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From the Department of Pathology, Kitasato University School of Medicine, Kanagawa 228-8555 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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Among these cyclins only cyclin D1 has been identified as a proto-oncogene, designated PRAD1. It is overexpressed in lung, breast, gastric, and esophageal carcinomas at a frequency ranging from 13 to 60% with or without amplification of the 11q13 region.10-15 Amplification and/or overexpression of cyclin E has also been reported in colorectal and breast carcinomas.16-21 Overexpression of cyclin A has been reported in several cases of cultured cell lines from alveolar epithelial cells of the lung.22 In addition, the cyclin A gene was found to be the unique insertion site of hepatitis B virus (HBV) in one clonal hepatoma. Cyclin A may thus play a role in the continuous proliferation of liver cells and ultimately in the pathogenesis of hepatocellular carcinoma.23,24 Based on these observations cyclins and CDKs are simply believed to be positive regulators of cell cycles and the pathological mechanisms of tumorigenesis and tumor cell proliferation in human lung carcinoma due to aberrant expression of various cell cycle regulators have not been fully analyzed. The histopathological heterogeneity of human lung carcinomas suggests that they may be caused by diverse cellular mechanisms. In this study we focused on the G1/S and S to G2 transitions in the cell cycle and examined the expression of cyclins A and E as well as their catalytic partner, CDK2, by immunohistochemistry. Furthermore, we performed immunoblotting analysis and in vitro kinase reaction assays to examine the expression of these molecules and their associated kinase activity in matched sets of tumor and normal tissues of the lung and in cultured cell lines of human lung carcinoma. Finally, we analyzed the data in relation to patient survival rates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For this study we examined 190 cases of primary lung carcinoma obtained from surgical material including biopsies and from autopsies in the Department of Pathology, Kitasato University Hospital, between 1980 and 1996. According to the World Health Organization (WHO) histological classification,25 these cases included 55 squamous cell carcinomas (SCC), 58 adenocarcinomas (AC), 36 small cell carcinomas (SmCC), and 41 large cell carcinomas (LCC).
Archival Tissue Samples and Immunohistochemistry
All archival tissue samples were routinely fixed in formalin and
embedded in paraffin. Deparaffinized sections were autoclaved (120°C,
2 atm, 20 min) in 20 mmol/L citrate buffer, pH
6.0.26
Immunostaining was performed with primary
antibodies at the following dilutions: anti-cyclin A (monoclonal,
Novocastra, Newcastle, United Kingdom), 1:500 dilution; anti-cyclin E
(monoclonal, Novocastra), 1:100; anti-CDK2 antibody (polyclonal, SANTA
CRUZ, Santa Cruz, CA), 1:2000. The specificity of these antibodies was
confirmed by immunoblotting (see Figure 4
). The conventional
streptavidin-biotinylated horseradish peroxidase complex method (LSAB
Kit, DAKO Japan, Kyoto, Japan) was used according to the
manufacturer's instructions. Colorization was performed by the
peroxidase-diaminobenzidine (DAB) method.
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The number of tumor cells with positive staining varied among the cases. The percentage of positive cells in each case was estimated by counting 500 tumor cells in 10 high-power fields and semiquantatively evaluated into one of the following five immunoreactivity groups: (a) immunoreactivity completely absent (negative, 0%); (b) <5%; (c) <30%; (d) <50%; and (e) up to 100%. In the present study, cases showing >5% of positive tumor cells were defined as "positive."
Patients' Follow-Up
Patients' outcome data were collected from hospital charts. Informative patient charts were available for 41 of SCC, 38 of AC, 17 of SmCC, and 29 of LCC cases. Follow-up period ranged from 2 months to 14 years after the pathological diagnosis.
Statistical Analysis
In immunohistochemical analysis, the ratios of positive cases to total cases among the four histological subtypes were compared and the differences were statistically examined by normal distribution analysis. Patients' survival curve was calculated using Kaplan-Meier analysis with the data obtained from hospital charts. The correlations between the rates of expression of cyclins or CDK2 and overall survival time were analyzed and the differences between the groups were tested for statistical significance with the Mantel-Cox test.
Cell Lines
Cultured cell lines from human lung carcinomas were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. These included Lu-130, Lu-134B, Lu-135 (small cell carcinomas) kindly provided by the National Cancer Center Research Institute (Tokyo) and PC-13 (poorly differentiated adenocarcinoma) provided by the Tokyo Medical College.
Paired Tumor/Normal Lung Tissue Collection
Fresh fragments of paired tumor and adjacent normal tissues were
obtained from surgically resected specimens and were used for
immunoblotting analysis, immunoprecipitation (IP), and in
vitro kinase reaction assay. These comprised 3 cases of SCC, 3 of
AC, 1 of SmCC, and 2 of LCC. (Histopathological and immunohistochemical
profiles of those cases are listed in Table 5
.)
