Relationship between p21/waf-1/cip-1 and apoptosis in cervical cancer during radiation therapy

doi:10.1016/S0360-3016(99)00026-7

International Journal of Radiation OncologyBiologyPhysics

Volume 44, Issue 2, May 1999, Pages 297-303

https://doi.org/10.1016/S0360-3016(99)00026-7 Get rights and content

Abstract

Purpose: p21/WAF-1/CIP-1 was not considered to be involved in the regulation of apoptosis, an important indicator of radiosensitivity. However, it has been reported recently that apoptosis was suppressed when p21 expressed. The purpose of this study was to clarify the relationship between p21 and apoptosis and to evaluate the role of p21 in cervical cancer during radiation therapy (RT).

Methods and Materials: Twenty-one patients with cervical cancer were treated by RT. Tissue samples were obtained from cervical tumors of all patients before RT, and 6 hours after the fifth dose of 1.8 Gy (5th Dose). Samples were subjected to nick end labeling for apoptosis and immunohistochemical staining for p21 and p53 antigen expression.

Results: The mean apoptotic index, p21 labeling index and p53 labeling index were 0.27%, 9.24% and 6.60%, respectively, before RT and increased significantly to 1.20%, 17.5% and 13.9%, respectively, after 5th Dose. The apoptotic index at 5th Dose was inversely correlated with the p21 labeling index (r = −0.50, p = 0.025). Furthermore, a positive correlation was observed between the p21 and p53 labeling indices both before RT and at 5th Dose (r = 0.52, p = 0.02; r = 0.63, p < 0.01, respectively).

Conclusion: Our results demonstrate that apoptosis and expression of p21 and p53 were induced in cervical cancer during RT. p21 expression was dependent on p53 expression and moreover, it is suggested−that p21 might be a potential suppressor of radiation-induced apoptosis in cervical cancer during RT.

Introduction

Since Kerr et al. (1) first reported apoptosis as a distinct mode of cell death different from necrosis, apoptosis has become the subject of intense and widespread research interest, including such areas as morphology, molecular biology and induction kinetics. The morphological features of apoptosis consist of rapid condensation and fragmentation of the nucleus without ballooning of the cell. At the molecular level, apoptosis is recognized by DNA fragmentation (2). Apoptosis, or programmed cell death, is not only a natural physiological process, but can also be induced by irradiation 3, 4, 5 and glucocorticoids (6).

In the fields of radiation biology and oncology, apoptosis is regarded as one of the important indicator of radiosensitivity 7, 8, 9. Thus, the evaluation of tumor radiosensitivity by the assessment of spontaneous apoptosis or radiation-induced apoptosis may be useful in designing the appropriate modality of radiation therapy (RT).

p53 is thought to be involved in the regulation of apoptosis, especially radiation-induced apoptosis 10, 11, 12. When a cell is irradiated, DNA damage induces the p53 protein to arrest the cell cycle at G1 to allow extra time for repair (13). If the repair fails, p53 may trigger deletion of the cell by apoptosis. Both functions of p53, in apoptosis and G1 arrest, inhibit erroneous DNA replication, which prevents carcinogenesis. Dysfunction of p53 plays an important role in carcinogenesis of colon cancer (14), gastric cancer 15, 16, and gallbladder cancer (17).

p21/WAF-1/CIP-1 (referred to in this article as p21) has been shown to mediate central functions of p53 18, 19. The expression of p53-dependent p21 regulates cell growth by arresting the cell cycle at G1 (19), thus p21 is considered to be a downstream protein of p53.

p21 can also be induced via a p53-independent pathway (20), suggesting there are at least two cell regulation pathways dependent on p21. In any event, in both pathways, p21 was not considered to be involved in the regulation of apoptosis until recently (21). However, it has been reported that apoptosis was suppressed when p21 expressed in colorectal cancer cells 22, 23, melanoma cells (24), and during myocyte differentiation (25), indicating p21 may have a role in apoptosis.

To the best of our knowledge, the relationship between p21 and apoptosis in human cancers during RT has not been studied. The purpose of the current study is to clarify the relationship between p21 and apoptosis and to evaluate the role of p21 in cervical cancer during RT.

