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Published ahead of print on October 3, 2003, doi:10.1165/rcmb.2003-0077OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 564-568, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0077OC

p53 N-Terminal Ser-15~P and Ser-20~P Levels in Squamous Cell Lung Cancer after Radio/Chemotherapy

Robert M. Mroz, Adam Holownia, Elzbieta Chyczewska, Lech Chyczewski and Jan J. Braszko

Department of Pneumology, Department of Clinical Pharmacology, and Department of Clinical Molecular Biology, Bialystok Medical University, Bialystok, Poland

Address correspondence to: Dr. Robert Mroz, Department of Pneumology, Bialystok Medical University, Zurawia 14, 15-540 Bialystok, Poland. E-mail: robmroz{at}wp.pl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Functional regulation of p53 protein, a critical regulator of cell cycle and apoptosis, was investigated in fiberoptic bronchoscopy biopsy samples taken from 23 patients suffering from recurrent squamous cell lung cancer by analyzing the expression and phosphorylation status of the p53 at Ser15 and Ser20 before and after treatment with radiotherapy/cisplatin/vinorelbine. Poly(ADP-ribose) levels as a marker of cellular DNA damage, expression of wild-type and mutated p53 protein, and Ki-67 expression as a marker of proliferation was also determined. Median p53 expression increased (61% increase) after therapy. p53 phosphorylated on Ser20 was also increased by ~ 57% in radiotherapy/chemotherapy patients, and these changes correlated with Ki-67 proliferation and with elevated (by 69%; P < 0.01) poly(ADP-ribose) levels. Our data indicate that apart from changes in p53 quantity, post-translational phosphorylation/dephosphorylation-mediated alterations, especially at Ser20 may play a role in p53 stabilization and, therefore, in antiproliferative activity of drugs inducing DNA damage and apoptosis.

Abbreviations: adenosine diphosphate, ADP • bovine serum albumin, BSA • enhanced chemiluminescence, ECL • ethylenediaminetetraacetic acid, EDTA • gray, Gy • immunoglobulin G antibody, IgG Ab • left lower lobectomy, LLL • left pulmonectomy, LP • murine double minute protein, MDM2 • sodium vanadate, Na3VO4 • sodium azide, NaN3 • saponine NP-40, NP-40 • p53 protein, p53 • p53 phosphorylated on serines 15, 20, 392, Ser15~P, Ser20~P, Ser392~P • p300 protein, p300 • phenylmethylsulfonyl fluoride, PMSF • phosphate-buffered saline + Tween, PBS-T • poly(ADP-ribose), PAR • proliferation-associated nuclear antigen Ki-67, Ki-67 • right lower bilobectomy, RLB • right pulmonectomy, RP • right upper lobectomy, RUL • sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE • tumor, node, metastasis, TNM • Western blot, WB


