Abstract
OBJECTIVE To investigate the expression of the mismatch repair proteins hMSH2 and hMLH1, and to examine the clinical significance of the intracellular expression site (ICES) in gastric carcinogenesis.
METHODS Specimens from 172 cases of gastric cancer, 151 tissues from paraneoplastic gastric mucosa and 34 from noncancerous gastric mucosa were collected in Dalain, China. An immunohistochemical method was used to determine the expression of the hMSH2, hMLH1 proteins and their ICES in the gastric mucosas.
RESULTS The rate of hMSH2 expression in gastric cancers, paraneoplastic gastric mucosas and noncancerous gastric mucosas were respectively 69.8%, 49.7% and 32.4%. The rate was significantly higher in gastric cancer compared to the latter two groups (P=0.000), but there was no obvious difference in the expression between the two latter groups (P=0.067). The hMLH1 protein expression rates were respectively 73.3%, 57.6% and 41.2% in the above three groups. The expression was significantly higher in the gastric cancer group compared to the two latter groups (P=0.000), while there was no significant difference between the latter groups (P=0.082). There was no obvious correlation between the hMSH2 and hMLH1 protein expression rates and related factors, such as gender, age and differentiated level of gastric cancer etc. The cell-nuclear expression of the hMSH2 protein was respectively 70.0%, 58.7% and 36.4% in the gastric cancer, paraneoplastic gastric mucosa and noncancerous gastric mucosa groups. The cytoplasmic expression rates were 30.0%, 41.3% and 63.6% in the three groups. The cell-nuclear expression rate of the hMSH2 protein gradually decreased in the gastric mucosas in the following order: cancer, paraneoplastic and noncancerous but cytoplasmic expression only increased slightly in these groups (r=0.161, P=0.020). There was no significant difference in the ICES of the hMLH1 protein among the three different gastric mucosas (P=0.659).
CONCLUSION Simultaneous determination of the expression and ICES of the mismatch repair proteins hMSH2 and hMLH1 in the gastric mucosa may be helpful in detecting early gastric cancer.
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INTRODUCTION
Integrity of the mismatch repair function is of fundamental importance for accurate copying of the cellular DNA and stability of the genome. Furthermore there is a close correlation between the loss of mismatch repair activity and oncogenesis and progression of several malignant tumors[1]. The mismatch repair proteins hMSH2 and hMLH1 are the key proteins for conducting mismatch repair activity. Under normal circumstances, these proteins are synthesized in the cytoplasm and then are conveyed to the cell nucleus where they are involved in the mismatch repair reaction. Therefore, expression of the proteins can be detected in the cytoplasm and the cell nucleus. Moreover, changes in their expression or the intracellular expression site (ICES) will affect the mismatch repair function.
Gastric cancer is one of the most common malignant tumors in China, and gastric carcinogenesis also is related to the loss of cellular mismatch repair function[2-4]. However, it is still unclear if gastric carcinogenesis is affected by the expression and ICES of the hMSH2 and hMLH1 proteins. In our study we performed in situ assays of ICES, hMSH2 and hMLH1 protein expression in gastric cancers, paraneoplastic gastric and noncancerous gastritic mucosa, in order to analyze the relationship among the ICES, expressed proteins, the occurrence and differentiation of gastric cancers, as well as the clinical significance.
MATERIALS AND METHODS
Specimens
The tissue specimens were collected in our hospitals as follows: 172 cases of gastric cancer, 151 paraneoplastic gastric mucosas and 34 noncancerous gastritic mucosas from patients who had undergone surgery and gastroscopic biopsy. In the gastric-cancer group, there were 127 male and 45 female patients with ages ranging from 33 to 89 years and a mean of 61.4±11.2. The tissues were treated with a 4% neutral formlin fixation, paraffin imbedding, sectioning at 3 to 4 μm followed by H&E staining. Pathological diagnoses were performed by two experienced pathologists. In the gastric-cancer group, there were 14 cases with well-differentiated carcinoma, 48 with moderately-differentiated cancer, 83 with poorly-differentiated cancer and 27 with colloid carcinoma. Pathological diagnoses of the paraneoplastic gastric mucosas and the noncancerous gastric mucosas were chronic superficial gastritis or chronic atrophic gastritis.
