Oncoprotein expression and inhibition of apoptosis during colorectal tumorigenesis

Abstract

AIM: To study bcl-2 and p53 protein expression and inhibition of apoptosis during colorectal tumorigenesis.

METHODS: Expression of bcl-2 and p53 was detected by immunohistochemical staining of 45 colorectal adenomas, 61 colorectal carcinomas, and 15 pathologically-confirmed normal colorectal biopsies.

RESULTS: The bcl-2 and p53 protein expression was uniformly negative in the normal mucosa specimens, whereas bcl-2 and p53 positive rates were significantly higher in the adenoma and carcinoma specimens (P < 0.01). Strong bcl-2 expression was often present in areas of severe dysplasia. In the colorectal adenoma specimens, expression of p53 increased with increasing size and dysplasia, being higher in adenomas ≥ 20 mm in diameter than in adenomas < 10 mm in diameter (77.8% vs 35.0%, P < 0.05). p53 protein expression was correlated with differentiation and Duke's staging. A significant inverse correlation was found between immunostaining of bcl-2 and p53 in adenomas but not in carcinomas. Furthermore, carcinomas with a high percentage of bcl-2 positive cells were significantly more likely to have low rates of apoptosis.

CONCLUSION: Bcl-2 expression appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis. p53 expression plays an important role in the development and malignant change of colorectal adenoma. Bcl-2 and p53 may represent useful markers of cell apoptosis.

Key Words: Colorectal neoplasms, Protein p53, Gene expression, Apoptosis, Bcl-2

INTRODUCTION

Colorectal cancer is a common malignant tumor and it is believed to arise from adenoma. A recent study showed that colorectal tumorigenesis is related not only to the balance of tumor genes and tumor suppressor genes, but also to cell apoptosis. Cell apoptosis is an important mechanism in cancer biology[1]. Although both of these genes are associated with the process of apoptosis, their relationship in the process has not yet been extensively investigated. Only recently has it been observed that bcl-2 can inhibit apoptosis triggered by wild-type p53[1].

Therefore, we analyzed bcl-2 expression in normal, adenoma and malignant colorectal epithelia and determined the time of bcl-2 activation and its relationship to alteration of p53 expression.

MATERIALS AND METHODS

Tissue samples

Sixty-one paraffin-embedded surgical samples of colorectal carcinoma, 45 colorectal adenomas removed by biopsy or polypectomy and 15 pathologically-confirmed normal colorectal biopsies obtained by colonoscopy were collected from patients being treated at the Guangzhou Military general Hospital. The biopsied tissues were fixed in 10% formalin and embedded in paraffin. Serial sections were made for immunohistochemical or histochemical hematoxylin-eosin (HE)-based staining of bcl-2 and p53 respectively. Colorectal adenoma and carcinoma were diagnosed according to the published criteria[2].

Immunohistochemistry

An immunohistochemical method involving a labeled streptavidin biotin (LSAB)[3] was used to detect bcl-2 and p53 protein in de-paraffinized tissue sections. The primary anti-bcl-2 mAb and anti-p53 mAb DO7 (1:20 and 1:40 respectively; Dako Corporation, Carpinteria, CA, United States) and the appropriate murine anti-human secondary antibodies (LSAB K-9002; Dako Corporation) were used.

Controls

A colorectal neoplasm with a high level of cytoplasmic bcl-2 and nuclear p53 immunoreactivity was used as a positive control for these oncoproteins. Negative control slides were processed with PBS alone, instead of the primary antibody, but included all other steps of the procedure.

Apoptotic index

The percentage of apoptotic cells was determined by microscopic examination (magnification of × 400) of HE-stained sections for the 61 colorectal carcinomas. For each slide, five fields of non-necrotic areas were examined and 100 nuclei in each field were scored as normal or apoptotic in appearance. Morphological features used to identify apoptotic nuclei included overall shrinkage and homogeneous dark-staining basophilia and acidophilia in cytoplasm[4].

Data statistics

Data were analyzed with the chi-square test, with exact probabilities using the 2 × 2 chi-square test, and correlation coefficient test. Statistical significance was defined as P < 0.05.

RESULTS

Expression of bcl-2

In histologically normal mucosa, bcl-2 staining was observed in basal epithelial cells of the colonic crypts overlying the muscularis mucosa, but was absent in the superficial portions of the mucosa. Bcl-2 staining in basal epithelial cells was intense and localized to the cytoplasm. In adenomas, dysplastic cells in adenomatous colonic glands showed diffuse positive staining for bcl-2. Severe epithelial dysplasia displayed intense staining. The bcl-2 positive rate was significantly higher in adenomas (73.3%) and carcinomas (73.8%) than in normal tissue (0%) (P < 0.01), but no significant difference was found between the staining in the adenomas and carcinomas. The expression of bcl-2 was not related to clinical and histopathologic features of the adenomas and carcinomas.

Expression of p53 protein

Expression of p53 protein was located in the cell nucleus, as evidenced by brown staining. Normal mucosa was uniformly negative for nuclear p53 immunostaining. The p53 positive rate was significantly higher in adenomas (53.3%) and carcinomas (55.7%) than in normal tissue (P < 0.01), but no significant difference was found between the staining in the adenomas and carcinomas. Expression of p53 increased with the size of adenoma and degree of dysplasia, and was higher in adenomas with ≥ 20 mm diameter than in adenomas with < 10 mm diameter (P < 0.05). The expression of p53 in adenomas was related to dysplasia (P < 0.05). In colorectal carcinoma, the expression of p53 was related to the differentiation and Duke's staging (Table 1).