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phosphorylation is a reversible, omnipresent, and important regulatory mechanism of signal transduction pathways altering conformational state of proteins and activating phosphorylated molecules. Both kinase and phosphatase activities and expressions are widely assayed in many diseases (1). The tumor-suppressor p53 protein (p53) is among the most vital proteins, regulating cell growth and apoptosis in the human body, representing leading defense against the uncontrolled growth of cancer (2). In most tissues and cell types DNA damage induces p53 stabilization and activates its transcription function (3); nevertheless, damaged and inactive p53 proteins have been found in more than half of human cancers (4). p53 activation depends on its phosphorylation at various sites, catalyzed by different protein kinases (5, 6). In vitro studies have identified many sites of p53 phosphorylation and have indicated kinase enzymes involved (7–10). In a previous study we have shown that DNA-damaging and proapoptotic multiple radiation/cisplatin/vinorelbine therapy alters tumor cell DNA status, increasing poly(ADP-ribose) levels, p53 expression, and p53 Ser392~P levels; however, at that stage no information was available on how p53 mutation affects its phosphorylation status (11). Recent studies have suggested that Ser15 and Ser20 of p53 is phosphorylated after ionizing radiation and chemotherapy, and this phosphorylation might be involved in the stabilization of p53 (12, 13). This mechanism may regulate p53 binding to other important proteins like p300 (14) and MDM2 (15), either by stabilizing the p300–p53 interaction (p53 activation) or via MDM2 binding inhibition (decreased p53 degradation). The level of p53 phosphorylation in human tumors required for physiologic response induction, the localization/relocalization of the phosphorylated p53, or the timing of phosphorylation in DNA damaging therapy, remain unknown. All published data reflecting p53 phosphorylation status and poly(ADP-ribose) levels come from animal and cell culture studies; therefore, we tried to evaluate these indices during the clinical course of cancer treatment. The aim of this study was to assess parameters reflecting the degree of DNA damage, proliferation rate, the total and mutated p53 levels, and its Ser15~P, Ser20~P status in recurrent squamous cell lung cancer before and after therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical Samples
Bronchial tissue was obtained during fiberoptic bronchoscopy from 23 male smoker patients, aged 47–71 yr (mean 63 yr) subjected previously to the surgical resection of primary lung tumor, admitted to the hospital due to clinical signs of recurrent lung cancer. All data presented in this paper come from patients suffering from recurrent squamous cell lung cancer, initially (before surgery) graded T1–3.N0–3.M0 (Tab I). All patients were treated with the postoperative chest radiation (30 daily 2-Gy fractions for a total of 60 Gy) and four cycles of chemotherapy with cisplatin/vinorelbine starting at 120 mg/m2 cisplatin every 3 wk and 30 mg/m2 vinorelbine on the 1st and the 8th day of each 21-d cycle (16).

Bronchoscopy Procedure
Bronchoscopy was performed before chemotherapy and at Days 22–24 of the last chemotherapy cycle and bronchial tissue samples were taken to assay. Before bronchoscopy, all subjects signed an informed consent form indicating their willingness to undergo bronchoscopy and bronchial biopsy. At bronchoscopy forceps biopsies were performed at an area of a suspected cancer (Table 1). In all 23 cases histology samples taken from the same area as for poly(-ADP-ribose) levels quantification and Western blot analysis of p53 protein before and after treatment revealed squamous cell carcinoma. Patients with no histologic evidence of squamous cell carcinoma, either before or after treatment, were excluded from further consideration (not included in this study).


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  		TABLE 1 Patient characteristics

 
Specimen Processing and Microscopic Procedures
Lung tissue was sonicated using Bandelin Sonoplus 200 W sonicator in ice-cold Tris-acetate buffer (50 mM; pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 0.3 M NaCl, and 1 mg/ml bovine serum albumin (BSA) and filtered through a 200-µM mesh filter to remove unfragmented tissue. Aliquots were taken to assay protein levels (BCA assay kit; Pierce, Rockford, IL) and homogenates were frozen in liquid nitrogen until assayed.

Poly(ADP-ribose)
Poly(ADP-ribose) (PAR) levels were quantified in dot blot using mouse IgG3 monoclonal antibody against PAR (Clone 10H; Alexis Biochemicals, Lausanne, Switzerland) and secondary peroxidase-coupled antibody (17). Briefly, equal amounts of tissue homogenate proteins were blotted into nitrocellulose membrane (Bio-Rad, Munich, Germany), dried, blocked with milk (Bio-Rad), and incubated overnight with the first antibody (1:500) with constant agitation. Then, the membrane was washed with PBS-T and incubated with peroxidase-conjugated anti-mouse secondary antibody (1:1,000) for 1 h. The blot was washed, revealed using enhanced luminescence (ECL) kit (Amersham Pharmacia, Freiburg, Germany) and quantified using ImageQuant software (Amersham). Unspecific staining was performed using the same isotype primary antibody.

Ki-67 Expression
Ki-67 expression was assayed in bronchial tissue cryostat sections by immunohistochemistry (18) using overnight incubation with polyclonal antibody anti-human Ki-67 (Dako, Glostrup, Denmark). Antibody working dilution was 1:500. Immunohistochemistry was performed using a commercial Elite ABC Kit (Vectastain; Vector Laboratories, Burlingame, CA) and the level of immunoreactivity of the Ki-67 antigen was expressed as percentages of the positive cancer cell nuclei.