Histological in situ assay of the hMSH2 and hMLH1 protein expressions
The immunohistochemical SP staining method was employed, and conventional deparaffinizing and hydrating of the sections and microwave coctoantigen repair conducted. Incubation of the mouse-antihuman monoclonal antibodies for hMSH2 (1:250 dilution, Zymed, clonal line: FE11) and for hMLH1 (1:50 dilution, Zymed, clonal line: 14) was performed overnight at 4°C. Other successional procedures were carried based on specification in the SP kit (the Fuzhou Maixin Biological Technology Co., Ltd.), with PBS as the negative control to replace the first antibody, and known positive sections as the positive controls.
Assessment of the results
The cells with pale-brown granules in the cell nucleus or cytoplasm were identified as positive. After assessment of 5 high-power fields or 500 cells, the specimen was scored as positive expression if the ratio of the positive cells to parenchymal cells was equal to or over 10%. Otherwise, there was negative expression. Based on the location of positive granules in the cells, the cases with a positive expression in the cell nucleus or a concurrent positive expression of the cell nucleus and cytoplasm were defined as positive nuclear-expression cases. The cases with only positive expression in the cytoplasm were identified as cytoplasmic-expression cases. A double-blind method was used to determine the results by two experienced pathologists who had no information concerning the patient clinical data.
Statistical analysis
The χ2 test was adopted and the Spearman rank correlation analysis used for correlation statistics, by SPSS13.0 statistical software. The value of P<0.05 indicated a statistical significance in the tests.
RESULTS
The expression of the hMSH2 and hMLH1 proteins in various gastric mucosas
The positive expression rate of the protein hMSH2 in gastric cancer was 69.8% (120/172), which was significantly higher than that in the paraneoplastic gastric mucosas with 49.7% (75/151) and noncancerous gastric mucosas with 32.4% (11/34) (P=0.000). However there was no significant difference between the expression of the two latter groups (P=0.067). The positive expression rate of the mismatch repair protein hMLH1 in the gastric cancers was 73.3% (126/172), which was significantly higher than that found in paraneoplastic gastric mucosas with 57.6% (87/151), and noncancerous gastric mucosas with 41.2% (14/34) (P=0.000). However there was no significant difference between the two latter groups (P=0.082, Fig.1). Table 1 shows there was no clear-cut correlation between the hMSH2 and hMLH1 expression rate and the related factors of gastric cancer patients, such as gender, age and level of cancer differentiation.
Expression of the mismatch repair proteins hMSH2 and hMLH1 in gastric cancer, paraneoplastic mucosa and gastric mucosa in patients with non-cancerous diseases.
Relationship between mismatch repair hMSH2 and hMLH1 protein expression and clinicopathologic features of the gastric cancer patients (% cases).
ICES of the hMSH2 and hMLH1 proteins in different gastric mucosas
In the cases with positive expression of the hMSH2 protein in gastric cancers, paraneoplastic gastric mucosas and noncancerous gastric mucosas, the nuclear expression rates were respectively 70.0% (84/120), 58.7% (44/75) and 36.4% (4/11), and the cytoplasmic expression rates were 30.0% (36/120), 41.3% (31/75) and 63.6% (7/11). The nuclear expression rates of the gastric cancers were apparently higher compared to that of noncancerous gastric mucosas (P=0.040), but there was no significant difference in comparison of the nuclear expression between gastric cancers and paraneoplastic gastric mucosas, or between paraneoplastic gastric mucosas and noncancerous gastric mucosas (P=0.105, P=0.203). The rate of hMSH2 nuclear expression gradually decreased, in order from gastric cancer and paraneoplastic gastric mucosa to noncancerous gastric mucosa. On the contrary, cytoplamic expression gradually raised (r=0.161, P=0.020).
In the cases with positive expression of the hMLH1 protein in gastric cancers, paraneoplastic gastric mucosa and noncancerous gastric mucosas, the nuclear expression rates were respectively 64.3% (81/126), 60.9% (53/77) and 64.3% (9/11), and the cytoplamic rates of expression were respectively 35.7% (45/126), 39.1% (34/77) and 35.7% (5/11). There were no significant differences in comparing the ICES of the hMLH1 protein among the three gastric mucosas (P=0.659).
