Analysis of cell cycle regulator proteins in non-small cell
V Esposito, A Baldi, G Tonini, B Vincenzi, M Santini, V Ambrogi, T C Mineo, P Persichetti, G Liuzzi,
V Montesarchio, E Wolner, F Baldi, A M Groeger
See end of article for
Dr A Baldi, Via G. Orsi 25,
80128 Naples, Italy;
Accepted for publication
4 July 2003
J Clin Pathol 2004;57:58–63
Background/Aims: Abnormalities of the proteins involved in cell cycle checkpoints are extremely common
among almost all neoplasms. This study aimed to investigate the expression of four components of the cell
cycle machinery—p21, p16, p53, and proliferating cell nuclear antigen (PCNA)—in non-small cell lung
Methods: The expression of p21, p16, p53, and PCNA was examined in 68 well characterised NSCLC
specimens using immunohistochemistry. The coregulation of these proteins and their influence on survival
were analysed using both univariate and multivariate analyses.
Results: By univariate analysis, the expression of all the proteins examined, except for PCNA, was
significantly correlated with survival. In multivariate analysis, the only immunohistochemical parameter
able to influence overall survival was p16, confirming the hypothesis that the RB–p16 tumour suppressor
pathway is inactivated in most lung cancer samples. Finally, the group of patients with NSCLC who were
negative for both p21 and p16 had a significantly shorter overall survival.
Conclusions: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in
lung cancers, and support the idea that functional cooperation between different cell cycle inhibitor
proteins constitutes another level of regulation in cell growth control and tumour suppression.
continues to be poor.1 2This situation exists because of
difficulty in reaching an early diagnosis and because several
aspects of lung cancer pathogenesis have not been clarified
yet. Nevertheless, great progress has been made in under-
standing the molecular and cellular pathogenesis of lung
cancer.3One area that has been the focus of much research is
cell cycle control. The precise regulation of the cell cycle is a
fundamental requirement for the homeostasis of the eukary-
otic cell. During the past decade, scientists have successfully
delved into the molecular machinery controlling the fine
regulation of the cell cycle, identifying and characterising
several genes and gene products involved.4A key role is
played by cell cycle kinases (CDKs), relatively small proteins
with an apparent molecular mass between 33 kDa and
43 kDa. The activity of these molecules is regulated by their
arrangement in a multimeric complex with larger proteins,
called cyclins because of their cyclical expression and
degradation during the cell cycle. Different CDK–cyclin
complexes, formed with precise timing throughout the cell
cycle, together with their phosphorylation/dephosphoryla-
tion, efficiently regulate the activity of the multimeric
holoenzyme. Conversely, CDK–cyclin complexes are nega-
tively modulated by the binding of a family of small proteins
called CDK inhibitors; namely the CIP (p21 and p27) and the
INK (p16) families.5 6The p53 tumour suppressor gene is also
involved in cell cycle checkpoints because it encodes a protein
that acts as a transcription factor for several cell cycle
regulatory proteins, including the p21 gene.7In contrast,
proliferating cell nuclear antigen (PCNA) is involved in
activation of DNA polymerase d, which is required for DNA
replication and repair.8 9Finally, the p53–p21 pathway also
inhibits DNA replication by merit of the interaction between
p21 and PCNA, without affecting the DNA repair function of
ung cancer is one of the most prevalent and lethal
tumours in western countries. Despite recent advances in
oncological treatment, the prognosis for this neoplasm
‘‘Cyclin dependent kinase (CDK)–cyclin complexes are
negatively modulated by the binding of a family of small
proteins called CDK inhibitors; namely the CIP (p21 and
p27) and the INK (p16) families’’
Although several of the factors involved in regulating cell
cycle control have been investigated in lung cancer, few
studies have examined multiple factors in the same tumour
series. Therefore, the aim of our study was to evaluate the
expression of the p53, p21, p16, and PCNA proteins in a large
series of non-small cell lung cancers (NSCLCs) to assess the
integrity of cell cycle checkpoints in these tumours, to
evaluate the coexpression of these proteins, and to examine
the relation between these cell cycle regulators and the
clinicopathological features of NSCLCs, including their ability
to predict survival in patients with NSCLC.
