Kawakami Kazuyuki, Ishida Yoshinori, Danenberg Kathleen D, Omura Kenji, Watanabe Go, Danenberg Peter V
Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California 90033, USA.
Jpn J Cancer Res. 2002 Nov;93(11):1221-9. doi: 10.1111/j.1349-7006.2002.tb01227.x.
The thymidylate synthase (TS) gene has a polymorphic repeated sequence in its 5'-untranslated region. The repeat length is associated with TS protein expression, which suggests that we may be able to predict the efficacy of 5-fluorouracil (5-FU)-based chemotherapy from a patient's TS genotype determined through analysis of normal tissue obtained non-invasively. However, it is not yet elucidated whether the TS genotype is identical in tumor and normal tissue. In this study, we investigated the TS genotype in 151 matched tumor and normal DNA samples isolated from colorectal cancer and adjacent normal tissues by PCR analysis. The results showed that TS genotypes are identical in normal and tumor tissues of homozygous individuals, suggesting that the repeat sequence is stable through carcinogenesis. However, in heterozygous samples, an imbalance between the 2R and 3R alleles in the tumor DNA was frequently observed, suggesting loss of heterozygosity (LOH) at the TS locus. Detailed LOH analysis revealed that 62% (31 of 50) of 2R/3R-heterozygous samples had LOH. Frequent LOH at the TS locus was confirmed by RT-PCR of TS mRNA and microsatellite analysis using the marker D18S59, located on 18p11.3. There was no difference in the expressions of TS mRNA and TS protein between LOH and non-LOH samples. However, when the heterozygous genotype bearing LOH was subdivided according to the number of repeats, the cancer tissue with 2R/loss genotype expressed a significantly lower level of TS protein than did that with 3R/loss genotype. The results suggest that the difference in TS genotype between tumor and normal tissue due to LOH should be considered when the genotype is analyzed with normal tissue, such as peripheral blood cells, because it is important for TS protein expression.
胸苷酸合成酶（TS）基因在其5'-非翻译区有一个多态性重复序列。重复长度与TS蛋白表达相关，这表明我们或许能够通过对非侵入性获取的正常组织进行分析来确定患者的TS基因型，从而预测基于5-氟尿嘧啶（5-FU）的化疗疗效。然而，肿瘤组织和正常组织中的TS基因型是否相同尚未阐明。在本研究中，我们通过PCR分析，对从结直肠癌及其相邻正常组织中分离出的151对匹配的肿瘤和正常DNA样本的TS基因型进行了研究。结果显示，纯合个体的正常组织和肿瘤组织中的TS基因型相同，这表明重复序列在癌变过程中是稳定的。然而，在杂合样本中，经常观察到肿瘤DNA中2R和3R等位基因之间的失衡，这表明TS基因座存在杂合性缺失（LOH）。详细的LOH分析显示，62%（50个样本中的31个）的2R/3R杂合样本存在LOH。通过对TS mRNA进行RT-PCR以及使用位于18p11.3的标记D18S59进行微卫星分析，证实了TS基因座存在频繁的LOH。LOH样本和非LOH样本之间的TS mRNA和TS蛋白表达没有差异。然而，当根据重复次数对存在LOH的杂合基因型进行细分时，具有2R/缺失基因型癌组织中TS蛋白的表达水平明显低于具有3R/缺失基因型的癌组织。结果表明，当使用外周血细胞等正常组织分析基因型时，应考虑由于LOH导致的肿瘤组织和正常组织之间TS基因型的差异，因为这对TS蛋白表达很重要。
