Heikkila R, Schwab G, Wickstrom E, Loke S L, Pluznik D H, Watt R, Neckers L M
Nature. 1987;328(6129):445-9. doi: 10.1038/328445a0.
Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in G0, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific antisense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically. We report here that the same c-myc complementary oligonucleotide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.
有丝分裂原或抗原引发的T淋巴细胞增殖涉及一系列基因激活事件。因此，当有丝分裂原激活的T细胞到达G1/S界面时，许多在G0期转录沉默的基因，如c-myc、白细胞介素-2（IL-2）、白细胞介素-2受体（IL-2R）和转铁蛋白受体（TfR）基因，已被转录激活。为了解单个基因在激活过程中的作用，必须能够特异性地干扰每个特定基因产物的表达或功能。通过这种方式，用抗体阻断IL-2R已证明，IL-2/IL-2R相互作用是激活的T细胞中诱导TfR表达所必需的。然而，当要阻断细胞内蛋白质的功能或表达时，尽管有可能，但将抗体引入活细胞的细胞质是一个限制因素。我们采用了另一种方法，即向大量细胞培养物中外源添加小的反义寡聚物。据报道，序列特异性反义寡脱氧核糖核苷酸可抑制细胞内病毒复制，而不干扰细胞蛋白质合成。同样，与珠蛋白mRNA起始密码子区域互补的寡聚物可抑制无细胞系统和兔网织红细胞中兔珠蛋白mRNA的翻译。最近，据报道，一种与人类c-myc mRNA起始密码子和四个下游密码子互补的十五聚脱氧核糖核苷酸可特异性抑制人类白血病细胞系HL-60的增殖。我们在此报告，相同的c-myc互补寡核苷酸可抑制人类T淋巴细胞中有丝分裂原诱导的c-myc蛋白表达并阻止进入S期。有趣的是，通过形态学母细胞转化、IL-2R和TfR基因的转录激活或3H-尿苷掺入的诱导评估，c-myc反义处理并未抑制从G0到G1的转变。
