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From the First Department of Pathology,*
Second
Department of Internal Medicine,
and
Department of Chest Medicine,
Chiba
University School of Medicine, Chiba, Japan, and Department of
Molecular and Cellular Biology,§
Medical
Institute of Bioregulation, Kyushu University, Fukuoka, Japan
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent progress in the study of the molecular biology of cancer has contributed to a better understanding of its molecular pathogenesis. Additionally, advances in cell cycle research have paved the way for the identification of proteins responsible for the regulation of cell proliferation. The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Among various Cdks, Cdk2 plays a pivotal role in the regulation of the cell cycle at the G1/S transition. For complete kinase activity, Cdks require physical association with regulatory subunits called cyclins.2-4 Cdk2 forms a functional complex with cyclin E during the G1/S phase; the expression of Cdk2 and abundance of the cyclin E/Cdk2 complex are maximal at the G1/S transition.5 Cdk2 activity is negatively regulated by interactions with inhibitors, among which p27Kip1 (p27) is the most potent.6,7 p27 is present in large amounts in quiescent cells and declines when cells proliferate in response to mitogenic signals such as growth factors and cytokines.8-10 Thus, the loss of p27 may contribute to oncogenesis and tumor progression. Unlike traditional tumor suppressor genes, the p27 gene rarely exhibits homozygous deletions or point mutations.11-14 Further, it was recently demonstrated that the absence of p27 expression is a powerful negative prognostic marker in non-small cell lung cancers (NSCLCs), as well as in colorectal and breast cancers.15-17 Thus, the potential function of p27 as a tumor suppressor provides new insight into the link between the cell cycle and oncogenesis in lung, colon, and breast tissues.
The proliferation index is a potent biological marker that estimates the growth of neoplasms quantitatively and can aid in determining the prognosis of patients with neoplasms.18-20 A variety of methods have been used for the estimation of the proliferation index of human cancers. Ki-67 is the most reliable antibody for assessing the growth fraction by immunohistochemistry.21 The Ki-67 antigen represents a DNA-binding nuclear protein of 345 and 395 kd, encoded by a gene on chromosome 10.22-24 It is expressed throughout the cell cycle in proliferating cells, but not in cells in the G0 phase or early G1 phase. It can thus be used to distinguish between growing and nongrowing cells.25 The value of Ki-67 antibody in the assessment of cell-proliferating activity has been widely documented for various human tumors, including lung cancer.26
Use of the Ki-67 antibody has methodological drawbacks, in particular, the restriction of its use in frozen tissue assays. Nevertheless, MIB-1, a recently developed antibody against an epitope of the Ki-67 antigen, can be used on paraffin material after antigen retrieval. Its reactivity has been shown to correlate strongly with Ki-67 staining and has the advantage of reacting with epitopes in routinely fixed, wax-embedded specimens.27 Thus, the MIB-1 labeling index (LI) is a useful prognostic factor and may enhance the accuracy of conventional morphological grading and pathological staging systems.19,28 The relationship between Ki-67 antigen expression and the estimated activities of cell cycle regulators that govern G1/S transition has not been well documented. Thus, it may be of interest to evaluate the correlation between the immunohistochemical labeling of cell nuclei with both MIB-1 and the expression of cell cycle regulators that govern G1/S transition in surgically resected NSCLCs.