Table 1 Relationship between bcl-2 and p53 in clinico-pathological features of colorectal adenoma and carcinoma.

Parameter	n	bcl-2		p53
		positive	%	positive	%
Adenoma size in mm
< 10	20	17	85.0	7	35.0
10-20	16	9	56.3	10	62.5
≥ 20	9	7	77.8	7	77.8a
Histological type
Tubular	20	14	70.0	10	50.0
Tubulovillous	7	5	71.4	4	57.1
Villous	18	14	77.8	10	55.6
Dysplasia
Mild	20	13	65.0	8	40.0
Moderate	18	15	83.3	10	55.6
Severe	4	4	100.0	3	75.0
Malignant change	3	1	33.3	3	100.0c
Serosal invasion
-	24	17	70.80	11	45.8
+	37	28	75.70	23	62.1
Well differentiated	13	10	76.90	4	30.8
Moderately differentiated	29	19	65.50	14	48.3
Poorly differentiated	12	11	91.67	10	83.3c
Mucinous	7	5	71.43	6	85.71
Lymph node metastasis
-	28	19	67.9	13	46.4
+	33	26	78.8	21	63.6
Duke's staging
A	17	12	70.6	5	29.4
B	9	5	55.6	7	77.8e
C	20	17	85.0	11	55.6
D	15	11	73.3	11	73.3a

^aP < 0.05, mild dysplasia vs malignant change; ^cP < 0.05, well differentiated vs poorly differentiated; ^eP < 0.05, Duke's stage B or D vs Duke's stage A.

Expression of bcl-2 and p53 proteins in colorectal tumors

A significant inverse relationship was found between the immunostaining of bcl-2 and p53 in the adenomas (P < 0.05) but not in the carcinomas (P > 0.05) (Table 2).

Table 2 Correlation between bcl-2 and p53 staining¹ in adenomas and carcinomas.

Pathology	n	Correlation coefficiency, r
Adenomas	45	-0.295
Carcinomas	61	-0.112

¹Positive label index.

Apoptotic index (AI)

The mean AI of 61 colorectal carcinomas was 2.76 ± 1.75 (range, 0.2%-10%). A significant inverse relationship was observed between the AI and the percentage of cells that were positive for bcl-2 (r = 0.74, P = 0.0001).

DISCUSSION

The bcl-2 proto-oncogene is a known inhibitor of apoptosis and may therefore allow for an accumulation of genetic alterations that propagates upon cell division. Over-expression of the bcl-2 gene was first described in follicular lymphoma and was then shown to result from a chromosomal 14:18 translocation. Our results indicate that basal epithelial cells of the normal colonic crypts express the bcl-2 protein, but its expression was found to be absent in superficial portions of the mucosa. The bcl-2 positive rate was significantly higher in adenomas (73.3%) and carcinomas (73.8%) than in normal tissue (0%) (P < 0.01), but no significant difference was seen between the expression in adenomas versus carcinomas, suggesting that abnormal bcl-2 gene activation is an early event in neoplastic development or progression. Thus, expression of bcl-2 may not be related to clinical and histopathologic features of colorectal cancer.

The p53 tumor suppressor gene is the most commonly mutated gene in human cancers and is a frequent abnormality found in colorectal cancers[5]. Our results showed p53 staining was higher in adenomas and carcinomas, with only minimal to undetectable in non-tumorous mucosa. Moreover, expression of p53 in the adenomas increased with the increasing size and extent of dysplasia. Expression of p53 in adenomas of ≥ 20 mm in diameter was higher than in adenomas of < 10 mm in diameter, and was higher in tissue with malignant changes than in those with mild dysplasia. In colorectal cancers, the expression of p53 was correlated with the differentiation status and Duke's stage. Our results are consistent with those previously reported by Kaklamanis[6], wherein nuclear p53 staining was detected in 42%-67% of adenomas[7,8]. Furthermore, our results showed a progressive increase in p53 alteration during colorectal tumorigenesis. Although p53 mutation is thought to be a late event in colorectal tumorigenesis, we suggest that it may be more common during malignant change of the polyps.

Spontaneous apoptosis is a feature of some human cancers and may regulate cell growth[9]. We found that the spontaneous apoptotic rate in colorectal carcinomas ranged from 0.2% to 10%. Moreover, our results indicated that an inverse relationship existed between the AI and bcl-2 expression in carcinomas. This result indicates that bcl-2 acts in vivo as an inhibitor of apoptosis in colorectal cancers.

Interestingly, bcl-2 and p53 staining was found in our study to be inversely correlated in adenomas but not in carcinomas. p53 nuclear staining was increased in adenomas at the areas of severe dysplasia, which showed mild staining for bcl-2. This result suggests a potential down-regulation of bcl-2 by mutant p53 in premalignant polyps. Similarly, an inverse correlation between bcl-2 and p53 staining has been reported in breast cancers and colorectal tumors[10,11]. Hence, a potential molecular basis for this effect may involve a p53-dependent negative response element on the bcl-2 gene through which p53 can directly or indirectly down-regulate bcl-2 expression, so that a colorectal epithelia malignant change may occur by inhibiting cell apoptosis.