Section snippets

Patients and tissue samples

Twenty-one patients with invasive cervical cancer were studied. All patients were treated by RT alone at the National Institute of Radiological Sciences Hospital from 1995 to 1996. The mean patient age was 57 years (range, 46–80). Clinical stages and histological subtypes are summarized in Table 1. Clinical staging was based on the criteria of the International Federation of Gynecology and Obstetrics (London) (26). The number of patients with Stage II, III, and IVA were 11, 8, and 2,

Apoptosis

Nuclei showing condensation or fragmentation, typical for apoptosis were stained by the TUNEL method (Fig. 1A). Some cells with morphologic features of necrosis, such as karyolysis, karyorrhexis, and pyknosis, were weakly stained by this method and were not included in the apoptosis count. Apoptotic cells were observed among the cancer cells in a scattered fashion, although few apoptotic cells were observed near the basement membranes. The apoptotic index did not show any association with age,

Acknowledgements

The authors thank Professor H. Aoyama, Hygiene and Preventive Medicine, Okayama University Medical School, for his helpful suggestions and scientific support. This study was supported by a grant from the Charged Particle Therapy Project (National Institute of Radiological Sciences, Chiba, Japan).

References (49)

E.L Levine et al.
Apoptosis as predictor of response to radiotherapy in cervical carcinoma
Lancet
(1994)
J.A Wheeler et al.
ASTRO research fellowshipApoptosis as a predictor of tumor response to radiation in Stage IB cervical carcinoma
Int J Radiat Oncol Biol Phys
(1995)
S.W Lowe et al.
p53-dependent apoptosis modulates the cytotoxicity of anticancer agents
Cell
(1993)
W.S El-Deiry et al.
WAF-1 a potential mediator of p53 tumor suppression
Cell
(1993)
C Deng et al.
Mice lacking p21CIP1/WAF1 undergo normal development but are defective in G1 checkpoint control
Cell
(1995)
J Searle et al.
The spontaneous occurrence of apoptosis in squamous carcinomas of the uterine cervix
Pathology
(1973)
R Komaki et al.
Apoptosis and mitosis as prognostic factors in pathologically staged N1 nonsmall cell lung cancer
Int J Radiat Oncol Biol Phys
(1996)
V Chyle et al.
Apoptosis and downstaging after preoperative radiotherapy for muscle-invasive bladder cancer
Int J Radiat Oncol Biol Phys
(1996)
D Suciu
Cellular death by apoptosis in some radiosensitive and radioresistant mammalian tissues
J Theor Biol
(1983)
R.M Macklis et al.
Cell cycle alterations apoptosis and response to low-dose-rate radioimmunotherapy in lymphoma cells
Int J Radiat Oncol Biol Phys
(1993)

T Nakano et al.

Histological and immunohistochemical prediction for local control of cervical squamous cell carcinoma treated with radiotherapy alone

Int J Radiat Oncol Biol Phys

(1990)

T Arai et al.

Local control prediction by histologic grading of radiation effects with drill biopsy specimens for uterine cervical cancer

Int J Radiat Oncol Biol Phys

(1993)

V Dulic et al.

p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest

Cell

(1994)

J.F.R Kerr et al.

ApoptosisA basic biological phenomenon with wide-ranging implications in tissue kinetics

Br. J. Cancer

(1972)

J.F.R Kerr et al.

Apoptosis

Cancer

(1994)

Kerr JFR, Searle J. Apoptosis: Its nature and kinetic role. In: Meyn RE, Winthers HR, editors. Radiation biology in...

L.C Stephens et al.

Apoptosis in irradiated murine tumors

Radiat Res

(1980)

R.M Macklis et al.

Cellular kinetics, dosimetry, and radiobiology of a-particle radioimmunotherapyInduction of apoptosis

Radiat Res

(1992)

A.H Wyllie

Glucorcorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation

Nature

(1980)

R.E Meyn et al.

Workshop reportRadiation-induced apoptosis, 5–8 May 1994, Round Top, Texas, USA

Int J Radiat Biol

(1994)

A.R Clarke et al.

Thymocyte apoptosis induced by p53-dependent and independent pathway

Nature

(1993)

S.W Lowe et al.

p53 is required for radiation-induced apoptosis in mouse thymocytes

Nature

(1993)

M.B Kastan et al.

Participation of p53 protein in the cellular response to DNA damage

Cancer Res

(1991)

D.J Koorey et al.