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For protein extraction, cells and tissues were lysed in high-salt lysis buffer (0.5% Nonidet P-40 (NP-40), 0.1% SDS, 50 mmol/L Tris-HCl, pH 8.0, 0.25 mol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml aprotinin, 5 µg/ml leupeptin).27 Fresh tissues were homogenized in high-salt lysis buffer on ice and the resultant lysates were sonicated on ice 4 times for 10 seconds each time.28 Lysates were clarified by centrifugation at 10,000 x g for 5 minutes.
Immunoblotting Analysis
Equal amounts (50 µg) of protein were used for immunoblotting. The same specific antibodies used in the immunohistochemical staining were used in the following dilutions: anti-cyclin A, 1:300; anti-cyclin E, 1:100; and anti-CDK2 antibody, 1:150. Each protein was detected by the sequential application of a specific primary antibody followed by an alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI, 1:6000 dilution). Colorization was performed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indol-phosphate (BCIP) (Bio-Rad, Gathersburg, MD) in 100 mmol/L Tris buffer, pH 9.6. For immunoprecipitation followed by immunoblotting, cells were lysed in NP-40 lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 0.5% NP-40, 0.15 mol/L NaCl, 50 mmol/L NaF, 1 mmol/L dithiothreitol (DTT), 1 mmol/L PMSF, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin).29 Lysates (200 µg of protein) were incubated with p13suc1-Sepharose for 4 hours at 4°C. The precipitates were used for further immunoblotting analysis for cyclin A or cyclin E. Alternatively, lysates were incubated with anti-cyclin A (diluted 1:300) or anti-cyclin E (diluted 1:100) antibodies for 1 hour at 4°C followed by an additional 1 hour incubation with protein G-Sepharose beads at 4°C.29 The immunoprecipitates were used for immunoblotting analysis for CDK2.
In Vitro Kinase Reaction
For immunocomplex kinase reactions for CDK2, cells were lysed in
NP-40 lysis buffer with addition of 1 mmol/L
Na3VO4.30
Lysates (250 µg of protein) were incubated with anti-CDK2 antibody
(diluted 1:150) for 1 hour followed by an additional 1 hour of
incubation with protein A-Sepharose beads at 4°C. For cyclin A- or
cyclin E-associated kinase reactions, lysates (250 µg of protein)
were incubated with anti-cyclin A (diluted 1:300) or anti-cyclin E
antibody (1:100) for 1 hour followed by an additional 1 hour of
incubation with protein G-Sepharose beads at 4°C. A
bacterially-expressed fragment of the retinoblastoma protein (pRB,
amino acids 385–928) fused to glutathione S-transferase (GST) was used
as a substrate (0.5 µg of protein) in 50 µl of kinase reaction
buffer (50 mmol/L Tris-HCl [pH 7.2], 10 mmol/L
MgCl2, 1 mmol/L DTT, 20 mmol/L
[
-32P]ATP(5 µCi;1 µCi = 37 kBq,
ICN, Irvine, CA).27,30
After incubation for 10
minutes at room temperature, the sample was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis followed by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Positive immunohistochemical staining for cyclin A, cyclin E, and
CDK2 was confined almost exclusively to the nuclei of the tumor cells
(Figure 1)
. The overall results of
the immunohistochemical analysis are summarized in Table 1
.
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and LCC (Figure 1d)
were
almost ubiquitously identified within the tumor nests, those in SCC
were identified predominantly in the basal areas of tumor nests and
well differentiated, keratinized areas, often localized in the central
portion of the nests, remained unstained (Figure 2a)
. In AC, 52% (30/58) stained
positively, and 70% of these (21/30) contained fewer than 30% total
positive cells (Table 2)
.
Furthermore, the proportion of positive tumor cells was inversely
correlated with the degree of morphological differentiation. In LCC,
positive staining was identified in all histological subtypes, ie,
ordinary, spindle, and giant cell types, and there was no predilection
for a certain subtype. The ratio of positive cases to total cases was
lower to a statistically significant degree in AC than in the other
three subtypes (Table 1)
.
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and Figure 1
). In
the SCC cases, 27% (15/55) stained positively, particularly tumor
cells in differentiated areas that were unstained by cyclin A staining
(Figure 2a)
. In AC, only 5% (3/58) cases of moderately and poorly
differentiated subtypes exhibited positive staining (Figure 1c
and
Table 3
). In SmCC, 17% (6/36) stained positively and none of these 6
cases contained more than 30% total positive cells (Figure 1c
and
Table 3
). In LCC, 37% (15/41) stained
positively (Figure 1d
and Table 3
). In several of these LCC cases,
positive tumor cells were found to be squamoid or large multinucleated
giant cells with rich cytoplasm which were unstained by cyclin A
staining (Figure 1d and 2b)
.
