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

p53, a tumor suppressor, inhibits cell proliferation by inducing cellular genes involved in the regulation of the cell cycle. MCG10, a novel cellular p53 target gene, was identified in a cDNA subtraction assay with mRNA isolated from a p53-producing cell line. MCG10 can be induced by wild-type but not mutant p53 and by DNA damage via two potential p53-responsive elements in the promoter of the MCG10 gene. The MCG10 gene contains 10 exons and is located at chromosome 3p21, a region highly susceptible to aberrant chromosomal rearrangements and deletions in human neoplasia. The MCG10 gene locus encodes at least two alternatively spliced transcripts, MCG10 and MCG10as. The MCG10 and MCG10as proteins contain two domains homologous to the heterogeneous nuclear ribonucleoprotein K homology (KH) domain. By generating cell lines that inducibly express either wild-type or mutated forms of MCG10 and MCG10as, we found that MCG10 and MCG10as can suppress cell proliferation by inducing apoptosis and cell cycle arrest in G(2)-M. In addition, we found that MCG10 and MCG10as, through their KH domains, can bind poly(C) and that their RNA-binding activity is necessary for inducing apoptosis and cell cycle arrest. Furthermore, we found that the level of the poly(C) binding MCG10 protein is increased in cells treated with the DNA-damaging agent camptothecin in a p53-dependent manner. These results suggest that the MCG10 RNA-binding protein is a potential mediator of p53 tumor suppression.

MCG10, a novel p53 target gene that encodes a KH domain RNA-binding protein, is capable of inducing apoptosis and cell cycle arrest in G(2)-M.

DNA polymerase η (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at γ-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.

DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

p73, a p53 family protein, shares significant sequence homolog and functional similarity with p53. However, unlike p53, p73 has at least seven alternatively spliced isoforms with different carboxyl termini (p73alpha-eta). Moreover, the p73 gene can be transcribed from a cryptic promoter located in intron 3, producing seven more proteins (DeltaNp73alpha-eta). DeltaNp73, which does not contain the N-terminal activation domain in p73, has been thought to be transcriptionally inactive and dominant negative over p53 or p73. To systemically analyze the activity of the DeltaN variant, we generated stable cell lines, which inducibly express DeltaNp73alpha, DeltaNp73beta, and various DeltaNp73beta mutants by using the tetracycline-inducible expression system. Surprisingly, we found that DeltaNp73beta is indeed active in inducing cell cycle arrest and apoptosis. Importantly, we found that, when DeltaNp73beta is expressed at a physiologically relevant level, it is capable of suppressing cell growth. We then demonstrated that these DeltaNp73beta activities are not cell type specific. We showed that the 13 unique residues at the N terminus are required for DeltaNp73beta to suppress cell growth. We also found that, among the 13 residues, residues 6 to 10 are critical to DeltaNp73beta function. Furthermore, we found that DeltaNp73beta is capable of inducing some p53 target genes, albeit to a lesser extent than does p73beta. Finally, we found that the 13 unique residues, together with the N-terminal PXXP motifs, constitute a novel activation domain. Like DeltaNp73beta, DeltaNp73gamma is active in transactivation. However, unlike DeltaNp73beta, DeltaNp73alpha is inactive in suppressing cell growth. Our data, together with others' previous findings, suggest that DeltaNp73beta may have distinct functions under certain cellular circumstances.

ΔNp73β Is Active in Transactivation and Growth Suppression

BACKGROUND
Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.

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Functional p53 Determines Docetaxel Sensitivity in Prostate Cancer Cells

The Unique NH2-terminally Deleted (ΔN) Residues, the PXXP Motif, and the PPXY Motif Are Required for the Transcriptional Activity of the ΔN Variant of p63

Xinbin Chen

Papers

MCG10, a novel p53 target gene that encodes a KH domain RNA-binding protein, is capable of inducing apoptosis and cell cycle arrest in G(2)-M.

DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

ΔNp73β Is Active in Transactivation and Growth Suppression

Functional p53 Determines Docetaxel Sensitivity in Prostate Cancer Cells

The Unique NH2-terminally Deleted (ΔN) Residues, the PXXP Motif, and the PPXY Motif Are Required for the Transcriptional Activity of the ΔN Variant of p63