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From the Department of Pathology,*
Kitasato University
School of Medicine, Kanagawa and the Department of
Pathology,
Saiseikai Central Hospital,
Minato-ku, Tokyo, 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 proliferation is strictly regulated by a cell cycle control mechanism which depends on the activities of the G1 cyclins and cyclin-dependent kinase (cdk) complexes.14-21 These complexes are regulated both positively and negatively. Positive regulators include the cyclins and the recently identified cdk-activating kinase (CAK) by which cdks are phosphorylated at specific threonine residues and activated.22-25 In addition, multiple negative regulators exist, including universal cdk inhibitors p21, p27, and p57 and cdk4/cdk6 inhibitors p16, p15, p18, and p19.17-19,25-27 Thus, cell proliferation is controlled by complex and redundant mechanisms.
Despite a large body of morphological observations, the mode of tumor extension, the pathological mechanisms of cell proliferation in human pulmonary carcinomas, and, more particularly, the participation of various cell cycle regulators have not been fully analyzed at the cellular level.
To elucidate these mechanisms in lung adenocarcinoma, we examined the expression of cell cycle regulator proteins in the early stage of pulmonary adenocarcinoma by immunohistochemistry, with special emphasis on the G1/S- and S-to-G2 cell cycle transitions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study examined 54 cases of primary well differentiated
adenocarcinoma of the lung, each less than 3 cm in maximum diameter and
classified into Stage I (T1M0N0) by the TMN
classification.28
These adenocarcinomas were
derived from surgically resected materials obtained in the
Departments of Pathology, Saiseikai Central Hospital and
Kitasato University Hospital between 1987 and 1997. All of these cases
were classified as type C ("localized bronchioloalveolar carcinoma
with foci of active fibroblastic proliferation") according to the
histopathological classification of early adenocarcinoma of the lung
(Figure 1)
.11
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All archival tissue samples were routinely fixed in formalin and embedded in paraffin. Deparaffinized sections were autoclaved (120°C, 2 atm., 20 minutes) in 20 mmol/L citrate buffer (pH. 6.0).29 Immunostaining was performed with primary antibodies at the following dilutions: anti-cyclin A (monoclonal, Novocastra, Newcastle, UK), 1:500 dilution; anti-p21, anti-p27 (monoclonal, Novocastra), 1:100; anti-cdk-activating kinase (anti-CAK, monoclonal, Novocastra), 1:200; anti-p53 (monoclonal, DAKO, Glostrup, Denmark), 1:100; anti-Ki-67 (monoclonal, DAKO), 1:100; anti-cdk2 (polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), 1:2000; and anti-epithelial keratin (AE-1, monoclonal, ICN, Lisle, IL), 1:200. The specificity of these antibodies was confirmed by immunoblotting (data not shown). The conventional streptavidin-biotinylated horseradish peroxidase complex method (LSAB kit, DAKO, Kyoto, Japan) was used as directed by the manufacturer's instructions. Colorization was performed by the peroxidase-diaminobenzidine method. In selected cases, serial sections 1 µm in thickness were made and stained alternately for several kinds of proteins known to interact mutually or to form a complex, including cdk2-cyclin A-p21/p27 or p53-p21.
Immunoreactivity Scoring
In each case, positive staining was evaluated in three areas: nonneoplastic lung tissue adjacent to the tumor, the peripheral region around the advancing border of the tumor nodule (designated the "periphery") where the tumor cells proliferate in emerging extension on the alveolar surface, and in the center of the tumor, often accompanied by granulation or loose fibrosis. The percentage of positive cells was estimated by counting 500 tumor cells in 10 high-power fields in each area and evaluating semiquantatively. In the present study, cases showing >5% positive tumor cells were defined as positive.
Statistical Analysis
Differences in labeling indices (LIs) between the tumor cells from the periphery and those from the center were calculated from the staining results, compared, and analyzed by paired comparison t-test. Degree of correlation between LIs obtained for two marker proteins was calculated by the Spearman's rank correlation coefficient test.