The aim of the present study was to determine: 1) whether any relationship existed between the proliferative potential represented by the MIB-1 LI and the immunoreactivity of p27 protein in surgically resected NSCLCs, and 2) the relationship between MIB-1 LI and Cdk2 activity as H1 histone kinase activity in tissue lysates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung cancer tissues and nonneoplastic lung tissues were obtained
from 63 patients with primary lung cancers (36 men and 27 women; median
age, 65 years) who underwent resection of NSCLC at the Hamamatsu
Medical Center (Hamamatsu, Japan) and East Matsudo City Hospital
(Matsudo City, Chiba, Japan). They had received no other therapy
before surgery. Twenty-nine patients were in stage I, 24 in stage II,
and 15 in stage IIIA.29
All cancers were
independently classified, according to the standard criteria of the
World Health Organization, by two pathologists; there were 37
adenocarcinomas (16 well differentiated, 16 moderately
differentiated, and 5 poorly differentiated) and 26 squamous cell
carcinomas (4 well differentiated, 9 moderately differentiated, and 13
poorly differentiated), as summarized in Table 1
.30
Forty-six of the 63
patients remain alive, with a median follow-up of 10 months (range, 1
to 32). For histological examination, portions of cancer tissues, as
well as surrounding normal lung tissues, were fixed in buffered
formalin overnight, dehydrated in graded ethanol at 4°C, and embedded
in paraffin. Fresh samples were taken from pure-neoplastic tissues
(excluding bronchial epithelium) and from nonneoplastic tissues for
Western blot analysis.
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Immunostaining for p27 and MIB-1 was performed on formalin-fixed, paraffin-embedded materials. Paraffin sections were deparaffinized with xylene, rehydrated, and microwaved for 20 minutes in 10 mmol/L citrate buffer (pH 6.0). Intrinsic and background stainings were blocked with 3% hydrogen peroxide in methanol before incubations with 0.1 mol/L phosphate-buffered saline at pH 6.0 containing either normal swine serum (1:10 dilution) for p27 staining or normal rabbit serum (1:10 dilution) for MIB-1 staining. Sections were then incubated overnight with a 1:200 (0.5 µg/ml) dilution of polyclonal p27 anti-body C-19 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a 1:100 dilution of monoclonal MIB-1 antibody (Immunotech S. A., Marceille, France), both in phosphate-buffered saline.15-17,31,32 After washing, slides were reacted with biotin-labeled anti-rabbit immunoglobulin G for p27 or anti-mouse immunoglobulin G for MIB-1 and then incubated with avidin-biotin-peroxidase complex (Dako Japan Ltd., Kyoto, Japan). 3,3'-Diaminobenzidine tetrahydrochloride substrate (Dojindo Laboratories, Kumamoto, Japan) was then added in the presence of hydrogen peroxide. Sections were counterstained with hematoxylin, dehydrated, and mounted. Negative controls using normal isotype-specific polyclonal rabbit immunoglobulin instead of primary antibody showed no evidence of staining. The pattern of p27 staining seen with the polyclonal antibody C-19 was confirmed using three other different p27 sera (one from Transduction Laboratories (Lexington, KY) and two from Santa Cruz Biotechnology). Specificity of the p27 staining was also assessed by preabsorption of the antibody with the recombinant p27 protein used.
Counting Procedure
Labeling indices for p27 and MIB-1 antibodies were determined in the same manner. Adjacent sections were used, and counting was performed in similar areas. After inspection, areas with the highest number of labeled cells were counted. Nuclei were considered positive if any nuclear staining was present. The degree of staining was scored independently by two different pathologists. Twenty high-power fields were chosen and scored for the percentage of cells showing nuclear staining of 1000 cells.
Preparation of Tissue Lysate
In tissues from 10 adenocarcinomas and 10 squamous cell carcinoma cases, we analyzed the expression of p27, its related Cdk2, and Cdk2 activity in both nonneoplastic and cancerous lung tissues. We confirmed histologically that these 20 samples did not show significant inflammatory involvement. Tissues were minced on dry ice into small pieces, and then 500 mg of each was quickly homogenized at 15,000 rpm with a Brinkmann Polytron homogenizer (PT 3000, Westbury, NY) in 1.0 ml of ice-cold lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4, 250 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L N-ethylmalemide, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 2 mmol/L sodium vanadate, 50 mmol/L sodium fluoride, 50 µg/ml leupeptin, 25 µg/ml aprotinin, and 10 µg/ml pepstatin. Lysates were sonicated and spun down at 15,000 rpm for 10 minutes at 4°C. Protein quantification, immunoprecipitation, Western blotting, and immunoprecipitation kinase assays were performed.
