Zhang L, Taub J W, Williamson M, Wong S C, Hukku B, Pullen J, Ravindranath Y, Matherly L H
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201, USA.
Clin Cancer Res. 1998 Sep;4(9):2169-77.
Reduced folate carrier (RFC) transcripts in human leukemias were measured by a competitive PCR assay. Total RNAs were reverse transcribed and amplified in the presence of competitive templates for RFC and beta-actin. RFC transcripts were normalized to transcripts for beta-actin. In a series of K562 sublines, a approximately 30-fold range of RFC transcripts measured by PCR assay closely agreed with results of Northern analysis and varied in proportion to RFC protein on Western blots and [3H]methotrexate transport. RFC transcripts varied over a 88-fold range in 49 specimens from 48 children with acute lymphoblastic leukemia (ALL). Median RFC transcripts were similar for 15 T-cell and 33 B-precursor ALL samples (RFC/beta-actin = 6.13 x 10(-3) and 7.92 x 10(-3), respectively) and for 41 diagnostic (7.20 x 10(-3)) and 8 relapse (5.58 x 10(-3)) samples. Whereas PCR measurements of RFC transcripts approximated changes in methotrexate transport in B-precursor ALL blasts (n = 10), for T-ALL blasts (n = 12) there was no apparent relationship between these parameters. For hyperdiploid B-precursor blasts (n = 11) with greater than 52 chromosomes and three to five copies of chromosome 21, the median RFC transcript level was approximately 3-fold higher than that for diploid B-precursor blasts. RFC transcripts were also elevated for two of three B-precursor specimens with acquired trisomy 21. Our results suggest that RFC gene expression is far more predictive of methotrexate uptake capacity in B-precursor than T-ALL and that increased copies of chromosome 21 in B-precursor ALL blasts are generally associated with increased RFC transcripts. Hence, the good prognosis for children with hyperdiploid B-precursor ALL treated with antimetabolite-based chemotherapy and the high levels of methotrexate and methotrexate polyglutamates accumulated may, in part, reflect elevated RFC gene expression and capacities for methotrexate transport.
采用竞争性聚合酶链反应（PCR）检测人白血病中还原型叶酸载体（RFC）转录本。在存在RFC和β-肌动蛋白竞争性模板的情况下，对总RNA进行逆转录和扩增。RFC转录本以β-肌动蛋白转录本进行标准化。在一系列K562亚系中，通过PCR检测测得的RFC转录本范围约为30倍，这与Northern分析结果密切一致，并且在蛋白质印迹和[3H]甲氨蝶呤转运方面与RFC蛋白成比例变化。在48例急性淋巴细胞白血病（ALL）儿童的49个样本中，RFC转录本变化范围达88倍。15个T细胞ALL样本和33个B前体ALL样本的RFC转录本中位数相似（RFC/β-肌动蛋白分别为6.13×10⁻³和7.92×10⁻³），41个诊断样本（7.20×10⁻³）和8个复发样本（5.58×10⁻³）也是如此。虽然PCR检测的RFC转录本变化与B前体ALL原始细胞（n = 10）中甲氨蝶呤转运变化相近，但对于T-ALL原始细胞（n = 12），这些参数之间没有明显关系。对于染色体数目大于52条且有三到五个21号染色体拷贝的超二倍体B前体原始细胞（n = 11），RFC转录本中位数比二倍体B前体原始细胞高约3倍。在三个获得性21号染色体三体的B前体样本中，有两个样本的RFC转录本也升高。我们的结果表明，RFC基因表达在预测B前体ALL中甲氨蝶呤摄取能力方面比T-ALL更具预测性，并且B前体ALL原始细胞中21号染色体拷贝数增加通常与RFC转录本增加相关。因此，接受基于抗代谢物化疗的超二倍体B前体ALL儿童预后良好，以及积累的高浓度甲氨蝶呤和甲氨蝶呤多聚谷氨酸，可能部分反映了RFC基因表达升高和甲氨蝶呤转运能力增强。
