Blinded by the Light: The Growing Complexity of p53

https://www.gastrojournal.org/article/S0016-5085(04)01593-8/pdf

Hepatocellular carcinoma in cirrhosis: incidence and risk factors.

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause.


This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress.


It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies.


Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent ‘probes' of RS, are also critically reviewed.


The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.





Keywords: Cell culture, free radical, reactive species, antioxidant, oxidative stress, oxidative damage, fluorescent probe, lipid peroxidation, superoxide


Introduction
Free radicals and other ‘reactive oxygen (ROS)/nitrogen/chlorine species' (for an explanation of these terms see Table 1) are widely believed to contribute to the development of several age-related diseases, and perhaps, even to the aging process itself (Halliwell & Gutteridge, 1999; Sohal et al., 2002) by causing ‘oxidative stress' and ‘oxidative damage' (terms explained in Table 2). For example, many studies have shown increased oxidative damage to all the major classes of biomolecules in the brains of Alzheimer's patients (Halliwell, 2001; Butterfield, 2002; Liu et al., 2003). Other diseases in which oxidative damage has been implicated include cancer, atherosclerosis, other neurodegenerative diseases and diabetes (Hagen et al., 1994; Chowienczyk et al., 2000; Halliwell, 2000a, 2001, 2002a, 2002b; Parthasarathy et al., 2000). If oxidative damage contributes significantly to disease pathology (Table 3 lists the criteria needed to establish this), then actions that decrease it should be therapeutically beneficial (Halliwell, 2001; Lee et al., 2002a; Liu et al., 2003). If the oxidative damage is involved in the origin of a disease, then successful antioxidant treatment should delay or prevent the onset of that disease (Halliwell, 1991, 2002a, 2002b; Galli et al., 2002; Steinberg & Witztum, 2002). To establish the role of oxidative damage (Table 3), it is therefore essential to be able to measure it accurately. For example, the failure of interventions with antioxidants such as vitamin E, β-carotene or ascorbate to decrease disease incidence in several human intervention trials may have simply been due to the failure of these compounds to decrease oxidative damage in the subjects tested (Halliwell, 1999a, 2000c; Levine et al., 2001; Meagher et al., 2001). In this review, we will examine the methods available to measure reactive species (RS) and oxidative damage, with a particular emphasis on those applicable to human studies.



Table 1

Nomenclature of reactive species





Table 2

Some key definitions





Table 3

Criteria for implicating RS as a significant mechanism of tissue injury in human disease




Measuring RS in vivo: basic principles

Some fascinating techniques such as L-band electron spin resonance (ESR) with nitroxyl probes and magnetic resonance imaging spin trapping are under development to measure RS directly in whole animals (e.g. Berliner et al., 2001; Han et al., 2001; Utsumi & Yamada, 2003), but no probes are currently suitable for human use. Most RS persist for only a short time in vivo and cannot be measured directly. There are a few exceptions: examples include H2O2 (discussed below), and perhaps, NO•, in the sense that serum levels of NO2− have been claimed to measure vascular endothelial NO• synthesis (Kelm et al., 1999), despite the fact that NO2− is quickly oxidized to NO3− in vivo (Kelm et al., 1999; Oldreive & Rice-Evans, 2001). Essentially, there are two approaches to detecting transient RS: 


attempting to trap these species and measure the levels of the trapped molecules and


measuring the levels of the damage done by RS, that is, the amount of oxidative damage.




Sometimes other approaches are used. They include measurements of erythrocyte antioxidant defences and of total antioxidant activity of body fluids; falls in these parameters are often taken as evidence of oxidative stress. Erythrocytes cannot synthesize proteins, however, and their antioxidant enzyme levels may drop as they ‘age' in the circulation (Denton et al., 1975). Thus changes in their levels are more likely to reflect changes in the rates of red blood cell turnover: if this slows down, the circulating erythrocytes will be older on average and so levels of antioxidant enzymes in them will appear to fall. Vice versa, if an intervention accelerates red cell removal or increases erythropoiesis, levels of antioxidants in red cells will seem to rise. Hence, such data should be interpreted with caution.

