Mad2 and p53 expression profiles in colorectal cancer and its clinical significance

Abstract

AIM: To investigate the expression of tumor suppressor gene p53 and spindle checkpoint gene Mad2, and to demonstrate their expression difference in colorectal cancer and normal mucosa and to evaluate its clinical significance.

METHODS: Western blot and immunohistochemistry methods were used to analyze the expression of Mad2 in colorectal cancer and its corresponding normal mucosa. The expression of p53 was detected by immunohistochemistry method in colorectal cancer and its corresponding normal mucosa.

RESULTS: Mad2 was significantly overexpressed in colorectal cancer compared with corresponding normal mucosa (P < 0.001), and it was not related to the differentiation of adenocarcinoma and other clinical factors (P > 0.05).The ratio of Mad2 protein in cancer tissue (C) to that in its normal mucosa tissue (N) was higher than 2, which was more frequently observed in patients with lymph gland metastasis (P < 0.05). p53 protein expression was not observed in normal mucosa. The rate of p53 positive expression in adenocarcinomas was 52.6%. There was a significant difference between adenocarcinomas and normal mucosa(P < 0.001), which was not related to the differentiation degree of adenocarcinoma and other clinical factors (P > 0.05).

CONCLUSION: Defect of spindle checkpoint gene Mad2 and mutation of p53 gene are involved mainly in colorectal carcinogenesis and C/N > 2 is associated with prognosis of colorectal cancer.

Key Words: $[Keywords]

INTRODUCTION

Accurate chromosomal segregation is essential for cell survival and genomic stability. Genomic instability is a common feature of human cancer and is thought to be an important contributor to the malignant phenotype[1,2]. At least two categories of instability have been described: CIN and MIN. CIN is characterized by an alteration in chromosome number and is commonly detected as aneuploidy. The fact that the majority of human cancer cells exhibit gains or losses of chromosomes suggests that CIN may contribute to tumorigenesis[3-5]. In yeast, a loss of mitotic checkpoint frequently leads to abnormal chromosome number, resulting in aneuploidy or polyploidy[6]. The mitotic checkpoint, known as the spindle assembly checkpoint, detects errors occurred in the spindle structure or in alignment of the chromosomes on the spindle, and delays chromosome segregation and anaphase onset until the defects are corrected. Two major groups of mitotic checkpoint genes, budding uninhibited by benomyl (BUB) 1-3 and mitotic arrest defect (MAD) 1-3, have been identified in budding yeast[7]. Mammalian homologues of the yeast mitotic checkpoint protein have also been characterized[8-10].

To date, the vast majority of studies about mitotic checkpoint have focused on cancer cell lines. Little data are available on Mad2 in colorectal adenocarcinoma. In this study, the authors used Western blot and immunohistochemical technique to examine the expression of Mad2 and p53 in colorectal cancer to elucidate the relation of Mad2 and p53 to carcinogenesis and clinical pathological factors.

MATERIALS AND METHODS

Specimens

Cancer tissues and corresponding normal tissues were obtained from the First Affiliated Hospital of Anhui Medical University from October 2000 to May 2001. No patient had been treated with anti-neoplasm therapy before tumor removal. Thirty eight patients (21 males, 17 females, aged between 28 to 81 years, median age 54.8 years) were as follows: 25 cases of well differentiated adenocarcinoma, 5 cases of moderately differentiated adenocarcinoma, 8 cases of poorly differentiated adenocarcinoma. Parts of the tissues were embedded in OCT, immediately frozen in liquid nitrogen. The other parts were fixed in 10% formalin, embedded in paraffin, cut into 4 μm serial sections.

Reagents

Mad2 polyclonal rabbit antibodes against human were provided by China Science and Technology University. HEPES, SDS and Polycrylamide were purchased from Sigma Biological Technology Ltd., USA. Immuno-blot nitrocellulose filter membrane was purchased from OSMONICS Biological Technology Ltd., USA. p53, goat anti-rabbit IgG-HRP and S-P immunohistochemical kit were purchased from Beijing Zhongshan Biological Technology Ltd.

Western blot

Both cancer and its corresponding normal frozen tissues were minced and homogenized in buffer (10 mM HEPES, 113 mM mannitol, 37 mM sucrose, 1 mM EDTA) for 5 min on ice. After spun at 15000 rpm for 20 min at 4 °C, supernatants were collected and frozen at -20 °C until use. Protein concentration was measured by modified Lowry protein assay. Samples containing 40 μg total protein were electrophoretically separated and transferred to an immuno-blot nitrocellulose filter membrane. The membranes were blocked overnight at 4 °C with 5% skim milk. The blots were incubated with rabbit polyclonal anti-Mad2 antibody at 1:200 dilution in 5% skim milk for 1 h at 37 °C and were incubated with goat anti-rabbit IgG-HRP at 1:50 dilution in 5% skim milk for 30 min at 37 °C. Following treatment with DAB, the blots were exposed to a gel image system and measured.