Paired Tumor/Normal Lung Tissue Collection
Fresh fragments of tissue from the peripheral and the central
regions of tumor nodules of well differentiated adenocarcinoma and
paired adjacent nonneoplastic lung tissues were obtained from
surgically resected specimens and were used for immunoblotting analysis
and in vitro kinase reaction assay. These consisted of five
cases; their immunohistochemical profiles are listed in Table 1
.
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For protein extraction, fresh tissues were homogenized in high-salt lysis buffer (50 mmol/L Tris-HCl (pH 8.0), 0.25 mol/L NaCl, 0.5% Nonidet P-40, 0.1% sodium dodecyl sulfate, 5 mmol/L EDTA, 50 mmol/L NaF, 0.5 mmol/L phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin)30 on ice and the resulting lysates were sonicated on ice four times for 10 seconds each time.31 Lysates were clarified by centrifugation at 10,000 x g for 5 minutes.
Initially, 50-µg aliquots of protein were used for AE-1 blotting. The same specific antibody used in the immunohistochemical staining was used in 1:200 dilution. Protein was detected by the sequential application of a primary antibody followed by an alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI, 1:6000 dilution). Colorization was performed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indol-phosphate (Bio Rad, Gaithersburg, MD) in 100 mmol/L Tris buffer (pH. 9.6).
In Vitro Kinase Reaction
For immunocomplex kinase reactions to detect the activity of cdk2
or p27-associated cdks, tissues were lysed in NP40 lysis buffer (50
mmol/L Tris-HCl (pH 7.4), 0.5% Nonidet P-40, 0.15 mol/L NaCl, 50
mmol/L NaF, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl
fluoride, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin) with addition
of 1 mmol/L
Na3VO4.30
To standardize the amount of extracts obtained from tumor cells or
nonneoplastic epithelial cells from the three areas, the protein
amounts from epithelial cells used for in vitro kinase assay
were determined by anti-AE1 (keratin) blotting. In brief, lysate
protein concentrations were quantitated by protein assay kit (Bio Rad,
Hercules, CA), 50 µg of extract were then subjected to anti-AE-1
immunoblotting analysis, and the results were quantified using a
GT6500ARTS scanner (Epson Co., Tokyo). All scans were kept as 16-bit
PICT files and incorporated into NIH Image software (version 1.56) for
densitometric analysis.32
From the results of
densitometric analysis, aliquots of lysates containing equivalent
amounts of AE-1 were taken and incubated with anti-cdk2 antibody
(diluted 1:150) for 1 hour followed by an additional 2-hour incubation
with protein A-Sepharose beads at 4°C. For p27-associated kinase
reactions, lysates were incubated with anti-p27 (diluted 1:100) for 1
hour followed by an additional 2-hour 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 was used as a substrate (0.5 µg protein) in 50 µl of
kinase reaction buffer (50 mmol/L Tris-HCl (pH 7.2), 10 mmol/L
MgCl2, 1 mmol/L dithiothreitol, 20 mmol/L
[
-32P]ATP(5 µCi;1 µCi = 37 kBq)
(ICN, Irvine, CA).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 in this study was confined
almost exclusively to the nuclei (Figure 2)
. The overall results of the LIs from
immunohistochemical analysis are shown in Figures 2 and 3
and described below.
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Proliferative activity was evaluated by determining the Ki-67 LI.
The Ki-67 LI was 10 to 20 times higher than the mitotic index, which
demonstrated the staining reliability of the
antibody.33
Nuclear positivity in the tumor cells
was identified in 90.7% (49/54) of the cases. In these 49 cases, the
LI of the tumor cells in the periphery ranged from 8 to 65%, whereas
that of cells in the center ranged from 1 to 45%. This difference in
LIs was statistically significant (P < 0.0001
by t-test, Figure 3
). There were three cases in which the
center exhibited a higher LI than the periphery. Those cases turned out
to contain small foci of tumor nests in solid proliferation,
corresponding to moderately or poorly differentiated adenocarcinoma in
those particular areas. Most of the cells in the nonneoplastic lung
tissue stained negatively except for occasional fibroblasts.
