Mutational and Functional Analysis of the p53 Tumor Suppressor Gene

我们正在研究p53介导的细胞凋亡的分子机制。我们以前的研究已经确定了一种新的凋亡途径，涉及p53与DNA解旋酶，XPB和XPD的功能和物理相互作用。 我们已经将这些研究扩展到RecQ解旋酶家族的其他成员，BLM和WRN，它们分别与癌症易感综合征Bloom和Werner有关。 WRN中的生殖系突变发现于患有早衰和癌症易感性综合征（称为Werner综合征（WS））的患者中。 p53通过其羧基末端在体内和体外与WRN蛋白结合。 WS成纤维细胞具有减弱的p53介导的凋亡反应，并且这种缺陷可以通过野生型WRN的表达来挽救。 这些数据支持的假设，p53可以通过特定的DExH-含有DNA解旋酶的调制诱导细胞凋亡，并可能有影响的癌症易感性观察WS患者。p53与BLM或WRN结合并抑制它们的DNA解旋酶活性。野生型p53大于p53突变体（248 W或273 H）阻止BLM或WRN对Holliday Junctions（HJ）的解旋。 通过磷酸化或Pab 421抗体修饰p53的羧基末端减少了p53与HJ的结合以及p53介导的对HJ的WRN或BLM解旋的抑制。 这些结果与p53有助于感知替代DNA结构的假设一致，例如，修复体的组装，以及DNA重组修复和凋亡的调节。 Bloom综合征（Bloom syndrome，BS）是一种常染色体隐性遗传的基因组不稳定综合征，以生长迟缓、免疫缺陷和癌症易感性为特征。 与来自XPB、XPD或WS个体的具有减弱的p53依赖性凋亡途径的细胞类似，p53介导的凋亡在BS成纤维细胞中也是有缺陷的。该凋亡途径可以通过野生型（wt）BLM基因的表达在功能上被拯救。 来源于BS供体的淋巴母细胞样细胞系（LCL）对γ-辐射或阿霉素诱导的细胞杀伤具有抗性，并且也可以被wt BLM拯救。 相比之下，BS细胞具有正常的Fas介导的细胞凋亡、正常的DNA损伤诱导的p53积累以及G1-S和G2-M细胞周期检查点。BLM定位于被鉴定为PML核体（NB）的核灶中，该核体是一种还含有早幼粒细胞白血病蛋白（PML）、Rb、SUMO-1等的结构，并且可能参与细胞凋亡。 来自携带p53生殖系突变的Li-Fraumeni综合征（LFS）患者的细胞具有减少的BLM病灶数量。 p53的诱导增加了BLM病灶的数量，但没有改变BLM水平或NB的数量。 这些结果表明p53的一种新的功能，并与以下假设相一致，即由p53介导的BLM向NB的核运输有助于其凋亡活性。

We are investigating the molecular mechanisms of p53-mediated apoptosis. Our previous studies have identified a novel pathway of apoptosis involving the functional and physical interaction of p53 with DNA helicases, XPB and XPD. We have extended these studies to other members of the RecQ helicase family, BLM and WRN, that are linked to cancer predisposition syndromes, Bloom and Werner, respectively. Germline mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53-mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients. p53 binds to BLM or WRN and inhibits their DNA helicase activities. Wild-type p53 greater than p53 mutants (248W or 273H) prevents the unwinding of Holliday Junctions (HJ) by BLM or WRN. Modification of the carboxyl terminal of p53 by phosphorylation or the Pab421 antibody reduces p53 binding to HJ and p53-mediated inhibition of WRN or BLM unwinding of HJ. These results are consistent with the hypothesis that p53 contributes to the sensing of alternative DNA structures, e.g., HJ, assembly of a repairsome, and modulation of DNA recombination repair and apoptosis. Bloom syndrome (BS) is an autosomal recessive genomic instability syndrome characterized by growth retardation, immune deficiency and cancer predisposition. Similar to cells from XPB, XPD or WS individuals who have an attenuated p53-dependent apoptotic pathway, p53-mediated apoptosis also is defective in BS fibroblasts. This apoptotic pathway can be functionally rescued by the expression of the wild-type (wt) BLM gene. Lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to either gamma-radiation or adriamycin-induced cell killing, and also can be rescued by the wt BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and a normal DNA damage-induced p53 accumulation and G1-S and G2-M cell cycle checkpoints. BLM localizes in nuclear foci identified as PML nuclear bodies (NBs), a structure that also contains the promyelocytic leukemia protein (PML), Rb, SUMO-1 and others, and may be involved in apoptosis. Cells from Li-Fraumeni syndrome (LFS) patients carrying p53 germline mutations have a decreased number of BLM foci. The induction of p53 increased the number of BLM foci, but did not alter either BLM levels or the number of NBs. These results indicate a novel function of p53 and are consistent with the hypothesis that, nuclear trafficking of BLM to NBs mediated by p53, contributes to its apoptotic activity.