Neutron activation increases activity of ruthenium-based complexes and induces cell death in glioma cells independent of p53 tumor suppressor gene

Novel metal complexes have received great attention in the last decades due to their potential anticancer activity. Notably, ruthenium-based complexes have emerged as good alternative to the currently used platinum-based drugs for cancer therapy, providing less toxicity and side effects to patients. Glioblastoma is an aggressive and invasive type of brain tumor and despite of advances is the field of neurooncology there is no effective treatment until now. Therefore, we sought to investigate the potential antiproliferative activity of phosphine-ruthenium-based complexes on human glioblastoma cell lines. Due to its octahedral structure as opposed to the square-planar geometry of platinum(II) compounds, ruthenium(II) complexes exhibit different structure–function relationship probably acting through a different mechanism from that of cisplatin beyond their ability to bind DNA. To better improve the pharmacological activity of metal complexes we hypothesized that neutron activation of ruthenium in the complexes would allow to decrease the effective concentration of the compound needed to kill tumor cells. Herein we report on the effect of unmodified and neutron activated phosphine ruthenium II complexes on glioblastoma cell lines carrying wild-type and mutated p53 tumor suppressor gene. Induction of apoptosis/authophagy as well as generation of reactive oxygen species were determined. The phosphine ruthenium II complexes tested were highly active against glioblastoma cell lines inducing cell death both through apoptosis and autophagy in a p53 independent fashion. Neutron activation of ruthenium compounds rendered them more active than their original counterparts suggesting a new strategy to improve the antitumor activity of these compounds.     

The p53 tumor suppressor gene

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specifity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

Expression of the human tumor suppressor p53 induces cell death in Pichia pastoris

The human tumor suppressor p53 is known as guardian of genome because of its involvement in many signals related to cell life or death. In this work, we report that human p53 induces cell death in the yeast Pichia pastoris. We showed a growth inhibition effect, which increased with the p53 protein expression level in recombinant Mut(s) (methanol utilization slow) strain of Pichia. However, no effect of p53 was observed in recombinant strain of Mut(+) (methanol utilization plus) phenotype. Interestingly, human p53 induces cell death in recombinant strains Mut(s) with characteristic markers of apoptosis such as DNA fragmentation, exposure of phosphatidylserine, and reactive oxygen species generation. Taken together, our results strongly suggest that human p53 is biologically active in this heterologous context. Thus, we propose that P.?pastoris could be a useful tool to better understand the biological function of human p53.     

The p53 tumor suppressor network in cancer and the therapeutic modulation of cell death

The molecular subversion of cell death is acknowledged as a principal contributor to the development and progression of cancer. The p53 tumor suppressor protein is among the most commonly altered proteins in human cancer. The p53 protein mediates critical functions within cells including the response to genotoxic stress, differentiation, senescence, and cell death. Loss of p53 function can result in enhanced rates of cell proliferation, resistance to cell death stimuli, genomic instability, and metastasis. The community of cancer scientists is now in possession of a vast repository of information regarding the frequency, specific mechanisms, and clinical context of cell death deregulation in cancer. This information has enabled the design of therapeutic agents to target proteins, including p53. The feasibility and impact of targeting cell death signaling proteins has been established in preclinical models of human cancer. The appropriate application of these targeted agents is now being established in clinical trials.     

抑癌基因p53与细胞自噬

细胞自噬(autophagy)是一种在进化上高度保守的代谢通路,它发生的分子机制和信号调控途径相当复杂,其中mTOR信号通路和Beclin1复合物发挥了最重要的调控作用,p53也是细胞自噬重要的调节因子。研究发现,p53可通过多种途径调节细胞自噬水平,这主要决定于它的亚细胞定位。在细胞核中,p53可通过多种方式上调细胞自噬;而在细胞质中,p53对细胞自噬具有负性调节作用,可抑制细胞自噬的发生。探究清楚p53与细胞自噬之间的调控关系将有助于人类正确认识由于细胞自噬功能异常所诱导的肿瘤的发生发展过程,从而最终攻克各种肿瘤性疾病。     

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs

Granzyme B (gzmB) of cytotoxic T lymphocytes (CTL) is essential for recovery from intracellular pathogens, but the molecular basis of its action is still unresolved. Here, we analyzed gzmB-mediated death pathways under physiological conditions using ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB(+)CTL). We show that gzmB(+)CTL abrogate target cell proliferation most likely by inducing cell death, independent of caspases and mitochondrial signaling. In addition, the data reveal that gzmB(+)CTL independently induce pro-apoptotic processes either via caspase-3/-7, leading to plasma membrane perturbance and ROS production or via Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit loss of DeltaPsi(m). Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB presumably to counteract evasion strategies of pathogens and to control tumors.     