Analysis of Total and Mutated p53 Protein and p53-Ser15~P or p53-Ser20~P Levels
Analysis of total and mutated p53 protein and p53-Ser15~P or p53-Ser20~P levels was performed using sequential immunoprecipitations followed by SDS-PAGE and/or WB (19, 20). Lung tissue was lysed for 1 h on ice, in homogenization buffer supplemented with 1% NP-40, 0.2% NaN3, and Na3VO4 (1 mM final concentration) and spun at 15,000 rpm for 10 min. All samples were equalized for protein (BCA assay kit; Pierce) and the precipitating antibodies were added. The first antibody was a mouse anti-p53 monoclonal antibody recognizing mutant human gene product only (IgG1 isotype, clone PAb 240; NeoMarkers; Lab Vision Corp., Newmarket, UK). Samples were incubated for 1 h with gentle rocking, following by 3 h incubation with protein G-sepharose beads (Sigma-Aldrich, Dorset, UK). After centrifugation the pellets were washed 5 times with lysing buffer. Then, a second antibody, monoclonal mouse IgG Ab against human p53 (Oncogen Research Products, Cambridge, MA) recognizing both wild-type and mutant gene product was added to the supernatant and incubated for 1 h, followed by addition of protein A-sepharose beads (Sigma). The beads were sedimented by centrifugation, washed 5 times with lysis buffer, resuspended in SDS-PAGE sample buffer (BioRad), equalized for p53 protein and run on SDS-PAGE using 10% separation gel and 4% stacking gel with loaded molecular weight markers (BioRad) and p53 standard (LabVision). Then, gels were stained with Comassie stain (BioSafe; BioRad) and analyzed for p53 expression. To determine p53-Ser15~P and Ser20~P pellets of immunoprecipitated p53 proteins (total) were subjected to SDS-PAGE as described, blotted on nitrocellulose membrane (BioRad), and revealed using rabbit polyclonal IgG antibody specifically recognizing p53 protein phosphorylated on Ser 15 or Ser20 (Cell Signaling Technology, Beverly, MA) a secondary peroxidase-coupled antibody and chemiluminescence detection system (Amersham), (21). The p53-Ser15~P and p53-Ser20~P bands were quantitated using densitometry, numerized, and compared.

Statistical Analysis
Statistical analysis was performed with the Statistica (Statsoft, Krakow, Poland) package using the Kolmogorof-Smirnof distribution test and the Wilcoxon signed rank test to compare median p53 expression in samples taken before and after treatment. Linear regression and correlation between p53, p53-Ser15~P, p53-Ser20~P, Ki-67, and PAR levels before and after therapy were calculated according to Pearson test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PAR
PAR was detected in all samples tested and levels were significantly increased in patients treated with radio- and chemotherapy. Representative spots are shown in Figure 1 (Figures 1A and 1B, respectively, for patients before and after treatment). Mean PAR content in row 1A was expressed as 100.0 ± 16.9 relative units and corresponding value in row 1B was 169.0 ± 27.0 (P < 0,01).



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  		Figure 1. Representative dot-blot spots of PAR assay in bronchial biopsy specimens from random recurrent squamous cell lung cancer patients (A) and the same patients treated with radiotherarapy/cisplatin/vinorelbine (B). Mean relative PAR content was higher (P < 0.01) in Group B (169.0 ± 27.0; n = 23) compared with Group A (100.0 ± 27.0; n = 23).

 
Ki-67 Expression
In 13 samples taken before therapy (56.5%) and 12 after therapy (52.2%), Ki-67 expression was considered positive. The median Ki-67 expression was 49.5 ± 17.7 (P < 0.05) before, and 33.9 ± 12.7 (P < 0,05) after therapy.

p53 Expression
p53 expression was found in 18 out of 23 patients before the therapy and in 19 samples taken after the therapy (Figure 2). Relative mean p53 band-intensities in row 1B compared with row 1A were increased by more than 61%, and this difference was statistically significant (P < 0.05). Most of the p53 (~ 77%) was identified by immunoprecipitation/PAGE assay as mutated proteins (Figure 3).