Cyclin A
The distribution of cells staining positive for cyclin A appeared
to parallel Ki-67 staining except in nonneoplastic lung tissue, where
positivity was identified only in the germinal center of lymphoid
follicles. Nuclear positivity in the tumor cells was identified in
72.2% (39/54) of the cases. The cyclin A LIs of tumor cells in the
periphery ranged from 7 to 68% and were higher to a statistically
significant degree (P < 0.001) than those in
the center, for which LIs ranged from 1.5 to 33% (Figures 2 and 3)
.
Cdk2
Cdk2 positivity was restricted to tumor cells (Figure 2)
. Nuclear
positivity in tumor cells was identified in 83.3% (45/54) of the
cases. In general, the cdk2 LI of the tumor cells was higher in the
periphery (9–69%) compared with the center (2–50%) to a
statistically significant degree (P < 0.05).
CAK
Nuclear positivity for CAK was ubiquitously identified in the
tumor cells and in the nonneoplastic cells, including bronchial
epithelial cells and alveolar macrophages. In the tumor cells,
CAK-positive staining was identified in 94.4% (51/54) of the cases and
the LI values among the tumor cells were quite similar at the periphery
and the center, ranging from 11 to 93%. Staining intensity was almost
identical in all areas and among various kinds of cells, except for a
few instances in which the LIs and staining intensity were slightly
higher in the periphery (Figure 3)
. Statistically, no obvious
difference in the LI between the periphery and the center of the tumors
was found (P > 0.1).
p21
Cells in the nonneoplastic lung tissue generally showed no or very
rare and faint staining. The exceptions were occasional intense
positivity in the infiltrating lymphocytes and macrophages. Nuclear
positivity in the tumor cells was identified in 72.2% (39/54) of the
cases. In the positive cases, the LI was approximately, 8 to 67% in
the periphery and 2 to 42% in the center (Figures 2 and 3)
. However,
the difference between p21 LIs for the two areas was not statistically
significant (P > 0.05).
p27
In nonneoplastic lung tissue, the bronchial and alveolar
epithelial cells and lymphoid cells showed intense nuclear staining for
p27 protein. In tumor, positive staining was demonstrated in 94.4%
(51/54) of the cases. Cells located in the periphery showed higher LI
(range, 12–88%) than those in the center (range, 2–68%) (Figures 2 and 3)
. The difference in p27 LIs between the two areas was
statistically significant (P < 0.0001). In
addition, staining in the periphery was more intense, although not as
intense as in nonneoplastic cells.
p53
63.0% (34/54) of the cases exhibited positive staining for the
p53 protein. No significant difference in LI was found between cells in
the periphery and those in the center of the tumors
(P > 0.1) (Figures 2 and 3)
.
Specific Correlations in Staining Patterns
The signal cascade in which many of these cell cycle regulators
are involved, and their interactions with one another have been
extensively studied in the past few years. Based on the known
relationship among some of these molecules, we statistically evaluated
the immunohistochemical results (LI) obtained in the periphery of the
tumor nodule as follows (Table 2)
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No specific relationship between p53 and p21 staining (ie, coexpression or reciprocal staining) was identified in either individual tumor cells or individual cases. The correlation coefficient as determined by Spearman's test between these two LIs was -0.171 (P > 0.1).
p21 and p27
Overall, there was a weak but significant inverse correlation
between these two LIs with a correlation coefficient of -0.573
(P < 0.001). Those cases which revealed high
p21 LI showed no or weak p27 staining in occasional tumor cells at the
periphery and vice versa. Furthermore, in serial sections, expression
of p21 and that of p27 were observed to be reciprocal at the cellular
level, not only in the tumor cells, but also in the bronchial or
alveolar epithelial cells and lymphocytes (Figure 4)
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The correlation between cyclin A and cdk2 LIs was statistically
significant with a coefficient of 0.74 (P <
0.001). Analysis of serial sections demonstrated that cells positive
for cyclin A staining also exhibited positive cdk2 staining at a high
frequency (Figure 5)
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Although cyclin A-cdk2 positivity was observed predominantly at
the periphery, positive CAK staining was observed ubiquitously, ie, in
both the periphery and the center (Figures 2 and 3)
. The correlation
coefficient between LIs of CAK and cyclin A was 0.12 and between CAK
and cdk2 it was 0.11. Neither was statistically significant.