Depending on the method that is used to measure it, the plasma or serum ‘total antioxidant capacity' (TAC) usually involves major contributions from urate, ascorbate and sometimes albumin −SH groups (Benzie & Strain, 1996; Halliwell & Gutteridge, 1999; Prior & Cao, 1999; Rice-Evans, 2000; Bartosz, 2003), although different methods measure different things (Schlesier et al., 2002; Bartosz, 2003). Thus, for example, if plasma albumin levels fall, TAC will fall. If urate levels rise, TAC will rise. The multiple changes in blood chemistry that occur in sick people mean that TAC changes should be interpreted with caution. TAC is also influenced by diet, often because consumption of certain foods may produce changes in plasma ascorbate and/or urate levels (Halliwell, 2003b).

/pdf/measuring-reactive-species-and-oxidative-damage-in-vivo-and-5a9iiwcjlt.pdf

Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles.

Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism.

/pdf/a-census-of-human-rna-binding-proteins-1mii7pfs5y.pdf

A census of human RNA-binding proteins.

The complex aspects linking the nucleolus and ribosome biogenesis to cancer are reviewed here. The available evidence indicates that the morphological and functional changes in the nucleolus, widely observed in cancer tissues, are a consequence of both the increased demand for ribosome biogenesis, which characterizes proliferating cells, and the changes in the mechanisms controlling cell proliferation. In fact, the loss or functional changes in the two major tumor suppressor proteins pRB and p53 cause an up-regulation of ribosome biogenesis in cancer tissues. In this context, the association in human carcinomas of nucleolar hypertrophy with bad prognoses is worthy of note. Further, an increasing amount of data coming from studies on both hepatitis virus-induced chronic liver diseases and a subset of rare inherited disorders, including X-linked dyskeratosis congenita, suggests an active role of the nucleolus in tumorigenesis. Both an up-regulation of ribosome production and changes in the ribosome structure might causally contribute to neoplastic transformation, by affecting the balance of protein translation, thus altering the synthesis of proteins that play an important role in the genesis of cancer.

/pdf/nucleolus-ribosomes-and-cancer-2ffdsg3li6.pdf

Nucleolus, ribosomes, and cancer.

In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin-fixed and paraffin-embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=-0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=-0. 99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6+/-5.6 SD and 95. 5+/-2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions.

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.

AgNOR staining and quantification

The importance of nucleolar changes in cancer cells is underestimated in tumour pathology. There is evidence that the nucleolus is the mirror of a series of metabolic changes that characterize cancer cells. Cell entry into the cell cycle is always associated with up-regulation of the nucleolar function and increased nucleolar size, which are also directly dependent on the rapidity of cell cycle progression. Furthermore, alterations of the major tumour suppressor retinoblastoma (Rb) and p53 pathways also contribute to the stimulation of nucleolar function and to nucleolar enlargement. High cell growth fraction, high cell growth rate and disruption of the Rb and p53 pathways are responsible for greater aggressiveness of cancer tissues. Therefore, the evaluation of nucleolar size allows one to obtain reliable information on the clinical outcome of the cancer: the greater the nucleolar size, the worse the tumour prognosis. Indeed, a series of studies carried out on numerous human tumours has shown that nucleolar hypertrophy (prominent nucleolus) was an independent predictive and prognostic parameter of a fatal clinical outcome.

What the nucleolus says to a tumour pathologist

Basal-like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up-regulation of stem cell regulatory genes We show here that tumour tissues expressing high levels of SLUG mRNA show a basal-like breast carcinoma phenotype and that such tumours also express high levels of stem cell-regulatory genes, ie CD133, Bmi1 Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal-like breast carcinoma We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX In this regard, we report that the exposure of SLUG-negative/luminal-like MCF-7 cells to a hypoxic environment promotes the onset of the basal-like breast carcinoma phenotype, together with up-regulation of the SLUG gene, which in turn blunts oestrogen receptor-α and boosts carbonic anhydrase IX gene expression Finally, we show that SLUG expression promotes the invasiveness of MCF-7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia-selected, MCF-7-derived cells in xenografts These data indicate that SLUG gene expression is part of a hypoxia-induced genetic programme which sets up a basal/stem cell-like, aggressive phenotype in breast cancer cells Copyright © 2007 Pathological Society of Great Britain and Ireland Published by John Wiley & Sons, Ltd

Davide Treré

Papers

Nucleolus, ribosomes, and cancer.

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.

AgNOR staining and quantification

What the nucleolus says to a tumour pathologist

The basal-like breast carcinoma phenotype is regulated by SLUG gene expression†