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  		Figure 2. Representative analysis of the p53 protein levels (wild type + mutated) in lung tissue taken from recurrent squamous cell lung cancer patients (in random order) before (A) and after (B) radio/cisplatin/vinorelbine therapy. The p53 protein was immunoprecipitated, run on SDS/PAGE, and revealed using Comassie stain. Positions of standard p53 protein (LabVision) are shown. Mean relative p53 levels were 61% higher after the therapy (P < 0.05).

 


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  		Figure 3. Representative picture from determination of mutated p53 protein (PAb 240) in recurrent squamous cell lung cancer patients after radio/cisplatin/vinorelbine therapy. The mutated p53 protein was immunoprecipitated with PAb 240, the precipitate was run on SDS/PAGE and stained with Comassie stain. Irrespective of the therapy, most patients (77%) had p53 protein identified as mutated.

 
p53 Phosphorylation on Ser15 and Ser20
Immunoprecipitation followed by immunoblot detection of p53 protein phosphorylated at Ser15 showed positive staining in 12 patients before therapy (mean relative expression 92.7 ± 41.3), and in 14 patients after therapy (mean relative expression 133.7 ± 86.8). The difference was not significant (Figure 4). Detection of p53 protein phosphorylated at Ser20 showed positive staining in 16 samples before therapy and in 18 samples taken after the therapy. In this group p53-Ser20~P levels were ~ 57% higher after therapy (P < 0.05) (Figure 5).



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  		Figure 4. Immunoprecipitation/Western blot analysis of the p53 protein phosphorylated at Ser15 in recurrent squamous cell lung cancer patients (in random order) before (A) and after (B) radio/cisplatin/vinorelbine therapy. The p53 protein was immunoprecipitated by monoclonal Ab recognizing both wild-type and mutated p53 and the precipitate was analyzed on an SDS/PAGE and WB. The transferred bands were hybridized with p53-Ser15~P Ab and revealed using ECL system. Mean relative p53-Ser15~P levels were similar in both groups.

 


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  		Figure 5. Immunoprecipitation/Western blot analysis of the p53 protein phosphorylated at Ser20 in recurrent squamous cell lung cancer patients (in random order) before (A) and after (B) radio/cisplatin/vinorelbine therapy. The p53 protein was immunoprecipitated by monoclonal Ab recognizing both wild-type and mutated p53 and the precipitate was analyzed on an SDS/PAGE and WB. The transferred bands were hybridized with p53-Ser20~P Ab and revealed using ECL system. Mean relative p53 Ser20~P level was ~ 57% higher in Group B (P < 0.05).

 
Regression Analysis
Regression analysis evidenced that p53 expression did not correlate with poly(ADP-ribose) levels or p53-Ser20~P levels before the therapy. After therapy a good positive correlation was found both between PAR levels and p53 expression (Figure 6A) and between p53 and p53-Ser20~P (Figure 6B). Moreover, negative correlation was found between Ki-67 levels and mutated p53 before therapy (r = -0.58), whereas after therapy negative correlations between Ki-67 and PAR (r = -0.62), Ki-67 and p53 (r = -0.49) (not shown), Ki-67 and p53-Ser15~P (r = -0.46), and Ki-67 and p53-Ser20~P (r = -0.39) (Figure 7), were found.



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  		Figure 6. (A) Regression and correlation analysis between p53 levels and PAR levels in recurrent squamous cell lung cancer patients evidenced no correlation (r = 0.05) before radiotherapy/cisplatin/vinorelbine treatment (not shown) and good correlation (r = 0.63, P < 0.05) after the therapy. (B) Regression and correlation analysis between p53 levels and p53-Ser20~P levels in recurrent squamous cell lung cancer patients showed no correlation (r = 0.05) before radiotherapy/cisplatin/vinorelbine treatment (not shown) and good correlation (r = 0.66, P < 0.05) after the therapy.