Immunohistochemistry

Immunohistochemical staining was performed using S-P method, anti-Mad2 antibody was diluted to 1:120. Anti-p53 was ready to use reagent. A negative control was dyed according to the above method with the primary antibody substituted by animal serum. The standard of positive Mad2 protein expression was stained with brown-yellow color mainly in cell plasma, p53 positive expression was stained in nucleus. A semi-quantitative evaluation was used to determine positively expressed cells by viewing 10 vision fields at × 400[11]. Negative (-) indicated cells were stained less than 10%, mild positive (+) showed 11%-25% cells were stained, moderately positive (++) demonstrated 26%-50% cells was stained, strong positive (+++) revealed over 50% cells were stained. The later three grades were all regarded as positive.

Statistical analysis

The data were analyzed by SPSS version 10.0. Fisher’s exact test or χ² test was used for statistical analysis. P < 0.05 was considered statistically significant.

RESULTS

Expression of Mad2 protein

We analyzed the results measured by gel image system. Mad2 protein in cancer tissue was significantly overexpressed compared with that in corresponding normal tissues (P < 0.001). (Figure 1)The calculated ratio of Mad2 protein in cancer tissues (C) to that in corresponding normal tissues (N) was 2.07 ± 1.87, ranging from 0.31 to 4.68, indicating that Mad2 expression was approximately 2-fold higher in cancer tissue than in normal tissue. Furthermore, we analyzed the association between the C/N ratio of Mad2 expression and clinicopathological parameters including histological differentiation and lymph node metastasis. The results are summarized in Table 1. No significant difference was found among well differentiated, moderately differentiated, and poorly differentiated adenocarcinomas. However, C/N > 2 was found to be related with lymph node metastasis (P < 0.05).

Table 1 Relationship between C/N ratio of Mad2 expression and histologic differentiation and lymph node metastasis.

Groups	n	Mad2		Positive (%)	P
		C/N > 2	C/N < 2
Differentiation
WD	25	10	15	40%
MD	5	2	3	40%
PD	8	2	6	25%	aP = 0.737
Lymph node metastasis
Absent	32	9	23	28%
Present	6	5	1	83.3%	bP = 0.01

^a: Comparison among groups of colorectal cancer,

^b: Comparison between absent and present, WD: Well differentiated adenocarcinoma, MD: Moderately differentiated adenocarcinoma, PD: Poorly differentiated adenocarcinoma.

Figure 1 Mad2 protein expression in normal tissue and colorectal Cancer. 1: standard molecular weight; 2: Mad2 in poorly differentiated adenocarcinoma; 3: Mad2 in correspond-ing norma l tissue; 4: Mad2 in well differ entia ted adenocarcinoma; 5: Mad2 in corresponding normal tissue; 6: Mad2 in moderately differentiated adenocarcinoma; 7: Mad2 in corresponding normal tissue.

The positive signals of Mad2 protein were brown-yellow stains mainly in cell plasma, and strength of color was directly proportional to positive percentage (Figure 2, Figure 3). Positive expression of Mad2 protein was detected in 33 out of 38 (86.8%) colorectal cancers, and 18 out of 38 (47.4%) normal tissues. There was a significant difference between colorectal cancer and normal tissues (P < 0.001). However, no significant difference was found among well differentiated, moderately differentiated, and poorly differentiated adenocarcinomas. The expression of Mad2 in colorectal cancer was not related with lymph node metastasis (Table 2).

Table 2 Relationship between expression of Mad2 protein and histologic differentiation and lymph node metastasis.

Groups	n	Mad2		Positive (%)	P
		+	-
Normal tissue	38	18	20	47.4%
Adenocarcinoma	38	33	5	86.8%	aP < 0.001
WD	25	23	2	92%
MD	5	5	0	100%
PD	8	5	3	62.5%	bP = 0.064
Lymph node metastasis
Absent	32	29	3	90.6%
Present	6	4	2	66.7%	cP = 0.169

^a: Comparison between normal tissue and adeocarcinama,

^b: Comparison among groups of colorectal cancer,

^c: Comparison between absence and presence.

Figure 2 Strongly positive expression of Mad2 in tubular adenocarcinoma. S-P × 400.

Figure 3 Weakly positive expression of Mad2 in normal mucosa S-P × 400.