Tumor suppressor genes and colorectal neoplasia

J Gastroenterol Hepatol

(1993)

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Citation Excerpt :
The substance p21/waf1/cip1 triggers G1 arrest or apoptosis in cells with the wild-type p53 gene (10). Niibe et al.(15) reported that overexpression of p21/waf1/cip1 correlated with p53 overexpression and apoptosis during cCRT for cervical SCC. In our study, no correlation was observed between the overexpression of p21/waf1/cip1 and p53.
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The number of patients with tumors positive for p53, p21/waf1/cip1, MIB-1, EGFR, HER2, and Bcl-2 was 26, 24, 49, 26, 13, and 11, respectively; no significant correlation was noted. The 5-year overall survival rates of HER2-positive and -negative patients was 76% vs. 44%, which was of borderline significance (p = 0.0675). No significant correlation was noted between overall survival and expressions of p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2. No correlation was observed between local control and expression of any of the proteins.
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Background: This study was aimed to develop possible predictive response of cervical carcinoma in stage IIIA and B patients by evaluating the changes in physical parameter, such as, membrane fluidity, biochemical parameters, such as, intracellular calcium, antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx)] and apoptotic cell death in cervical cancer cells from patients after treating with the first fractionated dose of 2 Gy in radiation therapy protocol. Methods: Biopsies of cervical carcinoma patients were collected before and 24 h after first fractionated radiation dose of 2 Gy. Cell suspensions and tissue of cervix cancer biopsies were used to measure various physical and biochemical parameters. Results and conclusions: A negative correlation was found to exist between observed fluidity of membrane/SOD level with the degree of apoptosis in cervical cells. On the other hand, a positive correlation was observed between intracellular calcium level and percent cellular apoptosis. These results suggest that changes in membrane fluidity, SOD and calcium level were involved in the mechanism of radiation induced cervical apoptosis as measured by TUNEL assay. Moreover, apoptotic sensitivity of these cells after the first dose of radiation treatment showed a direct correlation with the radiation treatment outcome in patients after completion of radiotherapy protocol (70 Gy) in the clinic suggesting that apoptotic index may form a basis for prognosis in radiotherapy in stage III cervix cancer patients.
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Because the incidence of adenocarcinoma of the uterine cervix has recently risen, the evaluation of radiotherapy (RT) for this disease has become an increasingly urgent matter. We analyzed the expression of the cell cycle-associated proteins p53, p27, p21/waf1/cip1, and cyclin D1 in cervical adenocarcinomas in correlation with the prognostic significance in tumors treated with RT alone.
The expression of p53, p27, p21/waf1/cip1, and cyclin D1 was studied using an immunohistochemical method in 53 cases of cervical adenocarcinoma treated only with RT. Patients received RT alone between 1965 and 1994. The mean patient age was 61.8 ± 12.6 years (range, 36–82 years). The number of patients with Stage I, II, III, and IVA disease was 6, 16, 28, and 3, respectively.
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Clinical InvestigationsRelationship between p21/waf-1/cip-1 and apoptosis in cervical cancer during radiation therapy

Abstract

Introduction

Section snippets

Patients and tissue samples

Apoptosis

Acknowledgements

Lancet

Int J Radiat Oncol Biol Phys

Cell

Cell

Cell

Pathology

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

J Theor Biol

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Cell

ApoptosisA basic biological phenomenon with wide-ranging implications in tissue kinetics

Br. J. Cancer

Apoptosis

Cancer

Apoptosis in irradiated murine tumors

Radiat Res

Cellular kinetics, dosimetry, and radiobiology of a-particle radioimmunotherapyInduction of apoptosis

Radiat Res

Glucorcorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation

Nature

Workshop reportRadiation-induced apoptosis, 5–8 May 1994, Round Top, Texas, USA

Int J Radiat Biol

Thymocyte apoptosis induced by p53-dependent and independent pathway

Nature

p53 is required for radiation-induced apoptosis in mouse thymocytes

Nature

Participation of p53 protein in the cellular response to DNA damage

Cancer Res

Tumor suppressor genes and colorectal neoplasia

J Gastroenterol Hepatol

Clinical Investigations
Relationship between p21/waf-1/cip-1 and apoptosis in cervical cancer during radiation therapy