Cell Death: Critical Control Points

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are shown.

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Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal.

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

Compared with many normal tissues, cancer cells are highly sensitized to apoptotic signals, and survive only because they have acquired lesions — such as loss of p53 — that prevent or impede cell death. We are now beginning to understand the complex mechanisms that regulate whether or not a cell dies in response to p53 — insights that will ultimately contribute to the development of therapeutic strategies to repair the apoptotic p53 response in cancers.

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Live or let die: the cell's response to p53

It has become evident that we cannot understand tumour growth without considering components of the stromal microenvironment, such as the vasculature. At the same time, the tumour phenotype determines the nature of the tumour vasculature. Much research is now devoted to determining the impact of angiogenesis on tumour development and progression, and the reciprocal influences of tumour products on the microvasculature. A more detailed understanding of the complex parameters that govern the interactions between the tumour and vascular compartments will help to improve anti-angiogenic strategies-- not only for cancer treatment, but also for preventing recurrence.

Tumorigenesis and the angiogenic switch

It is well established that induction of the p53 tumor suppressor protein in cells can lead to either cell cycle arrest or apoptosis. To further understand features of p53 that contribute to these cell responses several p53-null Saos2 and H1299 cell lines were generated that express wild-type or mutant forms of p53, or the cyclin-dependent kinase inhibitor p21/WAF1, under a tetracycline-regulated promoter. Our results show that the cellular level of p53 can dictate the response of the cell such that lower levels of p53 result in arrest whereas higher levels result in apoptosis; nevertheless, DNA damage can heighten the apoptotic response to p53 without altering the protein level of p53 in cells. We also demonstrate that arrest and apoptosis are two genetically separable functions of p53 because a transcriptionally incompetent p53 can induce apoptosis but not arrest, whereas induction of p21/WAF1, which is a major transcriptional target of p53, can induce arrest but not apoptosis. Finally, we show that a full apoptotic response to p53 requires both its amino and carboxyl terminus, and our data suggest that there is synergism between transcription-dependent and -independent functions of p53 in apoptosis. Thus, there are multiple independent cellular responses to p53 that together may account for the extraordinarily high frequency of p53 mutations in diverse types of human tumors. The implications of these results are discussed and a model is proposed.

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p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition Both G1- and G2-arrested cells were observed in all cell types, with different preponderances Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb G2 arrest was more prominent in pRb-negative cells The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRB is a critical determinant in blocking DNA replication and in preventing endoreduplication

Examination of the expanding pathways for the regulation of p21 expression and activity.

p73, a potential tumor suppressor, is a p53 homologue. Transient over expression of p73 in cells can induce apoptosis and p21, a cellular p53 target gene primarily responsible for p53-dependent cell cycle arrest. To further characterize the role of p73 in tumor suppression, we established several groups of cell lines that inducibly express p73 under a tetracycline-regulated promoter. By using these cell lines, we found that p73 can induce both cell cycle arrest and apoptosis. We also found that p73 can activate some but not all of the previously identified p53 cellular target genes. Furthermore, we found that the transcriptional activities of p53, p73 alpha, and p73 beta to induce their common cellular target genes differ among one another. These results suggest that p73 is both similar to and different from p53 in their signaling pathways leading to tumor suppression.

https://cancerres.aacrjournals.org/content/canres/58/22/5061.full.pdf

The potential tumor suppressor p73 differentially regulates cellular p53 target genes.

p53 is the most commonly mutated gene in human cancer. After activation by cellular stresses such as DNA damage or oncogene activation, p53, a sequence-specific DNA-binding protein, induces the expression of target genes which mediate tumor suppression. Two recently identified p53 homologues, p63 and p73, appear to function similarly to p53, that is, they both activate target gene expression and suppress cell growth when overexpressed; however, the p63 and p73 genes are rarely mutated in human cancer and do not adhere to Knudson's classical model of a tumor suppressor gene. Recently, exciting observations suggest nonoverlapping functions for the family members. Herein, we outline the recent literatures identifying and characterizing both the common and distinct target genes of the p53 family transcription factors in relation to their signaling pathways.

Xinbin Chen

Papers

p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRB is a critical determinant in blocking DNA replication and in preventing endoreduplication

Examination of the expanding pathways for the regulation of p21 expression and activity.

The potential tumor suppressor p73 differentially regulates cellular p53 target genes.

The common and distinct target genes of the p53 family transcription factors