Western Blot Analysis
Samples were boiled in 62.5 mmol/L Tris-HCl buffer, pH 6.8, containing 2% SDS, 5% 2-mercaptoethanol, 7% glycerol, and 0.01% bromphenol blue for 10 minutes, and 10 µl of each was electrophoresed by SDS-polyacrylamide gel electrophoresis (12.5%) and transferred to immobilon-P membranes (Millipore) (Nihon Millipore Ltd., Yonezawa, Japan). Transfer was at 60 mA for 3 hours at 4°C in transfer buffer (25 mmol/L Tris, 190 mmol/L glycine, and 10% methanol). Blots were then incubated with blocking buffer (50 mmol/L Tris, 200 mmol/L NaCI, 0.2% Triton X-100, and 3% bovine serum albumin) for 90 minutes at room temperature. Western blot analysis was performed with rabbit polyclonal anti-Cdk2 (M2) (Santa Cruz) and anti-p27 (C-19), followed by incubation with goat anti-rabbit coupled to horseradish peroxidase (Amersham, Buckinghamshire, England). Western blotting was developed using an enhanced chemiluminescence detection kit (Amersham).33
Northern Blot Analysis
Samples were lysed in 3 ml of 25 mmol/L sodium citrate solution (pH 7.0) containing 4 mol/L guanidinium isothiocynate and 0.1 mol/L 2-mercaptoethanol and then added to 1.7 ml of CsCl solution (5.7 mol/L CsCl (pH 7.2) and 0.1 mol/L ethylenediaminetetraacetic acid) and centrifuged at 80,000 x g for 36 hours at 16°C. The pellets were dissolved in Tris-ethylenediaminetetraacetic acid buffer, and RNA was precipitated with 1/10 volume of 3 mol/L sodium acetate (pH 5.2) and 2 volumes of ethanol at 80°C for 20 minutes. The concentration of RNA was determined by absorbance at 260 nm. Total RNA (10 µg) was separated on 1% formaldehyde agarose gels and transferred onto nitrocellulose membranes for 48 hours. Prehybridization was performed at 42°C for 16 hours in sodium citrate solution (750 mmol/L NaCl, 75 mmol/L sodium citrate (pH 7.0)) containing 1% Ficoll, 1% bovine serum albumin, 1% polyvinylpyrolidine, 50% formamide, 50 mmol/L sodium phosphate (pH 6.5), and 100 µg/ml salmon sperm DNA. The nylon filters were sequentially hybridized with 32P-labeled mouse p27 cDNA and human ß-actin cDNA by overnight incubation and washed using a standard protocol.34,35
Cdk2 Kinase Assay
Whole-cell lysates (300 µg/300 µl) obtained as above were
precleared by incubation with 30 µl of protein A-Sepharose (50%,
v/v; Pharmacia, Uppsala, Sweden) for 30 minutes, followed by
centrifugation at 15,000 x g for 5 minutes. The lysate
was then incubated with 1.5 µl of anti-Cdk2 antibody (M2) at 4°C
overnight. After this, 20 µl of protein A-Sepharose (50%, v/v) was
added and incubated for 30 minutes at 4°C. Immunoprecipitation was
achieved after a brief centrifugation at 3000 x g. The
activity of Cdk2 kinase was determined by the phosphorylation of H1
histone using the immunoprecipitates as reported
previously.36
Briefly, immunoprecipitates were incubated at
30°C for 30 minutes in 20 mmol/L Tris buffer, pH 7.0, 10 mmol/L H1
histone, 50 µmol/L ATP and 5.0 µCi 
-32P-ATP.
