Roy K, Chiao J H, Spengler B A, Tolner B, Yang C H, Biedler J L, Sirotnak F M
Program in Cell Biology and Genetics, Graduate School of Medical Sciences, Cornell University, New York, New York, USA.
Cancer Genet Cytogenet. 1998 Aug;105(1):29-38. doi: 10.1016/s0165-4608(98)00005-3.
A variant of the L1210 cell (L1210/R83) selected in the presence of the lipophilic antifolate, metoprine, and a concentration of the natural diastereoisomer of 5-formyltetrahydrofolate, lL5CHO-folateH4, suboptimum for growth exhibited a 35-fold increase compared to parental L1210 cells in one-carbon, reduced folate transport. This was evidenced by the increase in Vmax for [3H]MTX (methotrexate) influx and a commensurate increase in the amount of the 46 kilodalton (kDa) transport protein and reduced folate carrier (RFC-1) mRNA. The variant is resistant to lipophilic antifolates, but shows collateral sensitivity to classical folate analogues. Karyotype analysis of L1210/R83 cells revealed the presence of several new chromosome abnormalities. One of these was a large, submetacentric marker chromosome comprising a normal #10 and a longer, abnormally banded arm of uncertain origin which exhibited an interstitial, palely staining, HSR-like segment. The results of Southern and Northern blotting showed that the RFC-1 gene copy number and RNA transcript level were markedly increased (30-35 fold) in L1210/R83 cells. Fluorescence in situ hybridization (FISH) analysis revealed that the HSR-like segment in these cells was the site of amplified RFC-1 genes. Independent revertant subclones, obtained following growth in the absence of selection pressure, showed four- to 12-fold decreases in [3H]MTX influx Vmax and in amount of NHS (N-hydroxysuccinimide)-[3H]MTX affinity labeled one-carbon, reduced folate transporter compared to L1210/R83 cells. RFC-1 gene copy number also decreased, and the mean length of the HSR in these revertants declined 1.6- to 5-fold. Based upon genomic nucleotide sequencing, the RFC-1 gene in the normal mouse genome was localized to chromosome 10 in close association with the alpha 1 (Col18a1) collagen gene at 10B3(locus 41cM). The close association of these genes was confirmed by other data showing that the alpha 1 collagen gene was co-amplified in L1210/R83 cells. These results document the amplification at the site of a putative HSR in an L1210 cell variant of the RFC-1 gene regulating expression of the one-carbon, reduced folate transporter.
在亲脂性抗叶酸剂美托普林以及对生长来说亚最佳浓度的5-甲酰四氢叶酸的天然非对映异构体lL5CHO-叶酸H4存在的情况下筛选出的L1210细胞变体（L1210/R83），其一碳、还原型叶酸转运能力相比亲代L1210细胞提高了35倍。这通过[3H]甲氨蝶呤（MTX）流入的Vmax增加以及46千道尔顿（kDa）转运蛋白和还原型叶酸载体（RFC-1）mRNA量的相应增加得以证明。该变体对亲脂性抗叶酸剂有抗性，但对经典叶酸类似物表现出间接敏感性。对L1210/R83细胞的核型分析揭示存在几种新的染色体异常。其中之一是一条大的亚中着丝粒标记染色体，它由一条正常的#10号染色体和一条来源不明的更长、带异常条带的臂组成，该臂表现出一个间质、淡染的类均染区（HSR）片段。Southern和Northern印迹结果显示，L1210/R83细胞中RFC-1基因拷贝数和RNA转录水平显著增加（30 - 35倍）。荧光原位杂交（FISH）分析表明，这些细胞中的类HSR片段是RFC-1基因扩增的位点。在无选择压力下生长后获得的独立回复亚克隆显示，与L1210/R83细胞相比，[3H]MTX流入Vmax以及N-羟基琥珀酰亚胺（NHS）-[3H]MTX亲和标记的一碳、还原型叶酸转运体的量降低了4至12倍。RFC-1基因拷贝数也减少，并且这些回复克隆中HSR的平均长度下降了1.6至5倍。基于基因组核苷酸测序，正常小鼠基因组中的RFC-1基因定位于10号染色体，与10B3处的α1（Col18a1）胶原基因紧密关联（位点41cM）。这些基因的紧密关联通过其他数据得以证实，这些数据表明α1胶原基因在L1210/R83细胞中共同扩增。这些结果证明了在调节一碳、还原型叶酸转运体表达的RFC-1基因所在的L1210细胞变体中的一个假定HSR位点处发生了扩增。