Therapeutic targeting of the p53 tumor suppressor gene

The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.     

p53: the ultimate tumor suppressor gene?

Alterations in the gene encoding the cellular p53 protein are perhaps the most frequent type of genetic lesions in human cancer. At the heart of these alterations is the abrogation of the tumor suppressor activity of the normal p53. In many cases this is achieved through point mutations in p53, which often result in pronounced conformational changes. Such mutant polypeptides, which tend to accumulate to high levels in cancer cells, are believed to exert a dominant negative effect over coexpressed normal p53. Extensive research on p53, especially in the course of the last 3 years, has already provided much insight into the biological and biochemical mechanisms that underlie its capacity to act as a potent tumor suppressor. There are now many indications that p53 may play a central role in the control of cell proliferation, cell survival, and differentiation. Nevertheless, despite the purported importance of p53 for such crucial processes, mice can develop apparently without any defect in the total absence of p53. This raises the possibility that p53 may become critically limiting only when normal growth control is lost.     

Siva is an apoptosis-selective p53 target gene important for neuronal cell death

p53 plays a central role in neuronal cell death resulting from acute injury or disease. To define the pathway by which p53 triggers apoptosis, we used microarray analysis to identify p53 target genes specifically upregulated during apoptosis but not cell cycle arrest. This analysis identified a small subset of targets highly selective for the p53 apoptotic response, including Siva, a proapoptotic protein whose function is not well understood. Siva's expression pattern suggests that it plays an instructive role in apoptosis, and accordingly, we demonstrate that Siva is essential for p53-dependent apoptosis in cerebellar granule neurons. In addition, we determine that endogenous Siva is associated with the plasma membrane and that Caspase-8 and Bid are important for neuronal apoptosis. Our studies highlight the participation of membrane signaling events in p53's apoptotic program in primary neurons and have significant implications for understanding the mechanisms underlying pathogenesis after neuronal injury and in neurodegenerative diseases.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

Analysis of the p53 tumor suppressor gene by pyrosequencing

Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one or both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Furthermore, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.     

Akt versus p53 in a network of oncogenes and tumor suppressor genes regulating cell survival and death

The tumor suppressor protein, p53, and the oncoprotein, Akt, are involved in a cross talk that could be at the core of a cell's control machinery for switching between survival and death. This cross talk is a combination of reciprocally antagonistic pathways emanating from p53 and Akt, and also involves another tumor suppressor gene, PTEN, and another oncogene, Mdm2; such a connected network of cancer-relevant genes must be significant and demands a critical study. The p53-Akt network is shown in this report to possess the potential to exhibit bistability, a phenomenon in which two stable steady states of the system coexist for a fixed set of control parameter values. A hierarchy of qualitative networks and abstract kinetic models are analyzed and simulated on a computer to demonstrate the robustness of the bistable behavior, which, as argued in this study, is a likely candidate mechanism for a cellular survival-death switch. The analysis applies to cells that are neither p53-null nor Akt-null. The models presented here offer experimental predictions on the identity of control parameters of apoptotic thresholds and on network perturbations (including DNA damage and Akt inhibition) that are sufficient to generate switching between pro-survival and pro-death cellular states.     

Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides

Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We identified a human cancer cell line containing a PTC, for which high levels of readthrough were obtained in the presence of aminoglycosides. Using these cells, we demonstrated that aminoglycoside treatment stabilized the mutant mRNA, which would otherwise have been degraded by non-sense-mediated decay, resulting in the production of a functional full-length p53 protein. Finally, we showed that aminoglycoside treatment decreased the viability of cancer cells specifically in the presence of nonsense-mutated p53 gene. These results open possibilities of developing promising treatments of cancers linked with non-sense mutations in tumor suppressor genes. They show that molecules designed to induce stop-codon readthrough can be used to inhibit tumor growth and offer a rational basis for developing new personalized strategies that could diversify the existing arsenal of cancer therapies.