Modulating Molecular Functions of p53 with Small Molecules

Association of the p53 with many human cancers makes it a valuable therapeutic target. Stress-induced molecular interactions of p53 with other effector proteins are immensely intertwined with regulation of its functions in orchestrating a wide array of cellular responses, thereby defying analysis of the underlying molecular mechanisms with conventional molecular and cellular biology methods. Recent discoveries of small molecules that can selectively modulate the molecular interactions of p53 offer promising opportunities to address the challenge of dissecting these complex mechanisms and increase the hope for pharmacological control of p53 for clinical benefits of cancer patients.     

The ATDC (TRIM29) Protein Binds p53 and Antagonizes p53-Mediated Functions

The ataxia telangiectasia group D-complementing (ATDC) gene product, also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. ATDC has been proposed to form homo- or heterodimers and to bind nucleic acids. In cell cultures, ATDC expression leads to rapid growth and resistance to ionizing radiation (IR), whereas silencing of ATDC expression decreases growth rates and increases sensitivity to IR. Although ATDC is overexpressed in many human cancers, the biological significance of ATDC overexpression remains obscure. We report here that ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC represses the expression of p53-regulated genes, including p21 and NOXA. Mechanistically, ATDC binds p53, and this interaction is potentially fine-tuned by posttranslational acetylation of lysine 116 on ATDC. The association of p53 and ATDC results in p53 sequestration outside of the nucleus. Together, these results provide novel mechanistic insights into the function of ATDC and offer an explanation for how ATDC promotes cancer cell proliferation.Ataxia telangiectasia (AT) is an autosomal-recessive, complex, multisystem disorder (4, 33). One of the hallmarks for cells derived from AT patients is their unusual sensitivity to ionizing radiation (IR) and their failure to delay the cell cycle in S phase, termed radioresistant DNA synthesis. In addition, AT cells contain atypical cytoskeletal organization. An early attempt to complement the defect in an AT cell line (AT5BIVA) by transfection with a human cosmid library and selection by γIR resulted in the isolation of an AT cell line (1B3) that was partially resistant to IR (22). Subsequent isolation of the human DNA in the region of the integrated cosmid sequences in 1B3 cells resulted in the cloning of the ataxia telangiectasia group D-complementing (ATDC) gene (23).The ATDC gene is located at chromosome 11q23, where it is frequently associated with many different kinds of cancers. Analysis of the ATDC gene product revealed that it is a member of the tripartite motif (TRIM) protein family (also known as the RBCC family). This protein family is characterized by three zinc-binding domains, a RING, a B-box type 1, and a B-box type 2, followed by a coiled-coil region (5, 29, 42, 43, 47). Some TRIM proteins homo-multimerize through their coil-coil region, and the integrity of the TRIM motif is required for proper subcellular localization of TRIM proteins (43). Recently, it was discovered that one of the TRIM proteins is a component of the repressor binding site (RBS) binding complex found in EC and ES cells and functions in restricting retroviral replication (60).The ATDC protein has been shown to interact with a protein kinase C substrate and inhibitor, although the significance of this interaction is not exactly clear (6). Although early studies indicate that ATDC can complement the IR sensitivity of AT fibroblasts, later analysis reveals that ATDC does not affect radioresistant DNA synthesis and is most likely not mutated in any AT patients (29). Rather, the ATDC protein probably induces cell survival or confers cell growth advantage independently of IR. Although ATDC is overexpressed in a wide variety of different cancers (12, 17, 19, 26, 34, 38, 45, 66), its expression is highly cell type and tissue specific (6, 43) (see Fig. S1 and S2 in the supplemental material). Further, expression of ATDC in NIH 3T3 cells leads to more rapid growth and resistance to IR, whereas silencing of ATDC expression in BxPC-3 cells leads to decreased growth rate and increased sensitivity to IR (3).The beginning of a mechanistic understanding for the function of ATDC came recently from a study showing that ATDC promotes cell proliferation in vitro and enhances tumor growth and metastasis in vivo by stabilizing β-catenin via the Disheveled-2 protein (59). This finding is consistent with a previous report by the same group that pancreatic cancer cells overexpress ATDC at an average of 20-fold higher than epithelial cells from normal pancreas. In the present study, we propose an alternative, non-mutually-exclusive pathway by which ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC binds p53 and represses expression of p53-regulated genes, including p21 and NOXA. Intriguingly, we found that the ATDC-p53 interaction is regulated by posttranslational acetylation of ATDC. Our results provide novel mechanistic insights into the function of ATDC and further explanation of how ATDC promotes cancer cell proliferation.     

带组氨酸标签的斑马鱼p53真核表达载体的构建及表达鉴定

构建斑马鱼p53基因的真核表达系统,为下一步p53体内、外功能研究,以及为斑马鱼作为抗肿瘤药物筛选模型的构建和应用奠定基础。采用RT-PCR法从斑马鱼胚胎中扩增获得p53基因编码区,定向克隆到真核表达载pcDNA3.1上,构建真核表达质粒pcDNA3.1/his-p53,在起始密码前加入增强翻译的Kozak序列,并在终止密码前引入组氨酸标签便于检测和纯化,脂质体介导质粒转染HeLa细胞,RT-PCR和Western blotting检测基因表达情况。结果表明,成功构建了斑马鱼p53真核表达载体,RT-PCR扩增出1 100 bp的转录产物,表达产物能被抗his单克隆抗体特异性识别,Western blotting呈现53 kD左右单一条带。斑马鱼p53蛋白在HeLa细胞中成功表达。     

Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.     