Cyclin A-cdk2 and p21 or p27
p21 and p27 are universal inhibitors of cyclin-cdk complexes,
including cyclin A-cdk2, which was examined in this study. There was
occasional overlap of the staining patterns for cyclin A-cdk2 and p21
or p27 in the tumor cells. However, the correlations between the cyclin
A and the p21 LIs (
= 0.078) or the cyclin A and the p27 LIs
( = 0.312) were not statistically significant.
Cdk2- or p27-Associated Kinase Activity
Cdk2- or p27-associated kinase activity was examined in fresh
tissue samples from five selected cases which showed
immunohistochemical positivity for cyclin A, cdk2, and p27 (Table 1)
.
Protein amounts used for the assay were standardized by anti-AE1
blotting (Figure 6
, lower panel). This
standardization gave us equal amounts of protein from epithelial tumor
cells and normal epithelial cells, because almost all of the tumor
cells as well as nonneoplastic bronchial and alveolar epithelial cells
stained positive by AE-1 on immunohistochemistry (data not shown). We
found that the periphery exhibited significantly higher levels of cdk2
kinase activity compared with the center. As a control, matched
nonneoplastic lung tissues from the same case revealed no detectable
cdk2 kinase activity (Figure 6
, upper panel). Furthermore, the
periphery exhibited higher levels of p27-associated kinase activity
than the center. Adjacent nonneoplastic lung tissues from the same case
revealed no detectable p27-associated kinase activity (Figure 6
, middle
panel). We obtained similar results of kinase assay in all five cases
examined. Because the p21 LI was lower than the p27 LI in all samples
tested, we were unable to detect p21-associated kinase activity in any
area of the five samples (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To elucidate this mechanism, we selected 54 cases of small well differentiated adenocarcinoma. This was a very useful model for our experimental purpose because all cases showed tumor polarity without significant secondary changes such as necrosis or dense hyalinization. The clinicopathological implications could not be evaluated in this study because all of the cases were classified into Stage I and were expected to show a favorable prognosis. However, these studies were able to clarify several essential mechanisms in this process, particularly the involvement of cell cycle regulators.
First of all, Ki-67 staining revealed heterogeneous proliferative activity in the tumor nodules characterized by a significantly higher LI in the periphery.
Second, these actively proliferating, peripherally located cells stained positively for cyclin A and cdk2, as we previously demonstrated in squamous cell carcinoma of the lung.31 Presumably, their coexpression and consequent high kinase activity should serve to promote active proliferation.31 Indeed, in vitro cdk2 kinase assay revealed higher kinase activity in the periphery.
Third, this up-regulation of cdk2 activity is not induced through a CAK-dependent pathway. This is in contrast to a previous report in which the proliferative activity in human breast and urinary bladder carcinomas was attributed to up-regulated expression of CAK protein.38
Assuming that the cyclin A-cdk2 complex is not activated by the
positive regulator CAK, one alternative interpretation is that its
activity is up-regulated by the suppression of kinase inhibitors.
However, our results show that p21 and p27 exhibit higher LIs in the
periphery. To test the idea that these molecules do not function as
kinase inhibitors in these cases, we examined p27-associated cdk2
kinase activity in fresh tissue samples using a cdk2-specific protein
extraction procedure.30,39
Our results showed
that p27 probably forms complexes with cyclins-cdk2 and that these
complexes exhibit higher kinase activity in the periphery despite
higher p27 expression. As illustrated in Figure 7
, these results can be interpreted in
two ways.
