A novel mechanism of non-Aβ component of Alzheimer's disease amyloid (NAC) neurotoxicity. Interplay between p53 protein and cyclin-dependent kinase 5 (Cdk5)

The non-Aβ component of Alzheimer's disease (AD) amyloid (NAC) is produced from the precursor protein NACP/α-synuclein (ASN) by till now unknown mechanism. Previous study showed that like ASN, NAC peptide induced oxidative/nitrosative stress and apoptosis. Our present study focused on the mechanisms of PC12 cells death evoked by NAC peptide, with particular consideration on the role of p53 protein. On the basis of molecular and transmission electron microscopic (TEM) analysis it was found that exogenous NAC peptide (10 μM) caused mitochondria dysfunction, enhanced free radical generation, and induced both apoptotic and autophagic cell death. Morphological and immunocytochemical evidence from TEM showed marked changes in expression and in translocation of proapoptotic protein Bax. We also observed time-dependent enhancement of Tp53 gene expression after NAC treatment. Free radicals scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1 mM) and p53 inhibitor (α-Pifithrin, 20 μM) significantly protected PC12 cells against NAC peptide-evoked cell death. In addition, exposure to NAC peptide resulted in higher expression of cyclin-dependent kinase 5 (Cdk5), one of the enzymes responsible for p53 phosphorylation and activation. Concomitantly, we observed the increase of expression of Cdk5r1 and Cdk5r2 genes, coding p35 and p39 peptides that are essential regulators of Cdk5 activity. Moreover, the specific Cdk5 inhibitor (BML-259, 10 μM) protected large population of cells against NAC-evoked cell death. Our findings indicate that NAC peptide exerts its toxic effect by activation of p53/Cdk5 and Bax-dependent apoptotic signaling pathway.     

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.     

Inhibition of p53 activity in vitro and in living cells by a synthetic peptide derived from its core domain

Much effort was expended to develop anti-cancer drugs that restore the function of the p53 tumor suppressor protein. However, the p53 activity might be harmful to the organism by amplifying side effects of chemotherapy. Therefore, under certain conditions, inhibition of p53 can serve to prevent inappropriately triggered apoptosis in normal tissues. We have identified a short 22-mer peptide derived from the p53 core domain (peptide 14), which can inhibit p53 specific DNA binding. Upon introduction in living cells, peptide 14 inhibited the ability of p53 to transactivate a reporter gene. Moreover, peptide 14 blocked p53-induced apoptosis in two different cell lines. Peptide 14-mediated inhibition of p53 activity appears to operate via the binding of peptide to the core and/or C-terminal domains of the p53 protein. Our findings provide a basis for the development of a novel approach aimed at the inhibition of p53. This could be essential for the protection from cell death in tissues thus suppressing for example neurodegenerative process or side effects of radio- or chemotherapy.     

Inhibition of p53 Activity In Vitro and In Living Cells by a Synthetic Peptide Derived from its Core Domain

Much effort was expended to develop anti-cancer drugs that restore the function of the p53 tumor suppressor protein. However, the p53 activity might be harmful to the organism by amplifying side effects of chemotherapy. Therefore, under certain conditions, inhibition of p53 can serve to prevent inappropriately triggered apoptosis in normal tissues. We have identified a short 22-mer peptide derived from the p53 core domain (peptide 14), which can inhibit p53 specific DNA binding. Upon introduction in living cells, peptide 14 inhibited the ability of p53 to transactivate a reporter gene. Moreover, peptide 14 blocked p53-induced apoptosis in two different cell lines. Peptide 14-mediated inhibition of p53 activity appears to operate via the binding of peptide to the core and/or C-terminal domains of the p53 protein. Our findings provide a basis for the development of a novel approach aimed at the inhibition of p53. This could be essential for the protection from cell death in tissues thus suppressing for example neurodegenerative process or side effects of radio- or chemotherapy.     

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human ??diseases in a dish??, without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.     

p53: Guardian of reprogramming

The reprogramming of somatic cells to induced pluripotent stem (iPS) cells is one of the major discoveries of recent years. The development and application of patient specific iPS lines could potentially revolutionize cell-based therapy, facilitating the treatment of a wide range of diseases. Despite the numerous technological advancements in the field, an in-depth mechanistical understanding of the pathways involved in reprogramming is still lacking. Several groups have recently provided a mechanistical insight into the role of the p53 tumor suppressor pathway in reprogramming. The repercussions of these findings are profound and reveal an unexpected role of p53 as a "guardian of reprogramming", ensuring genomic integrity during reprogramming at the cost of a reduced efficiency of the process. Here we analyze the latest findings in the field and discuss their relevance for future applications of iPS cell technology.     