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. In addition, there was no obvious
correlation between the degree of differentiation and positive CDK2
staining. In the immunohistochemical investigation on serial sections
of all types of carcinomas, tumor cells expressing cyclin A also
displayed positive staining for CDK2 protein at a high frequency
(Figures 1 and 2c)
. However, well differentiated cells in SCC and
squamoid cells in LCC, both of which stained positively for cyclin E,
also revealed positive staining for CDK2 (Figure 2a and 2b)
.
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Staining results of cyclins A and E and CDK2 were evaluated for
their correlation with patients' survival rate. The cyclin A-positive
group revealed lower survival rate compared with negative group in
non-SmCC at a statistically significant level
(P < 0.02, Figure 3a
). In SmCC cases, correlation could not
be analyzed due to the paucity of cyclin A-negative cases. Conversely,
the cyclin E-positive group showed a significantly higher survival rate
compared with the cyclin E-negative group in both SmCC and
non-SmCC groups (Figure 3b)
. For CDK2 staining, no specific correlation
was found with patients' survival (data not shown).
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To further confirm the results observed by immunohistochemical
staining, immunoblotting analysis was performed using lysates obtained
from paired tumor and normal tissue samples of the lung as well as
cultured human lung carcinoma cell lines. Profiles of these cases in
immunohistochemical stainings are summarized in Table 5
.
Cyclin A blotting revealed one major band of 58 kd, suggesting the
absence of any rearrangement or chimeric protein formation (Figure 4a)
. In cell lines and tumor tissues that
exhibited higher numbers of positively stained cells on
immunohistochemistry (cases 2, 3, 6–9), significantly higher levels of
cyclin A expression were detected compared with normal lung tissue from
the same cases. The level of protein expression correlates well with
the number of positive cells revealed by immunohistochemistry (Figure 4
and Table 5
). All cell lines, 1 AC and 3 SmCC, also exhibited
higher level of expression.
Cyclin E blotting revealed two major bands of 49 and 43 kd,
corresponding to the two alternatively spliced forms of human cyclin E
(Figure 4b)
.31
Although expression of cyclin E
was elevated in SCC (cases 1–3) and LCC (cases 8 and 9), this was not
observed in the cases of AC (cases 4–6) and SmCC (case 7), consistent
with the lower numbers of positive cells detected by
immunohistochemistry. Although two cell lines of SmCC, Lu-134 and
Lu-135, exhibited noticeably higher levels of expression, other cell
lines, one AC (PC13) and one SmCC (Lu130), revealed levels of
expression similar to that in the tumor tissue.
Anti-CDK2 blotting revealed two major bands of 34 and 33 kd
corresponding to the hyper- and unphosphorylated forms of human CDK2
(Figure 4c)
.27
The levels of CDK2 expression
almost paralleled those revealed by anti-cyclin A blotting (Figure 4c)
.
Immunoblotting Analysis of CDK-Associated Cyclin A and Cyclin E
To evaluate the levels of cyclin A and cyclin E associated with
CDK in the cases which showed significantly higher level of expression
by immunoblotting analysis, we subjected p13suc1
precipitates prepared from the selected tissue samples and cell lines
to sequential immunoblotting analysis. As shown in Figure 5a
, the amount of cyclin A and cyclin E
associated with CDK which bound to
p13suc1-Sepharose was generally higher in cell
lines and in tumor samples compared with matched adjacent normal lung
tissues. To further confirm the association of CDK2 with cyclin A or
cyclin E, we subjected anti-cyclin A or anti-cyclin E
immunoprecipitates to sequential immunoblotting for CDK2. As shown in
Figure 5b
, we observed higher levels of CDK2-cyclin complexes in these
tumor tissues and in cell lines.
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CDK2-associated kinase activity was examined in selected tissue
samples and cell lines which showed significantly higher level of
protein expression by immunoblotting analysis after IP from the lysates
using anti-cyclin A, anti-cyclin E, and anti-CDK2 antibodies. Tumor
tissues and cell lines exhibited higher levels of cyclin A- or
CDK2-associated kinase activity compared to those obtained from matched
normal lung tissues (Figure 6a, b)
.
