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Molecular Oncology, Markers, Clinical Correlates |
Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Osaka 565-0871 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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Cell cycle arrest is another cell response, which occurs after DNA damage induced by irradiation. G1 arrest results from a p53-dependent pathway mainly (12) . p53 activates transcription of p21 (13) , a strong inhibitor for CDK2 and CDK4, both of which are required for transition from the G1 phase to the S phase (14) . Recently, the mechanism of G2-M arrest after irradiation has been elucidated as follows. ATM activates Chk1, which phosphorylates CDC25 on serine-216 and inactivates it (15 , 16) . Because CDC25 activates CDC2 by dephosphorylation of the tyrosine 15 and threonine 14 residues, inactivation of CDC25 results in G2-M arrest through inactivation of the cyclin B1/CDC2 complex.
Although both apoptosis and cell cycle arrest are caused by irradiation, it is not known whether cell cycle arrest facilitates or suppresses apoptosis. There are a few reports of cell cycle arrest stimulating apoptosis. However, a decline of G1 arrest attributable to p21 suppression (17 , 18) or cyclin D1 overexpression (19) is associated with apoptosis. In addition, radiation-induced apoptosis has been reported to be increased by inhibition of G2-M arrest by caffeine (20) but to be decreased by anticancer drugs that cause G2-M arrest (21) . These findings strongly suggest that cell cycle arrest is does not promote radiation-induced apoptosis but rather suppresses it. This can be explained as follows. Cell cycle arrest is indispensable for repairing DNA damaged by irradiation, and apoptosis results when DNA damage is not adequately repaired (22) . Thus, the cell after irradiation can survive if cell cycle arrest and DNA repair occur, but apoptosis will take place if there is not sufficient cell cycle arrest.
Although many biological markers for radiation sensitivity have been reported, p53 may be the only reliable marker for predicting radiation sensitivity both in vitro and in vivo (23 , 24) . p53 is involved in both apoptosis and cell cycle arrest, but its effect on apoptosis may override that on cell cycle arrest, even if cell cycle arrest might partially inhibit apoptosis. On the other hand, rapidly growing tumors are known to be more sensitive to radiation therapy than slowly growing ones (25) . Disorders of the cell cycle and cell cycle-regulating molecules are ubiquitous characteristics of cancer cells. Taken together, the molecules that down-regulate the cell cycle checkpoint and stimulate cell growth should be involved in increasing sensitivity to radiation therapy. In the present study, to investigate the implication of cell cycle checkpoints in sensitivity to clinical radiation therapy, we evaluated the expression of representative cell cycle regulating molecules, including not only p53 but also cyclin D1, Ki-67, cyclin B1, and CDC25B using immunohistochemical techniques.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preoperative Adjuvant Therapy.
Patients received a total dose of 40 or 60 Gy over 4 or 6 weeks, 2 Gy a
day, five fractions a week, in combination with continuous infusion of
5-fluorouracil (300 mg/m2 per day) and a single
infusion of cisplatin (10 mg/body per day) on each day. A 10-MeV high
energy linear accelerator was used to deliver radiation to the
mediastinum and the neck. The toxicities and their treatments were
described previously (26)
.
Immunohistological Staining Procedures.
Immunostaining for CDC25A, CDC25B, p53, cyclin B1, cyclin D1, and Ki-67
was performed using streptavidin-peroxidase complex methods described
previously (27)
. In brief, 4-µm paraffin sections were
deparaffinized, and endogenous peroxidase was blocked with 0.3%
H2O2 in methanol. The
sections were heated in 0.01 M citrate buffer (pH 6.0),
heated for 45 min at 95°C, then left covered with 10% normal goat or
rabbit serum for 20 min at room temperature. These prepared sections
were incubated overnight at 4°C with primary antibodies [CDC25A:
sc-97, 0.5 µg/ml (Santa Cruz Biotechnology, Santa Cruz, CA); CDC25B:
sc-326, 2.0 µg/ml (Santa Cruz); p53: DO-7, dilution 1:50 (Novocastra
Laboratories, Newcastle, United Kingdom); cyclin B1: clone 7A9,
dilution 1:20 (Novocastra); cyclin D1: M-20, 0.5 µg/ml (Santa Cruz);
Ki-67: clone Ki-S5, 1.0 µg/ml (DAKO, Carpinteria, CA)]. They were
then processed by the biotin-streptavidin method using a kit according
to the manufacturer’s instructions (Histo-Fine SAB-PO kit; Nichirei,
Japan). The color was developed with diaminobenzidine
tetrahydrochloride supplemented with 0.02% hydrogen peroxide, and the
nuclei were counterstained with Meyer’s hematoxylin.
