C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

SW480, a p53 double-mutant cell line retains proficiency for some p53 functions

During certain types of cellular stress, the p53 tumor suppressor protein binds to DNA and transactivates a variety of genes that regulate critical responses including apoptosis, cell cycle checkpoints, differentiation, and angiogenesis. In addition, functional p53 is known to be required for efficient nucleotide excision repair (NER) of bulky DNA adducts generated through exposure to environmental mutagens such as UV light. Nonetheless, we previously showed that the model p53-mutated human adenocarcinoma strain SW480 is proficient in the removal of UV-induced cyclobutane pyrimidine dimers (CPD) via NER. We undertook the present study to begin probing the molecular basis for this unexpected repair phenotype. Cytogenetic analysis indicated that SW480 is stable at the chromosomal level, i.e. manifests a karyotypic profile very similar to that revealed for this line as far back as 14 years ago. After fluorescence in situ hybridization (FISH), using a probe complementary to the p53 gene, we found that 98% of the SW480 interphase nuclei contains three copies of the gene, later revealed to be localized on intact short arms of three chromosomes 17. DNA sequence analysis further showed that all three p53 copies in SW480 carry two point mutations (R273H and P309S), and levels of the corresponding mutated p53 protein are about 20-fold higher than in the closely related p53 wild-type strain LoVo. Using an electrophoretic mobility shift assay (EMSA), we demonstrated that R273H/P309S p53 is able to bind with wild-type affinity to its consensus DNA sequence in vitro. Analysis of p21(Cip1/WAF1) expression and in vivo footprinting by ligation-mediated PCR (LMPCR) showed that, in wild-type LoVo cells, an exposure to cellular stress (e.g. UV or ionizing radiation) is necessary for p53 activation of the p21(Cip1/WAF1) promoter. In contrast, the R273H/P309S-mutated p53 protein in SW480 constitutively activates p21(Cip1/WAF1) in the absence of stress through an unknown mechanism. A similar phenomenon whereby mutated p53 in SW480 is able to induce NER-related proteins might explain the normal DNA repair phenotype previously observed in this strain. For now we conclude that, in general, results obtained using SW480 as a p53-deficient cell line should be interpreted very cautiously.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

应用免疫-PCR检测癌症的初步研究

探索一种简便、灵敏度高、特异性强且易推广的血清中突变型ｐ５３蛋白检测方法,用于肿瘤早期诊断。用突变型ｐ５３蛋白的单克隆抗体,与特定ＤＮＡ片段连接制成基因探针,用免疫－ＰＣＲ技术,对１１７例病人血清样品进行检测。在１１７例病人血清样品中,检测总阳性率为４１．８８％（４９／１１７）,在临床怀疑为癌症及肿瘤病人中阳性率达５２．１１％（３７／７１）。     

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.     

TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.     

雷公藤甲素诱导HeLa细胞p53依赖的自噬和凋亡

自噬与凋亡被认为是细胞程序性死亡的两种重要途径,二者的交互联系对阐明药物的抗肿瘤机理有重要价值.众多的研究表明,雷公藤甲素对多种肿瘤细胞都具有显著的抑制作用.细胞凋亡与自噬可被相同的因素所诱导,p53蛋白可以同时对二者起调控作用,在自噬与凋亡的交互作用(crosstalk)中扮演着重要角色.本文以He La细胞为模型,研究雷公藤甲素诱导He La细胞发生自噬和凋亡的机制,并通过抑制p53依赖的转录,研究雷公藤甲素诱导He La细胞p53依赖的自噬和凋亡交互联系.     

Dietary downregulation of mutant p53 levels via glucose restriction

The majority of human tumors express mutant forms of p53 at high levels, promoting gain of oncogenic functions and correlating with disease progression, resistance to therapy and unfavorable prognosis. p53 mutant accumulation in tumors is attributed to the ability to evade degradation by the proteasome, the only currently recognized machinery for p53 disruption. We report here that glucose restriction (GR) induces p53 mutant deacetylation, routing it for degradation via autophagy. Depletion of p53 leads, in turn, to robust autophagic activation and to cell death, while expression of degradation-defective mutant p53 blocks autophagy and enables survival to GR. Furthermore, we found that a carbohydrate-free dietetic regimen that lowers the fasting glucose levels blunts p53 mutant expression and oncogenic activity relative to a normal diet in several animal model systems. These findings indicate that the stability of mutant forms of p53 is influenced by the levels of glucose and by dietetic habits. They also unravel the existence of an inhibitory loop between autophagy and mutant p53 that can be exploited therapeutically.     

SAHA shows preferential cytotoxicity in mutant p53 cancer cells by destabilizing mutant p53 through inhibition of the HDAC6-Hsp90 chaperone axis

Mutant p53 (mutp53) cancers are surprisingly dependent on their hyperstable mutp53 protein for survival, identifying mutp53 as a potentially significant clinical target. However, exploration of effective small molecule therapies targeting mutp53 has barely begun. Mutp53 hyperstabilization, a hallmark of p53 mutation, is cancer cell-specific and due to massive upregulation of the HSP90 chaperone machinery during malignant transformation. We recently showed that stable complex formation between HSP90 and its mutp53 client inhibits E3 ligases MDM2 and CHIP, causing mutp53 stabilization. Histone deacetylase (HDAC) inhibitors (HDACi) are a new class of promising anti-cancer drugs, hyperacetylating histone and non-histone targets. Currently, suberoylanilide hydroxamic acid (SAHA) is the only FDA-approved HDACi. We show that SAHA exhibits preferential cytotoxicity for mutant, rather than wild-type and null p53 human cancer cells. Loss/gain-of-function experiments revealed that although able to exert multiple cellular effects, SAHA's cytotoxicity is caused to a significant degree by its ability to strongly destabilize mutp53 at the level of protein degradation. The underlying mechanism is SAHA's inhibition of HDAC6, an essential positive regulator of HSP90. This releases mutp53 and enables its MDM2- and CHIP-mediated degradation. SAHA also strongly chemosensitizes mutp53 cancer cells for chemotherapy due to its ability to degrade mutp53. This identifies a novel action of SAHA with the prospect of SAHA becoming a centerpiece in mutp53-specific anticancer strategies.     

Increased BNip3 and decreased mutant p53 in cisplatin-sensitive PDT-resistant HT29 cells

We have reported previously the isolation of three photodynamic therapy (PDT)-resistant human colon carcinoma HT29 cell lines that show increased expression of the Hsp27 and BNip3 protein and a decreased expression of the mutant p53 protein compared to parental HT29 cells. Since mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. We report here that the PDT-resistant HT29 cell lines showed a significant increase in cisplatin sensitivity and an increase in both spontaneous and cisplatin-induced apoptosis compared to parental HT29 cells. In addition, the cisplatin sensitivity of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells correlated with increased BNip3 and decreased mutant p53 protein levels, but not Hsp27 protein levels.     

突变型p53与其合成致死基因的研究进展

恶性肿瘤的靶向治疗已经成为现阶段肿瘤治疗的热点。随着人们对癌基因认知的加深,借助合成致死的方法靶向治疗肿瘤已成为针对肿瘤特异性治疗的新策略。p53基因突变在肿瘤的形成和发展过程中具有重要作用。因此,了解肿瘤中与突变型p53基因有合成致死关系的靶基因的作用方式,有助于指导由突变型p53基因诱发肿瘤的个性化治疗。与突变型p53基因具有合成致死关系的靶基因可分为细胞周期调控基因和细胞非周期调控基因,文章综述了这两类靶基因与突变型p53基因如何构成合成致死作用以及此作用的现实意义。