作者：JIANG Hai-Xing1, LIU Zhi-Ming2, ZHUANG Ya-Qiang3, YANG Ding-Hua2, JIANG Yi-Qiang4 and LI Jia-Quan2
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华人消化杂志981103 Abstract
AIM To investigate homozygous deletions of the multiple tumor suppressor gene 1 (MTS1/p16) in human gastric adenocarcinoma.
METHODS Using multiple PCR with primers for exon2, we analyzed the homozygous deletions of 45 primary gastric adenocarcinomas and 17 adjacent normal tissues surgically resected.
RESULTS The frequency of homozygous deletions of p16 gene in tumor tissues was 13.3% (6/45), but none in matched adjacent normal tissues. The deletions of p16 gene occurred exclusively in cases with metastasis to either lymph node or adjacent organs, and incidence of deletion in TNM stage Ⅲ and Ⅳ was significantly higher than those in stage Ⅰ and Ⅱ (23.1% vs 0% respectively), (χ2=5.0591, P<0.05).
CONCLUSION Homozygous deletion of p16 is involved in human gastric carcinogenesis, and might be a poor prognostic marker for gastric carcinoma.
INTRODUCTION
Recently, p16 gene, a putative tumor suppressor gene, has been found deleted at high frequency in a variety of tumor cell lines[1,2], nevertheless, the role of p16 in the tumorigenesis of primary cancer is unclear, since inactivation of p16 may have a selective growth advantage in vitro. In this study, we examined the homozygous deletions of p16 in primary gastric carcinoma to determine whether the loss of p16 plays a role in gastric carcinogenesis.
MATERIALS AND METHODS
Patients and tumor sampling
Fourty-five tumor samples and 17 matched normal tissues were obtained from patients with adenocarcinoma undergoing tumor excision. The tumors consisted of 5 papillary adenocarcinomas, 4 mucinous adenocarcinomas, 4 well differentiated adenocarcinomas, 32 poorly differentiated adenocarcinomas. Great care was taken to microdissect tumor specimens in order to eliminate non-neoplastic tissues, only tumors with greater than 80% neoplastic tissues were used to isolate DNA for further analysis. Tumor samples were snap frozen in liquid nitrogen and stored at -70℃ until use. According to the general rules for TNM classification (International Union against Cancer), five were stage Ⅰ, 14 in stage Ⅱ, 16 in stage Ⅲ and 10 in stage Ⅳ.
DNA extraction and polymerase chain reaction
DNA was isolated from the tissues as described[3]. Multiple PCR was performed in a final volume of 25μl containing 25ng genomic DNA, 50mm KCl, 10mm Tris-HCl (pH 8.3) 1.5mm MgCl2， 5% dimethysulfoxide, 100μm deoxyribonucleotide triphosphates, 2μ Taq polymerase, and 10pmol of each primers. Primers for exon 2 of p16 were (5＇-3＇) 2A:GCA AAT TGG AAA CTG GAA CGC (sense), 2B: TCT GAG CTT TGG AAG CTC (antisense). The exon 51 of Duchenne muscular dystrophy (DMD) served as an internal control, primers for exon 51 of DMD were (5＇-3＇) 51A: GAA ATT GGC TCT TTA GCT TGT GTT TC (sense), 51B: GGA GAG TAA AGT GAT TGG TGG AAA ATC (antisense). PCR conditions included a 5-min initial denaturation at 94℃, followed by 30 cycles of 1 min at 95℃, 1 min at 58℃, and 1 min at 72℃. PCR products were electrophoresed in a 1.5% agarose gel and stained with ethidium bromide. All the experiments were repeated to confirm the accuracy.
Statistical analysis
Comparison was made between two groups of patients in stage Ⅲ and Ⅳ, and stage Ⅰ and Ⅱ with an exact method of four-fold table. P<0.05 was considered statistically significant.
