Zhu Y M, Bradbury D, Russell N
Department of Haematology, Nottingham City Hospital.
Br J Haematol. 1994 Mar;86(3):533-9. doi: 10.1111/j.1365-2141.1994.tb04783.x.
We have previously shown that blasts from some patients with acute myeloblastic leukaemia (AML) grow autonomously in vitro and that this growth pattern is related to the production of autocrine growth factors including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 beta. However, another potential mechanism of autonomous growth involves the deletion of growth inhibitory molecules. One such inhibitory protein is the product of the retinoblastoma (Rb) gene which is expressed in normal haemopoietic cells and functions in cell cycle control and as a transcriptional repressor. Deletion of the Rb gene has been reported in various types of malignancy. We have examined the expression of the Rb gene product in blasts from 39 patients with AML by Western analysis. Using an antibody, Rb (Ab-2) which recognizes the C-terminal region, 11/39 samples (28%) failed to express Rb protein. The results of the Western blot analysis were confirmed by flow cytometry using a second antibody, Rb (Ab-1), against residues 300-380 of the Rb protein. Samples from seven of the 11 Rb-negative cases were studied for the expression of Rb mRNA; Rb mRNA was absent in four and three cases were positive. Analysis of the growth characteristics of these cells showed that blasts from 24/39 cases (62%) had partially or totally autonomous growth in a blast cell colony assay. Of these 24 samples with autonomous growth, 10 (41%) were Rb protein negative; in contrast, of the 15 cases with non-autocrine growth only one was Rb protein negative (7%, P < 0.05). Also, in Rb positive blasts, suppression of Rb protein production using an antisense oligonucleotide significantly increased proliferation of clonogenic AML blasts, confirming that the Rb protein acts as a negative regulator of growth in AML blasts. Our data suggest that deletion of Rb protein expression is frequently found in AML and is associated with the acquisition of autocrine growth characteristics, possibly as the consequence of derepression of genes involved in growth control and cytokine production.
我们之前已经表明，一些急性髓细胞白血病（AML）患者的原始细胞在体外能自主生长，且这种生长模式与自分泌生长因子的产生有关，这些自分泌生长因子包括粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-1β。然而，自主生长的另一种潜在机制涉及生长抑制分子的缺失。一种这样的抑制蛋白是视网膜母细胞瘤（Rb）基因的产物，它在正常造血细胞中表达，在细胞周期调控中发挥作用，并作为转录抑制因子。Rb基因的缺失已在各种类型的恶性肿瘤中被报道。我们通过蛋白质印迹分析检测了39例AML患者原始细胞中Rb基因产物的表达。使用一种识别C末端区域的抗体Rb（Ab-2），11/39个样本（28%）未能表达Rb蛋白。蛋白质印迹分析的结果通过流式细胞术得以证实，使用了针对Rb蛋白300 - 380位残基的第二种抗体Rb（Ab-1）。对11例Rb阴性病例中的7个样本进行了Rb mRNA表达研究；4例Rb mRNA缺失，3例为阳性。对这些细胞生长特性的分析表明，39例中的24例（62%）原始细胞在原始细胞集落测定中具有部分或完全自主生长能力。在这24个具有自主生长能力的样本中，10个（41%）为Rb蛋白阴性；相比之下，在15例非自分泌生长的病例中，只有1例为Rb蛋白阴性（7%，P < 0.05）。此外，在Rb阳性的原始细胞中，使用反义寡核苷酸抑制Rb蛋白产生显著增加了克隆性AML原始细胞的增殖，证实Rb蛋白在AML原始细胞中作为生长的负调节因子发挥作用。我们的数据表明，Rb蛋白表达缺失在AML中经常出现，并与自分泌生长特性的获得相关，这可能是参与生长控制和细胞因子产生的基因去抑制的结果。
