transition in the mammalian cell cycle, CDC2 and cyclin B are
well-known downstream molecules. CDC25B phosphatase acts as an upstream
effector of the CDC2/cyclin B complex. In contrast, various components
are currently known as gatekeepers at G1-S-phase
transition, including pRb, cyclin D1, cyclin E, CDK2, CDK4,
p21waf1/cip1, p27kip1, and
p16INK4, and colorectal carcinoma displays
altered expression of these molecules, as described above
(14, 15, 16, 17, 18, 19, 20, 21)
. Because CDC25A phosphatase is involved in the
complex process of G1-S-phase transition, CDC25A
expression alone may not be a sensitive marker. Ki-67 is a good
indicator of poor prognosis in certain types of tumors including
carcinomas of the liver, breast, and lung (40, 41, 42)
. In
contrast, the impact of Ki-67 on prognosis in colorectal carcinoma is
controversial, and many investigators have not found a positive
correlation in the past (43, 44, 45)
. These findings suggest
that features other than proliferation may play an important role in
determining the prognosis of patients with colorectal carcinoma.
Dukes’ staging system provides the most reliable information on
prognosis and is certainly useful for discriminating patients with
early-stage disease from those with very advanced stage disease.
However, its prediction of prognosis of patients with intermediate
levels of tumor invasion is less accurate. Several investigators have
reported that certain biological markers such as urokinase-type
plasminogen activator, erbB-2, vascular endothelial growth factor, and
E-cadherin (46, 47, 48, 49)
are useful for identifying those
patients with Dukes’ B tumors who are likely to show unfavorable
prognosis. We also found that CDC25B was an independent marker for poor
prognosis (Fig. 4B
). Dukes’B stage tumors are defined as
those without lymph node metastasis. We are not certain at present why
these localized tumors showed a difference in prognosis. One possible
explanation is that although pathological metastasis cannot be
detected, minimal cancer cells might invade blood and lymph vessels
because tumors of this stage spread beyond the propria muscularis
layer. CDC25B might enhance the probability of such occult metastasis,
i.e., micrometastasis. The results of the study reported by
Liefers et al. (50)
may support this hypothesis
because they showed that micrometastasis to regional lymph nodes was
indicative of poor prognosis only in stage II tumors. From a clinical
point of view, classification of patients with Dukes’ C stage tumor is
also important because clinical trials suggest that adjuvant
chemotherapy and treatment with mAbs could improve their survival rates
(51
, 52)
. Thus, CDC25B may serve as a good marker for
treating patients with appropriate adjuvant therapies because we found
that high expression of CDC25B alone was an independent indicator of
unfavorable prognosis in Dukes’ C stage disease (Fig. 4B
).
The present study also suggests that CDC25B might be a therapeutic
target for advanced colorectal carcinoma. Because high expression of
CDC25B is frequently noted in large tumors and in those with distant
metastasis, antisense CDC25B constructs or some specific inhibitors
might be of clinical use. Although we did not find a significant
correlation between CDC25B level and the efficacy of 5-fluorouracil
chemotherapy in Dukes’ B and C stage tumors (data not shown), it is of
interest that treatment with several reagents that interfere with
G2-M-phase transition, including the DNA
topoisomerase inhibitors camptothesin and etoposide or the
Vinca alkaloid vincristine, has already been found to be
clinically feasible. These reagents may suppress those
colorectal carcinomas exhibiting a high CDC25B expression.
In conclusion, we have shown in the present study that CDC25B is a novel prognostic marker in patients with colorectal carcinoma. The prognostic value of this protein is equivalent to that of lymph node metastasis and is independent of conventional clinicopathological parameters.
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FOOTNOTES |
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1 Supported by a Grant-in Aid for Cancer Research
from the Ministry of Health and Welfare of Japan and by an award from
the Osaka Medical Research Foundation for Incurable Diseases (to
H. Y.). 
2 To whom requests for reprints should be
addressed, at Department of Surgery II, Osaka University Medical
School, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan. Phone:
81-6-6879-3251; Fax: 81-6-6879-3259; E-mail: kobunyam{at}surg2.med.osaka-u.ac.jp
3 The abbreviations used are: CDK,
cyclin-dependent kinase; RT-PCR, reverse transcription-PCR; PBGD,
porphobilinogen deaminase, mAb, monoclonal antibody; T:N,
tumor:normal.
Received 9/15/99. Accepted 3/29/00.
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