环磷酰胺及塞替派诱导的人支气管上皮恶性转化细胞的p16^(INK4a)及p15^(INK4b)基因突变

Mutation of p16^(INK4a)and p15^(INK4b) genes of human bronchial epithelial cells malignantly transformed by cyclophosphamide and thiotepa

目的 了解环磷酰胺 (CP)和塞替哌 (TEPA)诱导永生化人支气管上皮细胞 (BEAS 2B)的恶性转化株内BEAS CP和BEAS TE细胞p16和p15基因的突变情况。方法 采用聚合酶链反应 (PCR)、聚合酶链反应 单链构象多态性分析 (PCR SSCP)及DNA序列分析技术。结果 BEAS CP细胞p15基因的外显子 1和外显子 2都出现了异常带型。BEAS TE细胞p16基因的外显子 1及外显子 2a出现了异常带型和迁移率滞后现象 ;p15基因外显子 1的 2条单链带之间出现异常条带。DNA序列分析发现BEAS CP细胞的p15基因外显子 1的 182位有C的插入 ,2 0 6位有G的插入 ;外显子 2的第 30 8位密码子有C→T(TCC→TTC)转换 (Ser→Phe) ,第 32 7位密码子有T→A(AAT→AAA)颠换 (Asn→Lys)。BEAS TE细胞的p16基因外显子 2a的第 12 5位密码子有G→A(CGC→CAC)转换 (Arg→His)。结论 p15基因在CP诱导BEAS 2B发生恶性转化的过程中发挥了重要作用。在TEPA诱导BEAS 2B细胞发生恶性转化的过程中可能涉及了p16基因的失活。

AIM To explore the molecular changes in genes p15 INK4b and p16 INK4a associated with cyclophosphamide(CP) and thiotepa(TEPA) mediated carcinogenesis. METHODS Polymerase chain reaction(PCR) amplification, single strand conformation polymorphism(SSCP) analysis and DNA sequencing. RESULTS Analysis of the genomic DNA from the BEAS-CP and BEAS-TE cells demonstrated mutations in the exon 2a of p16 gene, and exon 1 and exon 2 of p15 gene. For BEAS-CP cells, there were two single bases C and G pair insertions at 182th and 206th nucleotide in the exon 1 of p15 gene. Two bases substition (C→T and T→A) occurred in the exon 2 of p15 gene, one was TCC→TTC transition at codon 308 causing a serine to phenylalanine substitution, another one was AAT→AAA transversion at codon 327 leading to an asparagine to lysine substitution. For BEAS-TE cells, one mutation occurred in the exon 2a of p16 gene which was CGC→CAC transition at codon 125 resulting in an arginine to histidine substitution. CONCLUSION p15 gene plays an important role in the processes of cell transformation of BEAS-2B induced by CP and p16 gene is involved in the cell transformation of BEAS-2B induced by TEPA.