Phosphorylated H1 histone was analyzed by SDS-polyacrylamide gel
electrophoresis (10 to 20%) and autoradiography.36
Degradation Assay of p27
Recombinant mouse p27-glutathione-S-transferase fusion
protein was prepared using cDNA containing the full-length coding
region for mouse p27.34
Tissue homogenates were prepared as
described by Pagano et al8
with minor modifications. In
brief, each frozen human tissue sample was sectioned and quickly
homogenized with a Polytron homogenizer in 1 ml of ice-cold lysis
buffer (50 mmol/L Tris-HCl, pH 7.4, 250 mmol/L NaCl, 1 mmol/L
ethylenediaminetetraacetic acid, 10 mmol/L N-ethylmalemide,
0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100, 50 mmol/L
sodium fluoride, 2 mmol/L sodium vanadate, a protease inhibitor
mixture (10 µg/ml of p-amidinophenyl
methanesulfonyl fluoride hydrochloride, 10 mg/ml of pepstatin A, 10
mg/ml of chymostatin, 10 mg/ml leupeptin, and 10 mg/ml
antipain). The lysate was microfuged at 19,000 x
g for 5 minutes at 4°C to obtain supernatants. Purified
recombinant p27-glutathione-S-transferase fusion protein
(0.5 µg) was incubated at 37°C for different times in 30
µl volumes containing 300 µg of protein human tissue
homogenates, 50 mmol/L Tris-HCI (pH 8.0), 5 mmol/L MgCl, 1 mmol/L DTT,
2 mmol/L ATP, 10 mmol/L creatine phosphokinase, and 10 mmol/L creatine
phosphate. Adenosine-5'-(-thio)-triphosphate, a nonhydrolyzable ATP
analog, decreased p27 proteolysis; MG-115, a proteasome inhibitor, but
not E-64, also decreased p27 proteolysis. The reaction products were
analyzed by immunoblotting with anti-p27 antibody. Purified recombinant
glutathione-S-transferase was not degraded in these tissue
lysates.
Statistical Analysis
Differences between groups were analyzed by the Mann-Whitney test and relations between groups by Pearson's correlation coefficient. A rejection level of P < 0.05 was considered significant. This analysis was carried out using the StatView J 4.5 software statistical package (Abacus Concepts, Inc., Berkeley, CA).
Densitometry
For quantitative determinations of Western blot, Northern blot, and Cdk2 histone kinase activity, autoradiographic bands in the linear range were scanned with a DeskScan 4c (Hewlett-Packard, Tokyo, Japan) and analyzed by NIH Image 1.44 computer software. Values for Northern blots of p27 were calculated after normalization with ß-actin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the group of 63 lung cancer specimens and 51 nonneoplastic lung tissues that were reassessed with respect to their MIB-1 and p27 staining, an average of 763 cells (range, 280 to l,000 nuclei) were counted. The subjective assessment of the distribution of positively labeled cells was identical in all cases. Antibody against p27 stained nuclei in diffuse fashion in most of the cases, whereas MIB-1 stained nuclei in a variety of patterns, from discrete intranuclear blobs consistent with nucleoli to diffuse staining of the whole nucleus.
Positive immunoreactivity of p27 was detected in nonneoplastic tissues
such as the alveolar epithelium, bronchial epithelium and glandular
cells, fibroblasts in connective tissues, and lymphocytes in lymph
nodes. All of these were positively stained in the nuclei with a low to
absent background, but staining intensity varied among cell types.
Bronchial epithelium and lymphocytes in lymph nodes showed stronger
staining intensity than alveolar epithelium and fibroblasts in
connective tissues, and bronchial glandular cells showed modest
positivity. In lung cancers, p27 was expressed in the nucleus with a
low to absent background (Figure 1)
. The
number of cells expressing p27 and staining intensity varied from case
to case.
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The mean percentage of p27 LI was 17.9% (SD, 13; range, 0 to
58%) for all cancers. For adenocarconomas it was 19.8% (SD, 13.8;
range, 0 to 58%) and for squamous cell carcinomas 15.2% (SD, 10.6;
range, 1 to 48%). In normal bronchial epithelium, the p27 LI was
43.1% (SD, 6.7; range, 38 to 62%). The differences between cancers
and normal tissues were statistically significant
(P < 0.001) (Figure 2A)
. In contrast, differences in LI
between the histological types of cancers with different degrees of
differentiation were statistically not significant.
