Nambiar Prashant R, Giardina Charles, Guda Kishore, Aizu Wataru, Raja Rajiv, Rosenberg Daniel W
Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3101, USA.
Cancer Res. 2002 Jul 1;62(13):3667-74.
Considering the importance of the oncogene checkpoint function of the alternating reading frame(ARF)-p53 pathway, studies were undertaken to evaluate the status of this pathway in azoxymethane (AOM)-induced mouse colon tumors. A PCR-based analysis of ARF and p53 cDNAs in normal colon tissues and AOM-induced colon tumors failed to detect mutations in either of these two critical tumor suppressor genes. In addition, laser capture microdissection of tumors followed by PCR-based sequencing of exons 5-7 of genomic p53 showed that even the most pleomorphic cancer cells were p53 normal. A marked increase in ARF mRNA and protein levels was observed in colon tumors, indicating activation of the ARF-p53 pathway in these tumors. High levels of ARF protein stabilized p53 protein in the tumors, but the p53 protein showed little biochemical activity. Compared with a mouse colonocyte cell line that expresses high levels of wild-type p53 (YAMC), the p53 protein in tumors had no detectable DNA binding activity nor did it activate p21 expression. In fact, p21 levels were lower in tumor tissue relative to normal mucosa, even though p53 levels were approximately 30-fold higher in tumors relative to control. Within the A/J tumors, we also used a cDNA microarray approach to screen a panel of genes that are transcriptionally up- or down-regulated by functional p53. The expression patterns of these p53-regulated genes were consistent with a lack of functional p53. This work demonstrates that the ARF-p53 oncogene checkpoint can be overcome without p53 mutations and that the mechanism used to overcome this checkpoint involves the suppression of p53 transcriptional activating activity. The AOM colon cancer model may be well suited for studying tumor promotion events that precede p53 disruption.
鉴于交替阅读框（ARF）-p53 通路的癌基因检查点功能的重要性，开展了相关研究以评估该通路在偶氮甲烷（AOM）诱导的小鼠结肠肿瘤中的状态。对正常结肠组织和 AOM 诱导的结肠肿瘤中的 ARF 和 p53 cDNA 进行基于 PCR 的分析，未能检测到这两个关键肿瘤抑制基因中的任何一个发生突变。此外，对肿瘤进行激光捕获显微切割，随后对基因组 p53 的外显子 5 - 7 进行基于 PCR 的测序，结果表明即使是最异形的癌细胞，其 p53 也是正常的。在结肠肿瘤中观察到 ARF mRNA 和蛋白水平显著增加，表明这些肿瘤中 ARF-p53 通路被激活。肿瘤中高水平的 ARF 蛋白稳定了 p53 蛋白，但 p53 蛋白几乎没有生化活性。与表达高水平野生型 p53 的小鼠结肠上皮细胞系（YAMC）相比，肿瘤中的 p53 蛋白没有可检测到的 DNA 结合活性，也未激活 p21 的表达。事实上，尽管肿瘤中的 p53 水平相对于对照高出约 30 倍，但肿瘤组织中的 p21 水平相对于正常黏膜更低。在 A/J 肿瘤中，我们还使用 cDNA 微阵列方法筛选了一组受功能性 p53 转录上调或下调的基因。这些 p53 调节基因的表达模式与缺乏功能性 p53 一致。这项工作表明，ARF-p53 癌基因检查点可以在没有 p53 突变的情况下被克服，并且用于克服该检查点的机制涉及抑制 p53 的转录激活活性。AOM 结肠癌模型可能非常适合研究在 p53 破坏之前的肿瘤促进事件。
