Fracasso P M, Slapak C A, Nair S, Modrak D, Draper M P, LeCerf J M, Levy S B
Department of Medicine, New England Medical Center, Boston, MA, USA.
Oncol Res. 1997;9(4):183-91.
The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.
在两种不同的鼠源（DBA/2J）弗氏红白血病细胞系PC4和C7D的多药耐药亚系中，研究了多药耐药（mdr）基因的基因组结构和表达情况。这两个细胞系是通过从5 ng/ml开始逐步增加阿霉素浓度而获得的。在较高药物浓度（80 - 1280 ng/ml）下筛选出的PC4细胞系显示出所有三个mdr基因均有扩增，其中mdr3优先扩增。mdr2和mdr3基因的过表达伴随着它们的基因组扩增；然而，尽管mdr1基因有扩增，但未观察到其表达。在较高药物浓度（40 - 160 ng/ml）下筛选出的C7D细胞系中，观察到mdr1和mdr2有扩增和过表达，而mdr3没有。mdr1基因的表达增加先于基因扩增。母系细胞和耐药亚系之间，在微球菌核酸酶产生的、转录活性序列和蛋白质不同的染色质组分中，mdr特异性基因的分布有所不同。通过对PC4 - 80细胞（在80 ng/ml阿霉素中筛选并过表达mdr3）的染色质组分进行Southern分析，与亲本细胞相比，在富含活跃转录基因（如珠蛋白）的H1缺失单核小体组分中检测到mdr3基因富集（两到三倍）。使用一种基于PCR的新方法也检测到了mdr3富集，该方法检测了mdr3基因和重复序列。值得注意的是，尽管在PC4 - 20细胞系中未检测到mdr3表达，但从该细胞系中获得的H1缺失染色质组分显示出mdr3基因富集。这些研究表明，在选择对同一化疗药物耐药的遗传相关红白血病细胞系中，基因扩增和过表达的模式不同。mdr基因染色质组织的变化先于mdr3基因的过表达和扩增。