MATERIALS AND METHODS
Patients and tissue samples
We retrospectively evaluated surgical specimens from 68
patients with NSCLC who had undergone surgical resection
or biopsy in the departments of thoracic surgery, University
of Vienna, Austria and University of Rome, Italy. The case
series under investigation was representative of unselected
series of NSCLC. Tumour staging was performed according to
the international system for staging lung cancer.12The
patients consisted of 50 men and 18 women (median age,
58 years). All patients underwent surgery (50 patients) or
According to the international system for staging lung
cancer, there were 23 patients with clinical or pathological
... ............ ............. ............ ............. ...........
Abbreviations: CDK, cyclin dependent kinase; CI, confidence interval;
NSCLC, non-small cell lung cancer; PCNA, proliferating cell nuclear
stage I, 21 patients with pathological stage II, and 24 patients
with clinical or pathological stage III (18 IIIA and six IIIB).
The morphological classification of the carcinomas was
conducted according to the World Health Organisation
specifications: 35 were squamous carcinomas, 29 were
adenocarcinomas, and four were less frequent histotypes.
Postoperative radiotherapy was administered to 18 patients
with stage II and III disease, whereas 11 patients with stage
III disease received postoperative chemotherapy. During
follow up, all the 68 patients died of lung cancer. Table 1
summarises the main characteristics of the patients.
Briefly, sections from each specimen were cut at 3–5 mm,
mounted on glass slides, and dried overnight at 37˚C. All
sections then were dewaxed in xylene, rehydrated through a
graded alcohol series, and washed in phosphate buffered
saline. This buffer was used for all subsequent washes and for
the dilution of the antibodies. Tissue sections were heated
twice in a microwave oven for five minutes each at 700 W
in citrate buffer (pH 6), and then processed with the stan-
dard streptavidin–biotin–immunoperoxidase method (Dako
Universal kit; Dako Corporation, Carpinteria, California,
Biotechnology, Santa Cruz, California, USA) specific for p16
(sc-1661), p21 (sc-6246), and PCNA (sc-56) were used at a
1/100 dilution, whereas a monoclonal antibody specific for
p53 (D01; Dako Corporation) was used at a 1/500 dilution. All
the primary antibodies were incubated for one hour at room
temperature. Diaminobenzidine was used as the final
chromogen, and haematoxylin as the nuclear counterstain.
Negative controls for each tissue section were performed
leaving out the primary antibody. Positive controls included
in each experiment consisted of tissue previously shown to
express the antigen of interest. Two pathologists (FB and AB)
evaluated the staining pattern of the four proteins separately
and scored the protein expression in each specimen by
scanning the entire section and estimating the percentage of
tumour cell nuclei staining. All immunoreactive nuclei were
regarded as positive, irrespective of staining intensity.
To carry out statistical analysis, a dichotomised scoring
system was used, as follows: p53, p21, and p16 expression in
more than 5% of tumour cells was defined as positive
expression,13 14whereas the median value for the PCNA
labelling index in this tumour series was used as a cutoff
point, and tumours were classified as either less than or
greater than the median value.15Fischer’s exact test was used
to assess relations between ordinal data (correlation matrix
between immunostaining parameters). A univariate survival
Median age (range)
Squamous cell carcinoma
control proteins in the 68 tumours
Dichotomised expression levels of cell cycle
Cutoff point (%)NegativePositive
PCNA, proliferating cell nuclear antigen.