Methods for iPS cell generation for basic research and clinical applications

The induction of pluripotency can be achieved by forced expression of defined factors in somatic cells. The established cells, termed induced pluripotent stem (iPS) cells, have pluripotency and an infinite capacity for self-renewal in common with embryonic stem (ES) cells. Patient-specific iPS cells could be a useful source for drug discovery and cell transplantation therapies; however, the original method for iPS cell generation had several issues that were obstacles to their clinical application. Recent studies have brought about various improvements for iPS cell generation and uncovered several characteristics of iPS cells. Here we summarize the current status of iPS cell studies, with a focus on the improved methods that can be used to generate iPS cells, and also refer to the future challenges.     

n-Butylidenephthalide (BP) Maintains Stem Cell Pluripotency by Activating Jak2/Stat3 Pathway and Increases the Efficiency of iPS Cells Generation

In 2006, induced pluripotent stem (iPS) cells were generated from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient; maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required an expensive reagent-leukemia induced factor (LIF). Our goal is to find a pure compound that not only maintains ES and iPS cell pluripotency, but also increases iPS cell generation efficiency. From 15 candidate compounds we determined that 10 μg/ml n-Butylidenephthalide (BP), an Angelica sinensis extract, triggers the up-regulation of Oct4 and Sox2 gene expression levels in MEF cells. We used ES and iPS cells treated with different concentrations of BP to test its usefulness for maintaining stem cell pluripotency. Results indicate higher expression levels of several stem cell markers in BP-treated ES and iPS cells compared to controls that did not contain LIF, including alkaline phosphatase, SSEA1, and Nanog. Embryoid body formation and differentiation results confirm that BP containing medium culture was capable of maintaining ES cell pluripotency after six time passage. Microarray analysis data identified PPAR, ECM, and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phosphorylated Jak2 and phosphorylated Stat3 protein levels increased following BP treatment and suppressed with the Jak2 inhibitor, AG490. The gene expression levels of cytokines associated with the Jak2-Stat3 pathway were also up-regulated. Last, we used pou5f1-GFP MEF cells to test iPS generation efficiency following BP treatment. Our data demonstrate the ability of BP to maintain stem cell pluripotency via the Jak2-Stat3 pathway by inducing cytokine expression levels, at the same time improving iPS generation efficiency.     

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice

It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid^−/−) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid^−/− mice. Their induction efficiency was similar to that of wild-type (Aid^+/+) iPS cells. The Aid^−/− iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid^+/+ and Aid^−/− iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation.     

KSR-Based Medium Improves the Generation of High-Quality Mouse iPS Cells

Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP⁺ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP⁺ iPS cells. Remarkably, PD can rescue Nanog-GFP⁺ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells.     

Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a promising therapeutic tool for many diseases, including aged tissues and organs at high risk of failure. However, the intrinsic self-renewal and pluripotency of ES and iPS cells make them tumorigenic, and hence, the risk of tumor development hinders their clinical application. Here, we present a novel approach to limit their tumorigenicity and increase their safety through increased copy number of tumor suppressors. iPS containing an extra copy of the p53 or Ink4a/ARF locus show normal pluripotency, as determined by in vitro and in vivo differentiation assays. Yet, while retaining full pluripotency, they also possess an improved engagement of the p53 pathway during teratocarcinoma formation, which leads to a reduced tumorigenic potential in various in vitro and in vivo assays. Furthermore, they show an improved response to anticancer drugs, which could aid in their elimination in case tumors arise with no adverse effects on cell function or aging. Our system provides a model for studying tumor suppressor pathways during reprogramming, differentiation, and cell therapy applications. This offers an improved understanding of the pathways involved in tumor growth from engrafted pluripotent stem cells, which could facilitate the use of ES and iPS cells in regenerative medicine.     

肝癌细胞系中Yamanaka因子的内源和异位表达及其作用

目的检测肝癌细胞系中Yamanaka因子的表达,为肝癌细胞来源的诱导多能干细胞（iPS细胞）的建立和通过细胞重编程逆转肝癌的恶性表型提供实验依据。方法通过反转录PCR检测Huh7、SMMC-7721、HepG2等肝癌细胞系中4种Yamanaka因子Oct3／4、Sox2、Klf4和c-Myc的mRNA水平,通过Western印迹方法检测上述因子在蛋白质水平的表达,并构建上述因子的慢病毒表达载体,在肝癌细胞中过表达上述转录因子。结果4种Yamanaka因子在肝癌细胞系中均有不同程度表达,mRNA和蛋白质水平的检测结果一致。统计学分析显示,c-Myc的表达显著高于正常肝细胞。成功构建了Yamanaka因子的慢病毒表达载体,并诱导肝癌细胞呈现类似iPS细胞的形态。结论Yamanaka因子在肝癌细胞中有不同程度的内源表达,而外源高表达外源Yamanaka因子对于iPS细胞的诱导至关重要。     

The wild-type sequence (wt) p53(25-35) peptide induces HLA-DR7 and HLA-DR11-restricted CD4+ Th cells capable of enhancing the ex vivo expansion and function of anti-wt p53(264-272) peptide CD8+ T cells

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53(108-122) and p53(110-124), have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by approximately 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4- normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-gamma assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53(25-35) peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by approximately 15% of the population. In addition, the presence of anti-p53(25-35) CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53(264-272) CD8+ T cells, which from one donor were initially "nonresponsive" to the wt p53(264-272) peptide. The wt p53(25-35) peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.