Pan S S, Forrest G L, Akman S A, Hu L T
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Cancer Res. 1995 Jan 15;55(2):330-5.
An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive HCT 116 cells. Eight NQO1 cDNA clones were isolated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from HCT 116-R30A, are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC-resistant sublines showed 75-90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of cDNA fragment of nucleotide 298-617 revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO1 activity. Transcriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.
据报道，从对丝裂霉素C（MMC）敏感的HCT 116细胞衍生而来的MMC抗性亚系HCT 116-R30A中，MMC抗性与NAD(P)H：醌氧化还原酶（NQO1）活性降低之间存在关联。通过逆转录PCR从这两个亚系中分离出8个NQO1 cDNA克隆。两个克隆，来自HCT 116的pDT9和来自HCT 116-R30A的pDT20，全长为274个氨基酸。这两个克隆在核苷酸464处存在T到C的替换，导致该酶中第139位精氨酸被色氨酸取代。pDT9和pDT20的NQO1在大肠杆菌中表达、纯化，并显示具有一个分子量为30,000的蛋白质亚基。第139位氨基酸的变化导致等电点pH从9.5变为8.35，并且在还原MMC时活性降低60%。所有其他六个克隆与pDT9的不同之处在于缺失了外显子4。在Northern印迹分析中，我们在所有亚系中检测到由于可变聚腺苷酸化产生的两种NQO1 mRNA种类（1.2和2.7千碱基）。MMC抗性亚系相对于HCT 116细胞显示出75 - 90%的mRNA表达。对核苷酸298 - 617的cDNA片段进行逆转录PCR扩增，发现在HCT 116细胞中有两种全长mRNA，但在HCT 116-R30A细胞中只有一种全长mRNA。在两个亚系中都检测到了缺失外显子4的mRNA。这两种全长mRNA可能来自同一基因的两个等位基因或嵌合体，而缺失外显子4的mRNA是可变剪接的结果。在Western印迹分析中，我们在所有亚系中仅检测到一种分子量为30,000的蛋白质。MMC抗性亚系中这种蛋白质的大量减少（为HCT 116的5%）解释了其NQO1活性降低95%的原因。转录调控和转录后修饰可能是MMC抗性亚系中NQO1基因表达差异以及NQO1蛋白浓度低的原因。两个回复突变体中MMC抗性的逆转和NQO1的恢复进一步支持了以下假设，即细胞对NQO1的控制可以调节MMC的细胞毒性。