staining of cell cycle proteins in tumour
specimens: (A) p53, (B) p21, (C) p16,
and (D) proliferating cell nuclear
Prognostic role of p53, p21, p16 and PCNA in lung cancer 59
analysis for each prognostic variable on overall survival was
estimated according to the Kaplan–Meier method.16The
terminal event was death attributable to cancer or non-
cancer causes. The significance of the differences in survival
distribution among the prognostic groups was evaluated by
the log rank test.17The Cox proportional hazards model was
applied to the multivariate survival analysis.18The prognostic
variables on overall survival included sex, age, histological
types, pathological T factor, pathological N factor, clinical
tumour stage, p53, p21, p16, and PCNA. A p value , 0.05 was
regarded as significant in two tailed tests. SPSS software
(version 10.00, SPSS, Chicago, Illinois, USA) was used for
Immunohistochemical analysis of p53, p21, p16, and PCNA
protein expression was carried out on 68 primary NSCLC
specimens. All of the cell cycle associated proteins examined
were present in the nuclei of tumour cells, although a small
proportion of cells displayed cytoplasmic immunoreactivity in
addition to nuclear staining. Table 2 details the expression of
each protein, and fig 1 shows examples of positive
Clinicopathological data and cell cycle proteins
The cell cycle checkpoint proteins were analysed with respect
to detailed clinicopathological information available for all
patients in this cohort. A negative correlation was found
between lymph nodes status and p21 (p = 0.015) and p16
expression (p = 0.008), whereas a positive correlation was
found between lymph nodes status and PCNA (p = 0.022).
No correlations were detected with the other clinical features,
such as age, sex, clinical tumour stage, tumour grading, and
tumour histology. Finally, as expected, a positive correlation
was found between T and lymph nodes status (p , 0.0001).
Remarkably, no correlation was found between p16, p21, and
p53 differential expression. Table 3 summarises these results.
Overall survival and immunohistochemical and
We evaluatedthe prognostic
pathological parameters in patients with non-small cell lung cancer
Correlation matrix (and significance) between molecular markers and
PCNA, proliferating cell nuclear antigen.
non-small cell lung cancer in univariate analysis
Survival and pathological and immunohistochemical parameters in patients with
Number of patients
(months) 95% CI p Value
Stage III (A/B)
Pathological T factor
Pathological N factor
34.30 to 67.37
9.77 to 26.23
14.88 to 29.12
7.56 to 40.44
22.80 to 49.20
10.16 to 19.84
30.85 to 41.15
1.51 to 29.49
25.29 to 40.71
9.57 to 26.43
8.00 to 26.00
13.57 to 58.43
4.73 to 13.27
20.77 to 45.23
15.70 to 26.30
9.73 to 38.27
CI, confidence interval; NS, not significant; PCNA, proliferating cell nuclear antigen.
60Esposito, Baldi, Tonini, et al
immunohistochemical parameters both by univariate and
By univariate analysis, survival seemed to be influenced by
p53, p21, and p16. Patients expressing p53 had a worse
overall survival than did those negative for p53. In contrast,
p21 positive patients showed a better survival than did p21
negative ones. Moreover, patients with positive staining for
p16 had a better survival than did p16 negative patients. No
correlations were found between overall survival and cell
kinetics, as evaluated by PCNA. Among the clinical and
pathological parameters, the only two that influenced
survival in patients with NSCLC were lymph node status
and clinical tumour stage. There was a significant difference
in overall survival between patients with lymph node
involvement (N1–3) and those without lymph node metas-
tasis (N0). Furthermore, there was also a significant
difference in overall survival between patients with stage I–
II NSCLC and those with stage III NSCLC. Finally, no
differences were found between patients with stage I and
stage II NSCLC and between those with stage IIIA and stage
IIIB NSCLC. Chemotherapy and radiotherapy showed no
clinical impact on overall survival in our patients with
NSCLC. However, surgery influenced survival in univariate
analysis (median survival of the surgery group, 33 months
v mediansurvival of
17 months; = 0.005). Table 4 and fig 2A–D show the results
of the univariate analysis relating to the prognostic value of
the various parameters on overall survival in patients with
By multivariate analysis, the only clinical parameter that
influenced overall survival was tumour staging. When
comparing patients with stage I–II NSCLC with patients
with stage III NSCLC, the relative risk of death in those with
stage III disease was 3.45 (95% confidence interval (CI), 1.43
to 6.78; p = 0.001). The only immunohistochemical para-
meter that influenced overall survival was p16. The calculated
relative risk of death in p16 negative patients with NSCLC
was 3.149 (95% CI, 1.384 to 7.164; p = 0.006). Borderline
significance was recorded for p21 and p53. The relative risk of
death for patients overexpressing p53 was 1.771 (95% CI,
0.796 to 2.007; p = 0.053), whereas for p21 negative
patients it was 1.818 (95% CI, 0.912 to 3.407; p = 0.060).
