CA : A Cancer Journal for Clinicians

The terms 'antioxidant', 'oxidative stress' and 'oxidative damage' are widely used but rarely defined. This brief review attempts to define them and to examine the ways in which oxidative stress and oxidative damage can affect cell behaviour both in vivo and in cell culture, using cancer as an example.

Biochemistry of oxidative stress

https://www.cell.com/cancer-cell/pdf/S1535-6108(14)00037-3.pdf

Mutant p53 in Cancer: New Functions and Therapeutic Opportunities

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Chem. Pharm. Bull.(オピニオン)

The untranslated regions (UTRs) at the 3'end of mRNA transcripts contain important sequences that influence the fate of mRNA and thus proteosynthesis. In this review, we summarize the information known to date about 3'end processing, sequence characteristics including related binding proteins and the role of 3'UTRs in several selected signaling pathways to delineate their importance in the regulatory processes in mammalian cells. In addition to reviewing recent advances in the more well known aspects, such as cleavage and polyadenylation processes that influence mRNA stability and location, we concentrate on some newly emerging concepts of the role of the 3'UTR, including alternative polyadenylation sites in relation to proliferation and differentiation and the recognition of the multi-functional properties of non-coding RNAs, including miRNAs that commonly target the 3'UTR. The emerging picture is of a highly complex set of regulatory systems that include autoregulation, cooperativity and competition to fine tune proteosynthesis in context-dependent manners.

https://www.tandfonline.com/doi/pdf/10.4161/rna.20231

The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-β in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment.

C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances

p53 missense mutant proteins commonly show increased stability compared to wild-type p53, which is thought to depend largely on the inability of mutant p53 to induce the ubiquitin ligase MDM2. However, recent work using mouse models has shown that the accumulation of mutant p53 occurs only in tumour cells, indicating that stabilization requires additional factors. To clarify the stabilization of p53 mutants in tumours, we analysed factors that affect their folding and degradation. Although all missense mutants that we studied are more stable than wild-type p53, the levels correlate with individual structural characteristics, which may be reflected in different gain-of-function properties. In the absence of Hsp90 activity, the less stable unfolded p53 mutants preferentially associate in a complex with Hsp70 and CHIP (carboxy terminus of Hsp70-interacting protein), and we show that CHIP is responsible for ubiquitination and degradation of these mutants. The demonstration of a complex interplay between Hsp90, Hsp70 and CHIP that regulate the stability of different p53 mutant proteins improves our understanding of the pro-tumorigenic effects of increased Hsp90 activity during multi-stage carcinogenesis. Understanding the roles of Hsp90, Hsp70 and CHIP in cancers may also provide an important avenue through which to target p53 to enhance treatment of human cancers.

Chaperone-dependent stabilization and degradation of p53 mutants.

Cell death is an essential event in normal life and development, as well as in the pathophysiological processes that lead to disease. It has become clear that each of the main cellular organelles can participate in cell death signalling pathways, and recent advances have highlighted the importance of the endoplasmic reticulum (ER) in cell death processes. In cells, the ER functions as the organelle where proteins mature, and as such, is very responsive to extracellular-intracellular changes of environment. This short overview focuses on the known pathways of programmed cell death triggering from or involving the ER.

/pdf/endoplasmic-reticulum-stress-and-apoptosis-1cn1tmpjhm.pdf

Endoplasmic reticulum stress and apoptosis.

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false-positive and false-negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild-type protein expressed in normal cells, such that homozygous gene deletion or non-sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post-translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21(WAF1) and MDM2, and statistical correlations between MDM2/p21(WAF1) and p53 were found in tumours with wild-type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

Roman Hrstka

Papers

The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.

C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances

Chaperone-dependent stabilization and degradation of p53 mutants.

Endoplasmic reticulum stress and apoptosis.

Discriminating functional and non‐functional p53 in human tumours by p53 and MDM2 immunohistochemistry