Xu J, Zimmer D B
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688, USA.
Exp Hematol. 1998 Mar;26(3):265-72.
In clinical studies, both hydroxyurea and butyrate increase fetal hemoglobin expression and ameliorate the symptoms of sickle cell anemia. However, comparative studies of the effects of hydroxyurea and butyrate on the expression of the individual fetal hemoglobin genes, A gamma and G gamma, have not been performed. The present study reports the effects of hydroxyurea and butyrate on steady-state A gamma and G gamma mRNA levels in K562 cells. Because the high degree of homology between the A gamma and G gamma cDNA sequences precludes the use of large cDNA probes for detection of individual fetal hemoglobin gene products, we investigated the specificity of two 20-base oligonucleotide probes synthesized from the region of greatest sequence diversity between these genes. Hybridization experiments demonstrated that the A gamma oligonucleotide probe was specific for A gamma DNA and RNA sequences and the G gamma oligonucleotide probe was specific for G gamma DNA and RNA sequences. These oligonucleotide probes detected both A gamma and G gamma mRNAs in K562 cells. In K562 cells treated with 2 mM sodium butyrate for 168 hours, the G gamma mRNA level increased 3.6-fold, whereas the A gamma mRNA level was not significantly different from untreated cells. Similar results were obtained when K562 cells were treated with 80 microM hydroxyurea. The G gamma mRNA level increased 2.3-fold at 168 hours, whereas the A gamma mRNA level did not change. The above results demonstrate that both butyrate and hydroxyurea selectively increase G gamma expression. Selective regulation of individual fetal hemoglobin genes is also seen in human development, where approximately 70% of the total fetal hemoglobin in the fetus is G gamma. Therefore, understanding the mechanisms by which butyrate and hydroxyurea differentially regulate fetal hemoglobin gene expression may provide insights into the developmental regulation of hemoglobin expression as well as the mechanisms of action of pharmacological agents currently being used to treat sickle cell disease.
在临床研究中，羟基脲和丁酸盐均可增加胎儿血红蛋白的表达，并改善镰状细胞贫血的症状。然而，尚未对羟基脲和丁酸盐对单个胎儿血红蛋白基因（Aγ和Gγ）表达的影响进行比较研究。本研究报告了羟基脲和丁酸盐对K562细胞中稳态Aγ和Gγ mRNA水平的影响。由于Aγ和Gγ cDNA序列之间的高度同源性使得无法使用大的cDNA探针来检测单个胎儿血红蛋白基因产物，因此我们研究了从这些基因之间序列差异最大的区域合成的两种20碱基寡核苷酸探针的特异性。杂交实验表明，Aγ寡核苷酸探针对Aγ DNA和RNA序列具有特异性，而Gγ寡核苷酸探针对Gγ DNA和RNA序列具有特异性。这些寡核苷酸探针在K562细胞中检测到了Aγ和Gγ两种mRNA。在经2 mM丁酸钠处理168小时的K562细胞中，Gγ mRNA水平增加了3.6倍，而Aγ mRNA水平与未处理细胞相比无显著差异。当K562细胞用80 μM羟基脲处理时，得到了类似的结果。在168小时时，Gγ mRNA水平增加了2.3倍，而Aγ mRNA水平没有变化。上述结果表明，丁酸盐和羟基脲均可选择性地增加Gγ的表达。在人类发育过程中也可见到单个胎儿血红蛋白基因的选择性调控，胎儿体内约70%的总胎儿血红蛋白为Gγ。因此，了解丁酸盐和羟基脲差异调节胎儿血红蛋白基因表达的机制，可能有助于深入了解血红蛋白表达的发育调控以及目前用于治疗镰状细胞病的药物的作用机制。