Table 6 shows the results of the multivariate analysis relating
to the prognostic value of the various parameters on overall
survival in patients with NSCLC.
Finally, when we grouped the NSCLC cases based on the
p21 and p16 scores (group A, both positive; group B, p21
negative and p16 positive; group C, p21 positive and p16
negative; group D, both negative), we found that the group of
patients who were both p21 and p16 negative had signifi-
cantly shorter overall survival. Table 5 and fig 2E show these
the non-surgery group,
The ability of a cell to control its own replication is very
important for the maintenance of the structure and functions
of the organ it belongs to and of the organism as a whole.
Several pathologies have been linked to altered control of
cellular replication, and cancer is one of the most studied of
these. To date, many checkpoint proteins have been
examined in lung cancer, but few studies have investigated
multiple factors in the same tumours. We have analysed the
expression of four key proteins involved in cell cycle
checkpoints in a large series of well characterised NSCLCs.
When we looked at the correlation between the clinico-
pathological data and the expression of cell cycle proteins, we
found a negative correlation between lymph nodes status and
p21 and p16 expression, suggesting a possible role for these
two proteins in the progression of the disease. Interestingly,
proteins and clinical stages on overall survival of patients with non-small
cell lung cancer. (A) Positive expression of p53 was associated with
shorter patient survival, (B) positive expression of p21 was correlated
with improved outcome, (C) positive expression of p16 was associated
with improved outcome, (D) clinical stage III was correlated with shorter
patient survival, (E) patients lacking both p21 and p16 expression
(group D) had a significantly shorter overall survival (see table 5 for the
definition of the patient groups).
Kaplan–Meier survival curves showing the effects of cell cycle
Prognostic role of p53, p21, p16 and PCNA in lung cancer 61
no correlation was found between p16, p21, and p53
‘‘We found that the group of patients whose lung cancer
specimens were negative for both p21 and p16 had
significantly shorter overall survival’’
When we looked at the correlation between the expression
of the different proteins and survival using univariate
analysis, we found that all the cell cycle markers analysed,
except for PCNA, were significantly correlated with survival.
This result is in agreement with numerous studies published
about the cell cycle checkpoint proteins investigated here and
lung cancer.3 19–24As expected, lymph node status and clinical
tumour stage were also significantly correlated with survival.
Surprisingly, when we performed multivariate analysis, the
only immunohistochemical parameter that influenced overall
survival was p16. This result is in agreement with the
proposed hypothesis that the RB–p16 tumour suppressor
pathway is inactivated in most lung cancer samples.23Among
the clinical parameters, tumour staging was the only factor to
influence survival in multivariate analysis.
Finally, we grouped the lung cancer specimens based on
p21 and p16 status. Interestingly, we found that the group of
patients whose lung cancer specimens were negative for both
p21 and p16 had significantly shorter overall survival.