RESULTS
Figure 1 shows the result of the homozygous deletion in exon 2 of p16 in gastric carcinoma. Six (13.3%) of 45 tumors were found to have the homozygous deletion, in contrast, none was observed in the matched normal tissues. Table 1 shows the homozygous deletion of p16 and clinicopathological data in gastric carcinoma. The homozygous deletions all occurred in cases with metastasis to either lymph node or adjacent organs, including 5 poorly differentiated adenocarcinoma and one mucinous adenocarcinoma; two patients were in stage Ⅲ and four in stage Ⅳ; and the incidence of the deletion in stage Ⅲ and Ⅳ, and stage Ⅰ and Ⅱ was 23.1% (6/26) and 0% (0/19), respectively (χ2=5.0591， P<0.05).
Figure 1 G14 and G33 had homozygous deletion in exon 2. Band 480bp represents the amplified fragment of exon 2 for p16, band 388bp represents the amplified fragment of exon 51 for DMD. N:normal DNA; G:tumor DNA.
Table 1 Homozygous deletion of p16 and clinicopathological data in gastric carcinoma Case
Sex/age
Histology
TNM stage
Metastasis
LN
Organ
G11
M/50
P
Ⅳ
+
Liver
G14
M/62
P
Ⅲ
+
G32
M/58
P
Ⅳ
+
Liver
G33
M/53
P
Ⅳ
+
Spleen
G40
F/36
Mucinous
Ⅲ
+
G44
M/69
P
Ⅳ
+
Pancreas
P: poorly differentiated adenocarcinoma.
DISCUSSION
Recent advances in molecular biology have revealed that the genesis and/or progression of tumors results from accumulation of multiple genetic alterations, including inactivation of tumor suppressor genes and/or activation of protooncogenes. p16 is a cell cycle regulator that specifically binds to and inhibits Cdk4 (cyclin-dependent kinase 4), and its coding region encompasses three exons and two introns[4]. We have examined exon 2, which covers the majority of the coding region[5], to explore its role in gastric carcinogenesis.
The present observations indicated that homozygous deletion of p16 is involved in gastric carcinogenesis, suggesting a trend to metastases or a poor prognosis, the results are in agreement with the results published by Lu et al[6], uhose study revealed frequent deletion of p16 in primary gastric carcinoma using PCR and Southern blot analysis. But Lee and his colleagues[7] reported a result of conflict recently. They observed homozygous deletions of p16 from two of nine gastric carcinoma cell lines by the same method, but no deletions were detected in 36 primary gastric carcinomas. That specimens cantaining inflammatory and interstitial cells which may confound detection of homozygous deletions,and may give some interpretation to the different results. Recently, Dreyling and his colleagues[8]reported a method, the interphase fluorescence in situ hybridization, which could exclude the contamination by normal cells. By this method, they revealed a high frequent homozygous deletion of p16 in primary glioma.
We have not ruled out the existence of other structural abnormalities of p16, such as rearrangement, point mutation and aberrant methylation. Therefore, the 13.3% of mutation rate may be a minimal estimate. Besides, our results,as well as others also suggest that their genetic contribution may vary with the tumor type, since highly frequent p16 deletions were reported in primary esophageal cancers and other types of primary tumor[5,9].
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1Department of Gastroenterology, 2Surgery and 4Pathology,First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi Province, China
3Department of Surgery, The People＇s Hospital of Liuzhou, Liuzhou 545000, Guangxi Province, China
JIANG Hai-Xing, male, born on 1963-08-22 in Liuzhou, Guangxi Province, graduated from Guangxi Medical University as a postgraduate in 1992, now associate professor of internal medicine, majoring in clinical and basic research on gastroenterology and hepatology, having 23 papers published.
Correspondence to: Dr. JIANG Hai-Xing, Department of Gastroenterology, First Affiliated Hospital of Guangxi Medical University, 6 Binhu Road, Nanning 530021, Guangxi Province, China
Tel. +86*771*5312579, Fax. +86*771*5312523
Reveived 1998-06-29Original Articles
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