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure

5q-syndrome is a distinct form of myelodysplastic syndrome(MDS) where a deletion on chromosome 5 is the underlying cause.MDS is characterized by bone marrow failures,including macrocytic anemia.Genetic mapping and studies using various models support the notion that ribosomal protein S14(RPS14) is the candidate gene for the erythroid failure.Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis,similar to what is observed with other ribosomal proteins.However,due to the higher risk for cancer development in patients with ribosome deficiency,targeting the p53 pathway is not a viable treatment option.To better understand the pathology of RPS14 deficiency in 5q-deletion,we generated a zebrafish model harboring a mutation in the RPS14 gene.This model mirrors the anemic phenotype seen in 5q-syndrome.Moreover,the anemia is due to a late-stage erythropoietic defect,where the erythropoietic defect is initially p53-independent and then becomes p53-dependent.Finally,we demonstrate the versatility of this model to test various pharmacological agents,such as RAP-011,L-leucine,and dexamethasone in order to identify molecules that can reverse the anemic phenotype.     

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S- and M-phase cyclins (A and B), securin, and geminin (13, 25, 31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G₁/S- and M-phase progression and inducing rereplication (6, 21, 25, 31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1^m/m) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutagenesis screen designed to identify genes that are necessary for normal morphological development in embryos (1, 8, 9). Subsequent studies showed that the insertion was located between the first and second exons of the emi1 gene (2). The morphological defects in emi1^m/m embryos at 2 days postfertilization (p.f.) are described in the public access zebrafish model organism database (http://zfin.org). Briefly, abnormalities in emi1^m/m embryos can be identified as early as 20 h p.f. and include slightly smaller heads and a lack of ventral curving of the posterior presomitic mesoderm. By 25 h p.f., the tail is more ventrally curved than in normal embryos, and increased cell death is observed throughout the central nervous system. Mutant embryos have circulating blood cells, although the onset of circulation is delayed. We became interested in this mutant because it harbors defects in the numbers and morphology of granulocytes, an important myeloid cell type within the innate immune system.There is evidence that EMI1 may function in cancer pathogenesis, and a variety of human tumors express this factor very highly, although in some cases this may be a consequence of elevated proliferation rates (11, 18). In fact, the human homologue of emi1 resides within chromosome 6q25, a region often deleted in leukemia, which, together with the cell cycle-regulatory role of EMI1, suggested that this factor may also function as a tumor suppressor whose loss of function could promote genetic instability. Thus, in addition to investigating the role of zebrafish emi1 in zebrafish development, with particular emphasis on hematopoiesis, we examined mammalian cells to identify mechanisms that may be important in EMI1-related malignant transformation and explored a putative tumor suppressor role for this cell cycle-regulatory protein.     

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6–7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.     

Living with p53, dying of p53

The p53 tumor suppressor protein acts as a major defense against cancer. Among its most distinctive features is the ability to elicit both apoptotic death and cell cycle arrest. In this issue of Cell, Das et al. (2007) and Tanaka et al. (2007) provide new insights into the mechanisms that dictate the life and death decisions of p53.     

细胞命运抉择的数学模型:活性氧与p53的关系及其意义(英文)

生物体内的细胞生活在复杂的环境中。在生物体内,活性氧是普遍存在的。生物体内的活性氧可以诱导DNA损伤,最终破坏基因组稳定性。其中,对基因组损伤最严重的是DNA双链断裂损伤。肿瘤抑制因子p53是细胞内介导DNA损伤反应的重要因子。p53可以修复损伤DNA,保护轻度受损细胞。而当细胞受到严重损伤时,p53能够诱发细胞凋亡,从而维持机体稳态。p53的动力学对于细胞的反应性具有重要影响,然而对这方面却缺少系统的认识。因此在本文中,我们主要关注运用数学模型方法研究p53脉冲的动力学性质,从而揭示细胞内潜在的生死选择机制。     

Predicted Functions of MdmX in Fine-Tuning the Response of p53 to DNA Damage

Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation.     

Zebrafish Mms2 promotes K63-linked polyubiquitination and is involved in p53-mediated DNA-damage response

The ubiquitin-conjugating enzyme Ubc13 together with a Ubc/E2 variant (Uev) form a stable complex and mediate K63-linked polyubiquitination, which is implicated in DNA damage tolerance in yeast and mammalian cells. The zebrafish Danio rerio is a lower vertebrate model organism widely used in the studies of vertebrate development and environmental stress responses. Here we report the identification and functional characterization of two zebrafish UEV genes, Drmms2 and Druev1. Their deduced amino acid sequences indicate that the two UEV genes evolved separately prior to the appearance of vertebrates. Both zebrafish Uevs form a stable complex with DrUbc13 as well as Ubc13s from yeast and human, and are able to promote Ubc13-mediated K63 polyubiquitination in vitro, suggesting that their biochemical activities are conserved. Despite the fact that both zebrafish UEV genes can functionally replace the yeast MMS2 DNA-damage tolerance function, they exhibited differences in DNA-damage response in zebrafish embryos: ablation of DrMms2, but not DrUev1, enhances both spontaneous and DNA-damage induced expression of p53 effectors p21 and mdm2. In addition, DrUbc13 specifically binds Drp53 in an in vitro assay. These observations collectively indicate that zebrafish Mms2 and Ubc13 form a stable complex, which is required for p53-mediated DNA-damage response.