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MIB-1 LI: Proliferative Activity
In most lesions, heterogeneous staining was observed. However,
MIB-1 LI was significantly higher in lung cancers than in the control
group consisting of normal bronchial epithelium. The mean of MIB-1 LI
was 26.3% (SD, 14; range, 1 to 61%) for all cancers, 23% (SD, 15.2;
range 1 to 61%) for adenocarcinomas, and 31% (SD, 12.4; range, 10 to
51%) for squamous cell carcinomas. The mean LI for the normal
bronchial epithelium was 0.5% (SD, 0.8; range, 0 to 3.5%). The
difference of MIB-1 LI between cancerous and normal lung tissues was
statistically significant (P < 0.001) (Figure 2B)
. MIB-1 LI was not significantly different between lung cancers with
different histological features and different degrees of
differentiation. MIB-1 LIs of the two AAH lesions were 2.6% and
11.3%, respectively.
Relationship between p27 LI and MIB-1 LI
Pearson's relative coefficient estimation revealed a weak inverse correlation between p27 LI and MIB-1 LI, with a correlation coefficient of -0.459 (P < 0.001).
Relation to Clinical Findings
There was no significant difference in both MIB-1 LI and p27 LI that could be related to the degree of differentiation, nodal status, and clinical staging of the lung cancers.
Relationship between Immunohistochemistry and Western Blot Analysis
In tissues from 20 cases of lung cancer (10 adenocarcinoma and 10
squamous cell carcinoma), p27 levels were varied in each cancer.
Despite this, p27 staining was a good reflection of the level of p27
expression on Western blotting (Table 2)
.
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p27 mRNA was detected in all fresh cancerous samples. The levels
of p27 protein expression were not proportional to expression of p27
mRNA (Table 2)
.
Western Blot Analysis of Cdk2 and Cdk2 Activity
Eighteen of the 20 cancers exhibited increased levels of the
rapidly migrating, 33-kd phosphorylated form of Cdk2.34
Cdk2 activity was found at high levels in cancer tissues that showed
decreased expression of p27 (Figure 3A)
.
Densitometric analysis showed that the amounts of 33-kd Cdk2 expressed
in lung cancer tissues significantly correlated with Cdk2 activity
(r = 0.702, P < 0.001).
Statistical analysis also clearly showed that p27 LI has a strong and
inverse correlation with Cdk2 activity. The correlation coefficient was
-0.767 (P < 0.0001).
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Relationship between Cdk2 Activity and MIB-1 LI
Pearson's correlation coefficient demonstrated a strong
correlation between Cdk2 activity and MIB-1 LI with a correlation
coefficient of 0.767 (P < 0.0015) (Figure 4)
.
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Eight cases of 10 lung cancer specimens showed higher degradation
activity than corresponding nonneoplastic lung specimens. For these
specimens, p27 was reduced by approximately 50% within 120 minutes.
Figure 3B
shows two samples with high degradation activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reliability of the immunohistochemical assay for p27 LI in NSCLC was first described by Esposito et al.16 A tight correlation between immunohistochemistry and Western blot analysis of p27 in NSCLC was mentioned, although the data were not shown in their paper. In the present study, our data made a clear and strong correlation between the immunohistochemical assay for p27 LI and their Western blot analysis (r = 0.656, P < 0.0001). These results support the reliability of the immunohistochemical assay for p27 LI in NSCLC.