Numerous data from the literature suggest the existence of
a functional collaboration between distinct CDK inhibitor
genes.25Indeed it has recently been demonstrated that cell
cycle inhibition by p16 is associated with the post-transcrip-
tional induction of p21 and strong inhibition of cyclin E–
CDK2 kinase activity.26Moreover, it has been shown that
members of the p21 family of proteins promote the
association of D-type cyclins with CDKs by counteracting
the effects of p16 molecules.27Therefore, it has been proposed
that functional cooperation between different cell cycle
inhibitor proteins constitutes another level of regulation in
cell growth control and tumour suppression.25 28
Taking into account the complicated functional network
constituted by the cell cycle regulator proteins, it is evident
that knowledge of the level of expression of these factors, and
their coregulators, may be important in predicting patient
clinical response to treatment. Targeting multiple checkpoint
proteins may represent a good therapeutic strategy for the
development of new molecular treatments for lung cancer.
Our data support this hypothesis and the need for further
work aimed at investigating the simultaneous expression of
numerous cell cycle regulators in NSCLC.
This work was funded by grants from: International Society for the
Study of Comparative Oncology, Inc (ISSCO, President HE Kaiser)
Silver Spring, MD, USA; FUTURA-Onlus; Ministero della Salute;
MIUR; and Second University of Naples.
V Esposito, V Montesarchio, Third Division of Infective Diseases, D.
Cotugno Hospital, Naples 80100, Italy
A Baldi, F Baldi, Department of Biochemistry and Biophysic
‘‘F. Cedrangolo’’, Section of Anatomic Pathology, Second University
of Naples, Naples 80100, Italy
G Tonini, B Vincenzi, P Persichetti, Section of Oncology, Campus
BioMedico University, Rome 00100, Italy
M Santini, Department of Thoracic Surgery, Second University of Naples
V Ambrogi, T C Mineo, Department of Thoracic Surgery, Tor Vergata
University, Rome 00100, Italy
G Liuzzi, A.O. ‘‘L. Spallanzani’’, Rome 00100, Italy
E Wolner, A M Groeger, Department of Cardio-Thoracic Surgery,
University of Vienna, Vienna 1008, Austria
1 Jemal A, Chu KC, Tarone RE. Recent trends in lung cancer mortality in the
United States. J Natl Cancer Inst 2001;93:277–83.
2 Greenlee RT, Murray T, Bolden S, et al. Cancer statistics. CA Cancer J Clin
3 Zochbauer-Muller S, Gazdar AF, Minna JD. Molecular pathogenesis of lung
cancer. Annu Rev Physiol 2002;64:681–708.
4 Sherr CS. Cancer cell cycles. Science 1996;274:1672–7.
5 MacLachlan TK, Sang N, Giordano A. Cyclins, cyclin-dependent kinases and
cdk inhibitors: implication in cell cycle control and cancer. Crit Rev Eukaryot
Gene Expr 1995;5:127–56.
6 Grana X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins,
cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-
dependent kinase inhibitors. Oncogene 1995;11:211–19.
7 Kirsch DG, Kastan MB. Tumor-suppressor p53: implications for tumor
development and prognosis. J Clin Oncol 1998;16:3158–68.
8 Bravo R, Frank R, Blundell P, et al. Cyclin PCNA is the auxiliary protein of
DNA polymerase delta. Nature 1987;326:515–17.
survival in patients with non-small cell lung cancer
Multivariate Cox regression analysis of overall
RR of death 95% CIp Value
Stage III (A/B)
Pathological N factor
1.43 to 6.780.001
0.659 to 6.156
0.796 to 2.007
0.912 to 3.407
1.384 to 7.164
CI, confidence interval; NS, not significant; RR, relative risk.
and p16, in patients with non-small cell lung cancer
Survival according to staining patterns of p21,
(months)95% CIp Value
Group A (16 patients)
p21+ and p16+
Group B (21 patients)
p212 and p16+
Group C (19 patients)
p21+ and p162
Group D (12 patients)
p212 and p162
34.0022.04 to 45.96
11.006.73 to 15.27
CI, confidence interval.