However, there was no significant cyclin E-associated kinase activity
in lysates even from tumor tissues expressing higher levels of cyclin
E-CDK2 complexes compared with that in normal tissue, except
one case of tumor tissue (case 7) and a cell line from SmCC,
Lu135 (Figures 5 and 6a)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we clearly demonstrated diverse implications regarding the expression of cyclin A-CDK2 and cyclin E-CDK2 complexes, both of which have been simply believed to be positive regulators of cell cycle.
Positive immunohistochemical staining for cyclin A was detected in more than 80% of the cases of lung carcinoma. One possibility is that this positive cyclin A staining simply reflects an increase in the proportion of the cell populations in the S through G2/M phases. However, this is unlikely; non-neoplastic lung tissue barely exhibited positive staining for cyclin A, even though fluorescence activated-cell-sorting analysis of non-neoplastic lung tissues revealed that approximately 7% of these cells were in the S through G2/M phases (data not shown). From these observations, we speculate the positive cyclin A staining in immunohistochemistry represents overexpression.
Cyclin A was found to be overexpressed in most SCC, SmCC, and LCC cases. Furthermore, in SCC and AC, poorly differentiated subtypes tended to express cyclin A at a higher frequency and with a higher proportion of positive tumor cells. Analysis of serial sections revealed that many of the cells positive for cyclin A co-expressed CDK2, suggesting that these cells maintain their transformed and aggressive phenotype due to high cyclin A-associated CDK2 kinase activity. The distribution of cyclin A-positive cells in the basal areas of tumor nests of SCC which are interpreted as the proliferating zone, supports this notion. The lower frequency of positive cyclin A staining in AC suggests a lower cell proliferation rate, consistent with a previous report describing lower labeling index for BrdU and proliferating cell nuclear antigen (PCNA) in AC.34-36
In contrast, expression of cyclin E seemed inversely correlated with the proliferative activity of tumor cells, since well differentiated cells in SCC, which are assumed to have low proliferative activity, stained positively for both cyclin E and CDK2. Possibly, cyclin E plays a role in cellular differentiation, as described for the process of neuronal or osteoblastic cell line differentiation.27,37 Alternatively, as demonstrated in the present study, CDK2-cyclin E complexes accumulate in these well differentiated cells without catalyzing significant kinase activity, suggested in a previous report using senescent human diploid fibroblasts.38 If this is the case, the lack of kinase activity could be due to the action of CDK-cyclin inhibitor proteins p21, p27, p57, or others, and/or to changes in the phosphorylation state of CDK2 that modulate its activity.5,38-41 Although these hypotheses seemed applicable to most of the cases in the present study, we do not have reasonable explanation for a case of SmCC (case 7) and a cell line Lu135 both of which manifested higher cyclin E-associated kinase activity. We speculate that there was alteration in cyclin E gene resulting in stable and functional activation of cyclin E-CDK2 complex or functional inactivation of CDK2 inhibitors, and cyclin E may play a role for the tumor cells to get growth advantage in those particular cases.
Immunoblotting analysis detected cyclins A and E and CDK2 of an apparent wild-type molecular weight. Thus, the possibility of rearrangement, truncation, or chimeric protein formation as previously described for cyclins A and E,18,21,23,24 is less likely in the case of lung carcinoma.
The clinicopathological implications of amplification/overexpression of cyclins D1 and E in human tumors have been variously described in the literature as positive or negative prognostic factors.20,21,42,43 G1/S cyclins that are overexpressed but not genetically altered are presumably catalytically inactive and found predominantly in differentiated tumor cells, as are inactive cyclin E-CDK2 complexes detected in our present study, and eventually associated with better prognosis. Indeed, the immunohistochemical positivity of cyclin A was demonstrated to be related with shorter survival and cyclin E with longer survival compared with respective negative groups.
Altogether, our results show that expression of cyclin A-CDK2 complex and its associated kinase activity serve as novel markers that predict proliferative activity and eventually, patients' prognosis in lung carcinoma. Apparently, these complexes cause an increase in the population of S phase cells and drive the tumorous phenotype, particularly in SCC, SmCC, and LCC. Overexpression of cyclin E-CDK2 complexes, which normally play a crucial role in the G1/S transition, does not seem to be most critical in the cell cycle control in lung carcinoma, and is rather associated with better prognosis. However, based on the data that cyclin E expression was exclusively identified in the tumor cells, cyclin E-CDK2 complex appear to be expressed and accumulated in an inactive form in pararell with down-regulation of cyclin A-CDK2 expression when the tumor cells show differentiation or senescence after the phase of active proliferation.
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Acknowledgements |
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Footnotes |
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Supported in part by Grants-in-aid for Scientific Research 09770130 and 07670219 from the Ministry of Education, Science and Culture in Japan and by Kitasato University Research Grant for Young Researchers.
Accepted for publication June 10, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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