Evaluation of Staining.
The immunoreactivities of CDC25A and CDC25B were classified according
to the frequency of the positively stained cells as follows: negative
(-), <10%; weak (+) from 10 to 50%; and strong (2+), >50%.
Concerning p53 and cyclin D1 staining, when >10% of the cancer cells
showed positive staining, the tumors were evaluated as positive (+;
Refs. 27
and 28
). We calculated cyclin B1 and
Ki-67 indices as the percentage of cells with nuclear staining of the
total number of tumor cells.
Immunoblot Analysis.
The tissue samples were minced and homogenized with lysis buffer [50
mM Tris (pH 8.0), 150 mM NaCl, 0.5% NP40, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and
2 µg/ml leupeptin], and the extract was centrifuged at 14,000 rpm
for 25 min at 4°C. Aliquots (100 µg) of supernatant proteins were
separated by 10% PAGE, followed by electroblotting onto a
polyvinylidene difluoride membrane. Antihuman CDC25B rabbit polyclonal
antibody (Santa Cruz) was used at a concentration of 1.0 µg/ml.
Detection of the protein bands was performed using the Amersham ECL
detection system (Amersham, Arlington Heights, IL) according to the
manufacturer’s instructions.
Statistical Analysis.
For statistical evaluations, Student’s t test and
Spearman’s rank correlation test were used. P < 0.05
was accepted as statistically significant. This analysis was carried
out using the StatView J 4.5 software statistical package (Abacus
Concepts, Inc., Berkeley, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a). In the preradiation biopsy samples of cancerous tissue, CDC25B was
strongly expressed in the cytoplasm of cancer cells (Fig. 1
c) and classified as (2+) in 17.0% (8 cases), (+) in
46.8% (22 cases), and (-) in 36.2% (17 cases) according to the
frequency of the positive cells (Table 1)
. In the cancer tissues obtained by surgical treatment, CDC25B tends to
be expressed more in the deep infiltrating parts than in the
superficial parts (Fig. 1
d).
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. A band with strong intensity was located at
Mr 63,000, which corresponds to
the molecular weight of CDC25B. A strong band appeared in CDC25B (2+)
tumor samples, but the bands of the samples of CDC25B (-) tumors and
normal tissues were faint or totally absent from the blot. Thus, the
grades of CDC25B expression determined by immunohistochemistry
correlated well with those from immunoblot analysis.
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and slightly higher than in previous reports,
probably because of advanced clinical stages being more frequently
included in preoperative irradiation cases. Cyclin D1 displayed
overexpression (+) in 48.9% (23 cases; Table 1
). CDC25A was not
expressed in noncancerous epithelium, whereas it was overexpressed
frequently in esophageal cancer cells and classified as: 2+, 19 cases
(40.4%); +, 12 cases (25.5%); and -, 16 cases (34.1%; Table 1
; Fig. 1, g and h
). Cyclin B1 were specifically
expressed in the cytoplasm or nuclei of cells at the
G2-M phase, and Ki-67 was expressed in the nuclei
of all proliferating cells, except the G0 phase
(Fig. 1, e and f)
. Because both cyclin B1 and
Ki-67 were expressed to some extent in all normal and cancer tissues,
they were evaluated as an indices representing the percentage of
positively stained cells. These indices of cyclin B1 and Ki-67 in
esophageal cancers averaged 18.5 ± 2.0% and 38.0 ± 3.9%,
respectively, which were significantly higher than in the noncancerous
esophageal epithelium (8.3 ± 1.7% and 10.8 ± 1.9%,
respectively).
Radiation Sensitivity and Cell Cycle-regulating Molecules.
The expression of cell cycle-related molecules was compared with the
radiation sensitivity of tumors classified as RR (33 cases) and SR (14
cases), according to the presence of residual cancer cells.
p53 expression was not found in 10 of 14 SR cases (71.4%) but
was found in 30 of 33 cases (90.9%). Positive p53 expression, which is
regarded as an accumulation of abnormal protein, was strongly
associated with resistance to radiation therapy (P <
0.001). There was no difference of tumor size between p53 (-) and p53
(+) tumors (39.1 ± 5.6 mm versus 44.0 ± 4.9 mm;
P = 0.5851). The frequencies of strongly positive (2+),
weakly positive (+), and negative (-) CDC25B expression were 42.9% (6
of 14), 35.7% (5 of 14), and 21.4% (3 of 14) in SR cases but were
6.1% (2 of 33), 51.5% (17 of 33), and 42.4% (14 of 33) in RR cases.