 


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  		Figure 7. (A) Regression and correlation analysis between Ki-67 and p53-Ser15~P levels in recurrent squamous cell lung cancer patients evidenced poor correlation (r = 0.30) before radiotherapy/cisplatin/vinorelbine treatment (not shown) and negative correlation (r = -0.46, P < 0.05) after the therapy. (B) Regression and correlation analysis between Ki-67 and p53-Ser20~P levels in recurrent squamous cell lung cancer patients evidenced poor correlation (r = 0.21) before radiotherapy/cisplatin/vinorelbine treatment (not shown) and negative correlation (r = -0.39, P < 0.05) after the therapy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cancer therapy relies mostly on radiation and chemically-induced DNA damage, which usually results in apoptosis of targeted rapidly proliferating cells. Cell proliferation is strictly associated with the expression of the human Ki-67 protein. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle, but is absent from resting cells, makes it an excellent marker for determining the growth fraction of a given cell population through the cell division cycle (22). In our study positive Ki-67 expression characterized over 50% of patients before and after therapy. The percentage of Ki-67 positive cells decreased after therapy. Some cancer cells resist apoptosis due to significant acquired defects of DNA damage checkpoints. These defects often involve alterations in the structure and function of p53 tumor suppressor protein (23). Native p53 can induce cell-cycle arrest or apoptosis, and both protein functions appear to be involved in maintaining the integrity of the genome in vivo (24). p53 is present at very low levels in healthy cells in an inactive form, but with DNA damage its activity increases, mostly due to multisite phosphorylation and acetylation of the protein (25). Several kinase families phosphorylate p53 predominantly on its serine residues, and substitution studies indicate that some of their target amino acids located both in N- or C-terminal end of the protein are important in p53 function. Generally, N-terminal phosphorylation sites, e.g., Ser15 or Ser33 are thought to regulate transcription, Ser15 and Ser20 are implicated in murine double minute (MDM2) protein binding which targets p53 for degradation, whereas C-terminal modifications (both phosphorylation and acetylation) are thought to regulate principally the site-specific binding function of p53 (9, 10, 26). To estimate the clinical importance of p53 phosphorylation, we analyzed the p53 responses to DNA damage induced by ionizing radiation and chemotherapy in recurrent squamous cell lung cancer.

In our previous study with a similar group of patients, we showed increased p53-Ser392~P levels after radio/chemotherapy correlated to the grade of cell death (11). Now, we have focused on more detailed estimation of cell death and post-translational phosphorylation of N-terminal serines of p53 with respect to the wild-type and mutated protein. Phosphorylation of p53-Ser15 and p53-Ser20 was chosen because it may play a role in regulating p53-mediated apoptotosis (27). Moreover, N-terminal serines of p53 mapping to the p53-MDM2 interface, may be involved in p53 stabilization (9, 26, 28). Experimental data indicate that substitution of p53-Ser20 to alanine (p53-Ala20) abrogates the increase in p53 levels and increases negative regulation mediated by MDM2 protein (29) targeting p53 for degradation and regulating p53 protein half-life in vivo (30, 31). Although most of the p53 proteins in our study were found to be mutated, irradiation and chemotherapy resulted in significant accumulation of p53 protein within the cells and may strongly affect the oncogenic potential of cells. Correlation between p53 phosphorylation at Ser15 and/or Ser20 with p53 expression may confirm the involvement of N-terminal phosphorylation in p53 stabilization, whereas negative correlation between Ki-67 and p53-Ser15~P and Ki-67 and p53-Ser20~P may suggest that phosphorylation at Ser15 and Ser20 of p53 actually plays a role in the antiproliferative effect. It seems that alterations in p53 expression may be related to attenuation of MDM2 binding to p53 and it is possible that phosphorylation of p53 at Ser15 and/or Ser20 may be required for p53 upregulation following DNA damage, although the mechanism by which therapy activates p53 in vivo is still unknown. The possible pathways might involve Chk1/2, a DNA damage checkpoint kinases that phosphorylate Ser15 and/or Ser20 of p53 (32). Experimental study indicated that the expression of Chk2 in tumor cells led to increased p53 protein levels and arrested cell cycle in G1 phase (33).

Due to scarce experimental and clinical data, the signaling pathways leading to p53 activation in response to DNA damage are still unknown. It seems that p53 functional assays may be helpful in new anticancer treatment strategies.


   Acknowledgments
 
This study was supported by a Grant 3-46 624 from the Medical University of Bialystok.

Received in original form March 13, 2003

Received in final form August 24, 2003


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.