Relationship between the differentiation level of gastric cancers and the hMSH2 and hMLH1 ICES
In the cases with positive expression of hMSH2 of the well, moderately and poorly-differentiated carcinomas and colloid cancer, the nuclear expression rates were 66.7% (6/9), 65.7% (23/35), 72.4% (42/58) and 72.2% (13/18), while the cytoplamic rates of expression were 33.3% (3/9), 34.3% (12/35), 27.6% (16/58) and 27.8% (5/18), respectively. There were no clear-cut correlations between the differentiated level of gastric cancers and the hMSH2 protein ICES (P=0.906). In the cases with positive expression of hMLH1 of the well, moderately and poorly-differentiated carcinomas and colloid cancer, the nuclear expression rates were 80.0% (8/10), 54.3% (19/35), 61.9% (39/63) and 83.37% (15/18), and the rates of cytoplamic expression were 20.0% (2/10), 45.7% (16/35), 38.1% (24/63) and 16.7% (3/18), respectively. There were aslo no clear-cut correlations between the differentiated level of gastric cancers and the hMLH1 protein ICES (P=0.133).
DISCUSSION
hMSH2 and hMLH1, which form a major intrinsic protein of the recognition and the conjunctional complexes, directly participate in the mismatch repair reaction. When DNA base mismatching occurs, the recognition complex begins to identify the base mismatching, and the conjunctional complex cuts off the mismatched DNA fragments. Finally new DNA fragments are synthesized and catalyze by DNA polymerase, thus completing the mismatch repair reaction[5]. Oncogenesis of many malignant tumors takes place along with abnormal structure and function of the mismatch repair proteins, usually showing a decrease or increase in the level of the hMSH2 and / or hMLH1 protein expression. Decreased expression of these proteins caused by genetic mutation or methylation of the mismatch repair has been linked to carcinoma of the large intestine, and especially, in cases of hereditary nonpolypous colon carcinoma[6,7]. In general, a decrease in mismatch repair protein expression may bring about a decrease or loss of the mismatch repair function and DNA repair, thus resulting in canceration of the cells. However an increase in hMSH2 and/or hMLH1 protein expression frequently takes place in some malignant tumors, such as glioblastoma, brochoalveolar cell cancer, bladder cancer and endometrial cancer etc[8-10].
Our study also showed that there was an overexpression of the hMSH2 and hMLH1 proteins in gastric cancer cells. It has been suggested by some researchers that an increase in DNA base mismatching might accompany an excess proliferation of the tumor cells, resulting in a compensating increment of protein synthesis during cell mismatch repair[11]. However, it is still unclear if enhancement of the mismatch repair function is accompanied by a high expression of the mismatch repair proteins. Mismatch repair proteins are nucleoproteins, that function mainly in the cell nucleus where nuclear expression and the mismatch repair take place. Therefore in our study, the significance of the mismatch repair protein expression in noncogenesis of gastric cancer was considered, in combination with studies on the ICES of the mismatch repair proteins.
It was found that the nuclear expression rate of the hMSH2 protein was significantly higher in gastric cancer than in noncancerous gastric mucosa, which indicated that in gastric cancer cells, there was not only an increase in hMSH2 protein synthesis, but also an increment in conveying the protein into the cell nucleus, suggesting that the onset of gastric cancer might be accompanied by the occurrence of an increase in DNA base mismatches.
The paraneoplastic gastric mucosa and gastric cancer cells both are located in the same environment, undergo the same surrounding irritation and invasion, and share a common heretary background. Therefore canceration can more easily occur in paraneoplastic gastric mucosa compared to noncancerous gastritic mucosa. Our study showed that, in comparing the expression rates and ICES of the hMSH2 and hMLH1 proteins between paraneoplastic gastric mucosa and noncancerous gastritic mucosa, there was no significant difference in the hMSH2 and hMLH1 protein expression. However, there was an increasing tendency in nuclear expression of the hMSH2 protein in paraneoplastic gastric mucosa compared to noncancerous gastritic mucosa. This suggests that there was enhanced base mismatching of the gastric mucosa before cancerization of the cells. Our present study showed there was no obvious increment in hMSH2 protein synthesis, but an increase in transfer of the protein to the cell nucleus. Therefore a concurrent assessment of expression and ICES of the mismatch repair proteins hMSH2 and hMLH1 in the gastric mucosa might be helpful for an early warning of gastric cancer.
Footnotes
This work was supported by a grant from the Knowledge Innovation Program of the Chinese Academy of Sciences (No. DICPkaaaabc).
- Received August 16, 2007.
- Accepted October 10, 2007.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
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