Thus, overexpression of CDC25B was significantly associated with
radiation sensitivity (P = 0.0168). CDC25A (2+) was
observed in 35.7% (5 of 14) of SR and 42.4% (14 of 33) of RR cases.
Cyclin D1 was overexpressed in 57.1% (8 of 14) of SR and 45.5% (15 of
33) of RR cases. The cyclin B1 index was 17.0 ± 2.6% and
18.8 ± 1.8% in SR and RR, and the Ki-67 index was 40.5 ±
5.2% and 36.9 ± 3.3%, respectively (Table 2)
. Thus, no difference were observed for the cyclin D1, cyclin B1, and
Ki-67 status in SR and RR. The cyclin B1/Ki-67 index was calculated to
evaluate the proportion of G2-M cells and
classified as high (>0.7), moderate (between 0.4 and 0.7), and low
(<0.4). The frequencies of high, moderate, and low cyclin B1/Ki-67
index were 7.1, 42.9, and 50.0% in the SR group but 24.2, 51.6, and
24.2% in the RR group. Thus, RR cases showed higher cyclin B1/Ki-67
index than SR cases, although the difference was not statistically
significant (P = 0.0575). Cyclin B1/Ki-67 index was
lower in CDC25B (2+; 33.2%) than in CDC25B (+; 59.0%) or CDC25B (-;
56.0%). Although this difference was not statistically significant, it
might suggest the involvement of CDC25B in G2-M
progression in vivo.
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As mentioned above, CDC25B expression could be underestimated in the
biopsy sample because its expression was stronger in the deep
infiltrating parts. Thus, CDC25B expression of the whole tumor was
evaluated for a total of 48 RR cases with postradiation residual tumors
(including additional 15 cases without preoperative biopsy) and 43
cases without radiation (Table 3)
. The trend in Table 1
is more evident here; thus, the frequency of
CDC25B (2+) was 4.2% in the former and 51.2% in the latter, and that
of CDC25B (-) was 45.8 and 14.0%, respectively (P <
0.001).
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, Thus, all four cases with p53 (-)/CDC25B (2+), but only 2 of 20
cases with p53 (+)/CDC25B (+/-) showed SR (Table 5)
. Multivariate analysis by the logistic regression model, including
p53, CDC25B, and cyclin B1/Ki-67 indices, revealed that p53 and CDC25B
were independent significant markers for predicting radiation
sensitivity (P < 0.0001 and P =
0.0076, respectively).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CDC25 is a family of protein phosphatase, which dephosphorylates and activates CDKs. Three members of the cdc25 gene family have been identified in human cells (29 , 30) . CDC25A activates CDK2 and facilitates the G1 checkpoint (31) , whereas CDC25B and CDC25C promote G2-M transition by activating the CDC2/cyclin B1 complex (16 , 32 , 33) . Recent studies have shown the frequent overexpression of CDC25A and CDC25B, but not of CDC25C, in various types of cancers (34, 35, 36) . The current study showed consistent CDC25B and CDC25A overexpression in esophageal SCC, thus suggesting their role as an oncogene that triggers G2-M or G1-S transition.
Cell death after irradiation was roughly classified into "interphase death" and "mitotic death" (37) . The latter was strongly associated with G2-M arrest, which is necessary to allow time for DNA repair to escape from apoptosis. Recently, the mechanism of G2-M arrest after irradiation has been revealed to involve many molecules, such as ATM, cyclin B1, CDC2, Chk1, and CDC25. Among them, only the alteration of CDC25B has been observed frequently in various cancers. We investigated CDC2 and CDC25C expression by immunohistochemistry, but they were relatively invariable among esophageal cancers and not associated with radiation sensitivity (data not shown). According to this scheme, the high activity of CDC25B theoretically can prevent G2-M arrest after irradiation. Therefore, overexpression of CDC25B in esophageal cancers might reduce the duration of G2-M arrest, which would thus not allow enough time for the repair of DNA damage caused by irradiation, and result in increased apoptosis and sensitivity to radiation therapy.
The implication of G2-M arrest in radiation sensitivity has not yet been demonstrated in clinical human samples in vivo. The number of cyclin B1-positive cells, which are only found in the G2-M phase, was not correlated with radiation sensitivity. Because the portion of cells resting in the G0 phase was relatively high and varied among solid tumors (38) , the relative proportion of the G2-M phase in proliferating cells was demonstrated as the cyclin B1/Ki-67 index. Interestingly, the cyclin B1/Ki-67 index tended to be higher in tumors with radiation resistance. This might be interpreted as showing that G2-M progression in these tumors was slower than that in the others, would provide a longer time for DNA repair, and eventually might lead to escape from apoptosis. p53 is partly involved in this pathway by sequestering CDC25 through up-regulation of the 14-3-3 protein (39) . However, in general, cells that lack wild-type p53 still demonstrate strong G2-M arrest, implying that p53 is involved more in G1 arrest than in G2-M arrest.