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is that the inhibitory effects of up-regulated
p21 or p27 on kinase activity in the periphery are overcome by the
greater up-regulation of cyclin A-cdk2 activity (Figure 7a
, middle). In
the central region of the tumor nodules, expression of p21/p27 is
lower, but cyclin A-cdk2 expression is also lower, resulting in low
kinase activity (Figure 7a
, right). In nonneoplastic lung tissue,
cyclin A-cdk2 is not significantly detectable; thus, p21/p27 inhibition
is more effective (Figure 7a
, left). This may be a reasonable
interpretation of the results of immunohistochemistry.
The second interpretation (Figure 7b)
is that the functions of p21,
p27, or both change depending on the stoichiometry of their
interactions with cyclin A-cdk2.40
In the
nonneoplastic tissue, p21 and p27 are up-regulated and exert their
function as kinase inhibitors, inactivating the cyclin A-cdk2 kinase
complex (Figure 7b
, left). However, in the proliferating cells of the
periphery, expression of p21/p27 is intermediate. Here, they function
as positive regulators by, for example, serving as assembly factors for
the cyclin-cdk complex (Figure 7b
, middle). In contrast, low expression
of p21/p27 in the central region of the tumor results in low cyclin
A-cdk2 complex formation due to insufficient assembly factor activity
(Figure 7b
, right).
We support the latter hypothesis for several reasons. First, although p21 has been characterized as a negative regulator of the cell cycle, we have successfully established cell lines constitutively expressing p21 derived from rat pheochromocytoma PC12 cells.30 Furthermore, the one among those cell lines which exhibited the highest expression of p21 protein also showed a slightly enhanced proliferation rate compared with parental cells. These results are consistent with the idea that at some expression level, p21 can function as an assembly factor and activator of cdk rather than a kinase inhibitor. This is also supported by Zhang et al, who demonstrated that addition of recombinant p21 protein to cyclin A-cdk2 complex in vitro resulted in a dose-dependent, biphasic change in cdk2-kinase activity; activity initially increased at lower p21 concentrations and then rapidly disappeared as p21 concentration was increased.40 From this, we imagine that p21 and p27 could function as assembly factors of cdk and cyclin to form stable ternary or quaternary complexes, similar to the way p37/MAT1 functions to promote assembly of cyclin H and cdk7 into the CAK complex.41,42
Our analysis of serial sections further revealed that expression of p21 and p27 are inversely correlated in a statistically significant manner, suggesting that they function as reciprocal and redundant cdk regulators.
Finally, the expression of p21 seemed to be regulated by a p53-independent pathway in adenocarcinoma of the lung, similar to what has been described in several other kinds of human tumors including pancreatic or non-small cell lung carcinomas.43-45
From a pathological viewpoint it appears that despite their lower proliferative activity, centrally located tumor cells still retain their malignant potential as ascertained by such criteria as vascular invasiveness.13,46 Although one explanation for this biological aggressiveness may be the abundance of capillaries and lymph vessels in the center, the discrepancy between proliferative capability and high invasiveness should be elucidated by future study.
In conclusion, we propose the following hypothesis for the mechanism of extension of pulmonary adenocarcinoma at the cellular level:
Peripherally located tumor cells maintain their proliferative potential and extend outward on the alveolar surface. During this process, high kinase activity manifested by cyclin A-cdk2 complexes plays a crucial role.31 Cyclin A and cdk2 are associated and form a stable complex31 and p21/p27 expression is mildly suppressed below the level at which p21 and p27 can exert an inhibitory effect on cdk.
As the advancing border of the tumor extends, centrally located tumor cells adapt themselves to the fibrotic process, probably by host response. During that process, p21/p27 expression is down-regulated, possibly by up-regulation of some type of suppressor of p21/p27. Concomitantly, cyclin and cdk are also down-regulated, thus resulting in an overall decrease in kinase activity.
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Acknowledgements |
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Footnotes |
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Supported by grant-in-aid for Scientific Research (no. 09770130) from the Ministry of Education, Science and Culture in Japan and by a Mitsui Life Social Welfare Foundation Research Grant.
Accepted for publication November 20, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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