Haviernik Peter, Schmidt Martina, Hu Xinrong, Wolff Linda
Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA.
Oncogene. 2003 Mar 20;22(11):1600-10. doi: 10.1038/sj.onc.1206268.
Cyclin-dependent kinase inhibitors p16(INK4a) and p15(INK4b), encoded by the CDKN2A and B loci, play an important role in negative regulation of the cell cycle. Furthermore, p19(ARF) also encoded by the CDKN2A locus, has been shown to regulate positively the p53 pathway leading to growth arrest and apoptosis. All three genes have been inactivated in human tumors. In myeloid cells, p15(INK4b) mRNA is upregulated during cytokine-induced differentiation and/or growth arrest, and hypermethylation of the p15(INK4b) gene promoter region is a common event in acute myeloid leukemia. In the present study, we examined murine monocyte/macrophage tumors with deregulated c-myc for evidence of Ink4 gene inactivation. p15(Ink4b) mRNA and protein were detected in the majority of leukemias, and p16(Ink4a) mRNA and protein were highly expressed in two of them. pRb was in a hypophosphorylated state in most of the neoplasms indicating that the Cdk inhibitors that were expressed in the cells were functional. The observed expression of p15(Ink4b) is inconsistent with their proliferation state, although it might be expected to be expressed owing to the maturity of the cells. These data suggest, therefore, that deregulated c-Myc bypasses the pRb restriction point and cell cycle arrest in these tumors. An examination of p19(Arf) exons revealed deletions of the gene in up to 94% of the tumors. Since this gene shares exon 2 with p16(Ink4a), it is often difficult to determine which gene is the relevant tumor suppressor. However, the loss of only the p19(Arf)-specific exon 1 beta was observed in a tumor that had normal p16(Ink4a) protein expression. In addition, the p19(Arf)-specific exon was deleted in another tumor that expressed a functional chimeric protein, p15Ex1-p16Ex2-3; it was demonstrated here that this fusion protein is capable of inducing G1 arrest. These data overall supports the hypothesis that the critical inactivation event in these hematopoietic neoplasms is elimination of p19(Arf), and not Ink4 function.
由CDKN2A和B基因座编码的细胞周期蛋白依赖性激酶抑制剂p16(INK4a)和p15(INK4b)在细胞周期的负调控中起重要作用。此外，同样由CDKN2A基因座编码的p19(ARF)已被证明可正向调节p53通路，导致生长停滞和细胞凋亡。这三个基因在人类肿瘤中均已失活。在髓系细胞中，p15(INK4b) mRNA在细胞因子诱导的分化和/或生长停滞期间上调，并且p15(INK4b)基因启动子区域的高甲基化是急性髓系白血病中的常见事件。在本研究中，我们检查了c-myc失调的小鼠单核细胞/巨噬细胞肿瘤，以寻找Ink4基因失活的证据。在大多数白血病中检测到p15(Ink4b) mRNA和蛋白，其中两例中p16(Ink4a) mRNA和蛋白高表达。在大多数肿瘤中，pRb处于低磷酸化状态，表明细胞中表达的Cdk抑制剂具有功能。观察到的p15(Ink4b)表达与其增殖状态不一致，尽管由于细胞成熟可能预期其会表达。因此，这些数据表明，失调的c-Myc绕过了这些肿瘤中的pRb限制点和细胞周期停滞。对p19(Arf)外显子的检查发现，高达94%的肿瘤中该基因存在缺失。由于该基因与p16(Ink4a)共享外显子2，通常很难确定哪个基因是相关的肿瘤抑制因子。然而，在一个p16(Ink4a)蛋白表达正常的肿瘤中，仅观察到p19(Arf)特异性外显子1β的缺失。此外，在另一个表达功能性嵌合蛋白p15Ex1-p16Ex2-3的肿瘤中，p19(Arf)特异性外显子被删除；在此证明该融合蛋白能够诱导G1期停滞。这些数据总体上支持了这样的假设，即这些造血肿瘤中的关键失活事件是p19(Arf)的缺失，而不是Ink4功能的缺失。