Because CDC25B was expressed more strongly in deep infiltrating
parts than in superficial parts, according to immunohistochemical
tests, its expression might be underestimated in biopsy specimens. For
example, CDC25B (2+) was observed in 51% of the surgical specimens of
esophageal cancers (Table 3)
; however, only half of them were evaluated
as CDC25B (2+) with preoperative biopsy samples (data not shown). We
compared CDC25B expression in surgical specimens with or without
preoperative CRT (Table 3)
. The frequency of complete disappearance of
tumors by CRT was observed in, at most, 20–40% of the cases
(26)
. We concluded that a remarkably low level of CDC25B
expression after CRT might imply that its expression is reduced during
CRT. However, it is not clear whether this results from the clonal
selection of cells with low CDC25B expression or suppression of the
expression by radiation therapy.
G1 progression and total cell proliferation were also evaluated by the investigation of cyclin D1 and Ki-67 expression. Cyclin D1, which is frequently overexpressed in SCC of the esophagus (40) , is the most crucial regulator of the G1 checkpoint (41) . There have been a few reports showing cyclin D1 overexpression to be associated with high susceptibility for radiation therapy in vitro (42 , 43) . Such a trend was observed but was not statistically significant in this study. Rapidly growing tumors have been reported to be more radiosensitive in clinical samples (25) . The total cell proliferative activity was evaluated by Ki-67 expression, which was strongest in the S phase, and observed in cell cycle phases other than G0. Consistently, the Ki-67 index was slightly higher in RR tumors than in SR tumors; however, the difference was not statistically significant in the present study.
p53 is commonly acknowledged to be a definite indicator for radiation sensitivity in various cancers (23 , 24) . In this study, p53 immunoreactivity was considered to be attributable to the accumulation of abnormal p53 protein. Although this idea is generally accepted, we should remember that negative immunoreactivity of p53 does not always reflect normal p53 function, because there may be homozygous deletion, stop codon mutation, or acceleration of protein degradation (44 , 45) . In fact, a functional assay of p53 using fission yeast showed that 70–90% of cancers display loss of the p53 function in various tumors, including SCC of the head and neck and uterus (46 , 47) . In contrast, radiation therapy is clinically effective in more than half of these cancers. This strongly suggests the existence of an alternative pathway independent of p53, which regulates radiation sensitivity. The G2-M checkpoint, in which CDC25B is involved, is a candidate for this p53-independent pathway.
The relationship between cell cycle and radiation effect is not fully understood. Prolonged cell cycle arrest is considered to induce terminal differentiation and loss of proliferative activity, and those cells would undergo cell death through a different mechanism from checkpoint.
From the clinical standpoint, it is very important that the present study was performed using biopsy samples, because our results can be directly used for the treatment for esophageal cancers to predict the effect of CRT and select patients for this treatment. Furthermore, our results suggest the use of compounds that can up-regulate G2-M transition, such as caffeine, as radiosensitizers especially in patients with low CDC25B expression. Thus, clinical application of these biological markers is promising and of urgent priority.
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FOOTNOTES |
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1 Supported by Grants-in-Aid for Scientific
Research (C) 09671303 and 10671185, Grants-in-Aid for Encouragement of
Young Scientist 09770936, and Grant 12213078 for Cancer Research (to
H. Y.) from The Ministry of Education, Science, Sports and Culture,
Japan. 
2 To whom requests for reprints should be
addressed, at Department of Surgery and Clinical Oncology, Graduate
School of Medicine, Osaka University, 2-2-E2, Yamadaoka, Suita, Osaka
565-0871, Japan. Phone: 81-6-6879-3251; Fax: 81-6-6879-3259; E-mail: dokiy{at}surg2.med.osaka-u.ac.jp
3 The abbreviations used are: ATM, ataxia
telangiectasia-mutated; CDK, cycle-dependent kinase; SCC, squamous cell
carcinoma; RR, resistant to radiation; SR, sensitive to radiation; CRT,
chemoradiation therapy.
Received 3/13/00; revised 9/20/00; accepted 9/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Radiation induces upregulation of Bax protein and apoptosis in radiosensitive cells in vivo. Oncogene, 12: 187-192, 1996.[Medline]
is a p53 regulated inhibitor of G2/M progression. Mol. Cell, 1: 3-11, 1997.[CrossRef][Medline]
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