Correlation between Mad2 protein and p53

The percentage of positive p53 expression was 52.6% (20/38) in colorectal cancer(Figure 4), no such protein expression was observed in normal tissue. We analyzed the correlation between Mad2 protein and p53 expression by χ² test. There was a significantly positive correlation between the expressions of Mad2 and p53(P < 0.05).

Figure 4 Strongly positive expression of p53 in tubular adenocarcinoma. S-P × 400.

DISCUSSION

Kinetochores are linked to both chromosomes and microtubules, and play an important role in the generation of mitotic checkpoint signals. The function of kinetochores was to ensure that chromosomes were not segregated until each of them was aligned and attached to the spindle[12,13]. The majority of protein associated with mitotic checkpoint function have been shown to localize at kinetochores unattached to microtubules[14,15]. It has been proposed that the mitotic checkpoint proteins, especially Mad2 be crucial for generating the “wait” signal to prevent the onset of anaphase after microtubule disruption[16-18]. Previous studies indicated that mutations of mitotic checkpoint protein gene or lower expression of mitotic checkpoint protein might lead to the failure of mitotic checkpoint function, which might in turn lead to tumorigenesis, especially aneuploid cancer. Li and benezra reported that one breast cancer cell line with a mitotic checkpoint defect expressed Mad2 at a level one-third less than that of diploid normal cell lines[8]. Recently, Takahashi et al[19] reported mitotic checkpoint function was impaired in 4 (44%) out of 9 human lung cancer cell lines. In the present study, the expressions of Mad2 and p53 protein were examined in colorectal cancer and its corresponding normal tissues. Mad2 expression in colorectal cancer was higher than that in its corresponding normal tissues. Our results on the expression of Mad2 were not consistent with the reduced Mad2 expression in a breast cancer cell[8]. We speculate it might be the surrounding in which cell lived that led to the expression difference. In addition, positive expression of Mad2 was found in normal tissues to some extent. In budding yeast, the amount of Mad2 protein appeared to be constant during the cell cycle, although the level of Clb2p, a mitotic cycle cyclin, changed regularly in response to cell progression[20]. Similarly, a certain amount of Mad2 protein is thought to be required to maintain the normal checkpoint system in human cell. Loren et al[21] showed that subtle differences in Mad2 protein level markedly altered checkpoint function. Therefore, inactivation of Mad2 would be sufficient to create a haplo-insufficient effect and the loss of mitotic checkpoint control. The most convincing evidence of the role of mitotic checkpoint defect in CIN in mammalian cells obtained in two recent studies showed that disrupting of Mad2 expression resulted in CIN. CIN cells became aneuploidy, a hallmark of cancer associated with aggressive tumor behavior and a poor prognosis[21,22]. Very recently, Poelzl et al[23] reported that estrogen receptor β, which might be involved in the regulation of cellular proliferation, interacted with Mad2 directly and specifically. The Mad2 protein also interacted with the cytoplasmic domain of insulin receptors, which was thought to be a regulator of cellular growth[24,25]. Mad2 protein overexpressed in cancer tissues was exclusively present in the cytoplasm of cancer cells. We speculate that cytoplasmic Mad2 protein may enhance the positive regulatory action of estrogen receptor β and insulin receptor on cellular proliferation. We also discovered that there was a significantly positive correlation between the expressions of Mad2 and p53 (P < 0.05). Orr-Weaver et al[17] thought aneuploidy resulted from defect of Mad2 and other unknown factor could increase the rate at which tumor suppressors, such as p53, were lost. p53 gene was the most frequently mutated gene in human cancer[26-28]. One of its roles was to ensure that when DNA damaged cells were arrested in G1, it attempted to repair their DNA before it was replicated[29,30]. In some cell types, p53 induced apoptosis when overexpressed and was required for apoptosis in response to severe DNA damage[31,32]. p53 might directly activate death genes such as BAX, or down-regulate survival genes such as BCL-2[33-35]. So when p53 gene mutates, aneupoidy from defect of Mad2 might escape from apoptosis and go on its mitosis, finally develop to malignant tumor.

The C/N ratio of Mad2 protein was found to be associated with lymph node metastasis. A C/N ratio greater than 2 was observed more frequently in patients with lymph node metastasis. Therefore, a C/N ratio > 2 may be clinically an important indicator for lymph node metastasis of colorectal cancer.

In conclusion, the protein level of Mad2 is higher in colorectal cancer tissues than in normal tissues. The higher the protein level in colorectal cancer tissues is, the more frequently lymph node metastasis. Since the difference in the protein level between colorectal cancer and normal mucosa is easily detected by immunohistochemistry, Mad2 protein might be a good marker of lymph node metastasis of colorectal cancer.