Ki-67 is the most reliable antibody for assessing growth by immunohistochemistry.21 High proliferative activity, as measured by Ki-67 antigen, has been shown to correlate with reduced p27 in lymphoid neoplasms, carcinomas of the oral cavity, and endocrine tumors, including pituitary, thyroid, and parathyroid gland hyperplasia.31,32,38 In contrast, studies showing no correlation between tumor cell proliferation and p27 have been reported in colorectal and breast cancers.39,40 A relation between Ki-67 and p27 protein was not previously analyzed in primary NSCLC.15,16,31,32 Therefore, we performed the study to investigate the association of proliferative activity (as measured by the expression of Ki-67) with alterations of p27 in primary NSCLC. Our results revealed that the expression for p27 and Ki-67 antigen had a weak but significant inverse correlation in primary NSCLC (r = -0.459, P < 0.001). This may indicate that p27 protein contributes to, or reflects, increased cell proliferation in primary NSCLC; however, within the limited population studied here, there was variability in this relationship from case to case. Thus, other mechanisms that influence the changes in proliferation in the NSCLC cannot be excluded.
Of the various histological types of human lung carcinoma, the most prevalent in Japan is adenocarcinoma. Little is known about the lesions preceding this type of lung cancer.41 Some investigators regard AAH lesions as precursors of adenocarcinomas, particularly bronchiolo-alveolar carcinomas.42-45
In the present study, the value of p27 LI and MIB-1 LI in AAH lay between those in normal bronchial epithelium and adenocarcinomas, but only two such cases were available for analysis. A decrease in the number of cells expressing p27 may play a role during progression from normal lung tissue to AAH and then to adenocarcinomas, although larger numbers of AAH lesions will have to be analyzed.
All cancerous lung tissues showed p27 transcripts at various levels and enhanced degradation activity of p27 compared with nonneoplastic lung tissues. We used samples from lung tissues as a source of ubiquitinating enzymes and proteasomes. Our results support the view that p27 abundance is mostly regulated at a posttranscriptional level by ubiquitin-proteasome-mediated proteolysis, and also that the p27 degradation pathway in NSCLC is enhanced as reported in previous studies.16,39
Our second objective was to analyze the relationship between MIB-1 LI and Cdk2 activity in lung cancer tissues. There have been a few reports measuring Cdk2 activity in fresh-frozen tumor tissues.46 We analyzed the expression of Cdk2 by Western blotting and Cdk2 activity by H1 histone kinase assay in nonneoplastic lung tissues and lung cancer tissues. To our knowledge, this is the first report demonstrating Cdk2 activity in lung cancer tissues. Densitometric analysis of the Western blots showed that the amounts of 33-kd Cdk2 expressed in lung cancer tissues were significantly correlated with Cdk2 activity (r = 0.702, P < 0.001). p27 LI and Cdk2 activity also showed a tight inverse correlation (r = -0.784, P < 0.0001). These results indicate that the proliferative activity of lung cancer tissues was closely related to Cdk2 activation in these tissues. It would be of value to study whether p27 degradation is directly linked to Cdk2 activation in NSCLC.5,47 In addition to the elimination of p27, Cdk2 activation requires the binding with cyclin E and the phosphorylation of threonine 160 by Cdk-activating kinase.48 These Cdk regulators may also play a role in the proliferation of NSCLC.
p21Cip1/Waf1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, which includes the p21, p27, and p57 proteins.6,7 A recent study has shown that p21 protein and mRNA in the majority of lung neoplasms are expressed at higher levels than in the corresponding normal tissues and that their expression is independent of p53 gene and protein alterations.35 The role of p21 in the proliferation of NSCLC will require further investigation.
The present study clearly demonstrates that decreased p27 expression correlates with increased Cdk2 activity and increased proliferative activity measured by MiB-1 LI in a series of NSCLCs. We also show that the degrading activity of p27 in NSCLCs was higher than in nonneoplastic lung tissues. The results support the conclusion that p27 and Cdk2 have an essential role in the proliferation of non-small cell cancers. The molecular mechanism of p27 degradation in proliferating cells has not been well documented. Clarification of the mechanism by which degradation of p27 in NSCLC is increased may eventually yield a new form of therapy for this kind of neoplasia.
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Acknowledgements |
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Footnotes |
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Accepted for publication May 8, 1998.
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