Vonlanthen S, Heighway J, Tschan M P, Borner M M, Altermatt H J, Kappeler A, Tobler A, Fey M F, Thatcher N, Yarbrough W G, Betticher D C
Department of Clinical Research, University of Bern, Switzerland.
Oncogene. 1998 Nov 26;17(21):2779-85. doi: 10.1038/sj.onc.1202501.
The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT-PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1alpha methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT-PCR results were balanced, and sites within exon 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a<p19ARF) predicted methylation of exon 1alpha (P=0.0006) as well as downregulation of p16INK4a. p19ARF downregulation was inversely correlated with p53 overexpression (P=0.025), whilst negative immunostaining for p16INK4a was inversely correlated with pRb down-regulation (P=0.003) and directly correlated with p53 overexpression as assessed by immunostaining (P=0.015). Our results show that: (1) p16INK4a and p19ARF expression are altered in almost half of resectable NSCLC; (2) methylation within exon 1alpha is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p53 alteration is consistent with a linked pathway.
CDKN2基因座表达两种不同的mRNA转录本，分别命名为α和β。α转录本的蛋白质产物是细胞周期抑制剂和肿瘤抑制因子p16INK4a。β转录本在一个交替阅读框（ARF）中进行翻译，在人类中编码一种15 kDa的蛋白质（p19ARF）。对p16INK4a的免疫组织化学和蛋白质印迹分析表明，该蛋白在大量肿瘤中表达下调，但对p19ARF表达的了解较少。我们通过免疫染色（n = 49）和多重逆转录聚合酶链反应（n = 28）检测了可切除的非小细胞肺癌（NSCLC）中p16INK4a和p19ARF的表达。为了研究p16INK4a下调的机制，采用基于聚合酶链反应的检测方法分析了外显子1α的甲基化情况。在通过免疫染色检测的49个肿瘤中，分别有24个和20个肿瘤p16INK4a和p19ARF表达为零至低水平。p19ARF主要定位于肿瘤细胞核，但在淋巴细胞、软骨细胞、成纤维细胞和上皮细胞的细胞核中也有不同程度的表达。p16INK4a正常的肿瘤中，p19ARF表达未降低。在16个p16INK4a表达为零至低水平的肿瘤中，11个肿瘤外显子1α内至少一个位点发生完全甲基化，这些肿瘤p19ARF表达正常。相反，在5个也缺乏p19ARF的肿瘤中未观察到外显子1α甲基化。在正常肺组织中，未检测到p16INK4a和p19ARF表达，多重逆转录聚合酶链反应结果平衡，外显子1α内的位点高度甲基化。在肿瘤中，多重逆转录聚合酶链反应数据失衡（p16INK4a < p19ARF）预示外显子1α甲基化（P = 0.0006）以及p16INK4a下调。p19ARF下调与p53过表达呈负相关（P = 0.025），而p16INK4a免疫染色阴性与pRb下调呈负相关（P = 0.003），与免疫染色评估的p53过表达呈正相关（P = 0.015）。我们的结果表明：（1）在几乎一半的可切除NSCLC中，p16INK4a和p19ARF表达发生改变；（2）外显子1α内的甲基化是p16INK4a下调的常见但非唯一机制；（3）p19ARF与p53改变的负相关与相关途径一致。