Take home messages
N In univariate analysis, the expression of p53, p21, and
p16 in patients with non-small cell lung cancer
(NSCLC) was significantly correlated with survival
N In multivariate analysis, only p16 influenced overall
survival and those patients who were negative for both
p21 and p16 had a significantly shorter overall
N Thus, loss of control of cell cycle checkpoints is common
in lung cancer, and functional cooperation between
different cell cycle inhibitor proteins may be another
level of regulation in cell growth control and tumour
62 Esposito, Baldi, Tonini, et al
9 Prelich G, Tan C, Kostura M. Functional identity of proliferating cell nuclear
antigen and DNA polymerase delta auxiliary protein. Nature
10 Waga S, Hanno GJ, Beach D, et al. The p21 inhibitor of the cyclin dependent
kinases controls DNA replication by interaction with PCNA. Nature
11 Li R, Waga S, Hannon GJ, et al. Differential effects of the p21 CDK inhibitor
on PCNA-dependent DNA replication and repair. Nature 1994;371:534–37.
12 Mountain CF. Revisions in the international system for staging lung cancer.
13 Zirbes TK, Baldus SE, Moenig SP, et al. Prognostic impact of p21/waf1/cip1
in colorectal cancer. Int J Cancer 2000;89:14–18.
14 Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16
protein loss in sporadic gastric carcinomas. Lab Invest 2000;80:689–695.
15 Paradiso A, Rabinovich M, Vallejo C, et al. p53 and PCNA expression in
advanced colorectal cancer: response to chemotherapy and long-term
prognosis. Int J Cancer 1996;69:437–41.
16 Kaplan EL, Meier P. Nonparametric estimation from incomplete observations.
J Am Stat Assoc 1958;53:457–81.
17 Peto R, Pike MC, Armitage P, et al. Design and analysis of randomised clinical
trials requiring prolonged observation of each patient. Br J Cancer
18 Cox DR. Regression models and life tables. J R Stat Soc 1972;34:187–220.
19 Caputi M, Esposito V, Baldi A, et al. p53-independent expression of p21 cdk
inhibitor in non small cell lung cancer: relationship to survival. Am J Respir Cell
Mol Biol 1998;18:213–17.
20 Mitsudomi T, Hamajima N, Ogawa M, et al. Prognostic significance of p53
alterations in patients with non-small cell lung cancer: a meta-analysis. Clin
Cancer Res 2000;6:4055–63.
21 Shoji T, Tanaka F, Takata T, et al. Clinical significance of p21 expression in
non-small-cell lung cancer. J Clin Oncol 2002;20:3865–71.
22 Zhou JX, Niehans GA, Shar A, et al. Mechanisms of G1 checkpoint loss in
resected early stage non-small cell lung cancer. Lung Cancer 2001;32:27–38.
23 Kaye FJ. RB and cyclin dependent kinase pathways: defining a distinction
between RB and p16 loss in lung cancer. Oncogene 2002;21:6908–14.
24 Groeger AM, Caputi M, Esposito V, et al. Independent prognostic role
of p16 expression in lung cancer. J Thorac Cardiovasc Surg
25 Franklin DS, Godfrey VL, O’Brien DA, et al. Functional collaboration between
different cyclin-dependent kinase inhibitors suppresses tumor growth with
distinct tissue specificity. Mol Cell Biol 2000;20:6147–58.
26 Mitra J, Dai CY, Somasundaram K, et al. Induction of p21 and inhibition of
cdk2 mediated by the tumor suppressor p16. Mol Cell Biol
27 Parry D, Mahony D, Wills K, et al. Cyclin D–CDK subunit arrangement is
dependent on the availability of competing INK4 and p21 class inhibition. Mol
Cell Biol 1999;19:1775–83.
28 Geradts J, Fong KM, Zimmerman PV, et al. Correlation of abnormal RB,
p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic
abnormalities, and clinical features in 103 primary non-small cell lung
cancers. Clin Cancer Res 1999;5:791–800.
Prognostic role of p53, p21, p16 and PCNA in lung cancer63