TRIMming p53's anticancer activity.

Several TRIM proteins control abundance and activity of p53. Along this route, TRIM proteins have a serious impact on carcinogenesis and prognosis for cancer patients. In the past years, a significant increase has been made in our understanding of how the TRIM protein family controls p53 activity.

TRIM25 has a dual function in the p53/Mdm2 circuit.

P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identified TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context.

p53 is active in murine stem cells and alters the transcriptome in a manner that is reminiscent of mutant p53.

Since it was found that p53 is highly expressed in murine embryonic stem cells, it remained a mystery whether p53 is active in this cell type. We show that a significant part of p53 is localised in the nucleus of murine embryonic stem cells and that the majority of this nuclear p53 is bound to DNA. According to its nuclear localisation, we show that p53 alters the transcriptional program of stem cells. Nevertheless, the anti-proliferative activity of p53 is compromised in stem cells, and this control is due, at least in part, to the high amount of MdmX that is present in embryonic stem cells and bound to p53. Instead of the anti-proliferative activity that p53 has in differentiated cells, p53 controls transcription of pro-proliferative genes in embryonic stem cells including c-myc and c-jun. The impeded anti-proliferative activity of p53 and the induction of certain proto-oncogenes by p53 in murine embryonic stem cells can explain why stem cells proliferate efficiently despite having high levels of p53.

Activation of NF-κB after chronic ethanol intake and haemorrhagic shock/resuscitation in mice.

BACKGROUND AND PURPOSE: Chronic ethanol abuse and haemorrhagic shock are major causes of global mortality and, separately, induce profound hepato- and immune-toxic effects via activation of NF-κB. Here, we assessed the effects of chronic ethanol intake upon the pathophysiological derangements after haemorrhagic shock with subsequent resuscitation (H/R), with particular attention to the contribution of NF-κB. EXPERIMENTAL APPROACH: Transgenic NF-κB(EGFP) mice, expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-κB cis-elements were fed a Lieber-DeCarli diet containing ethanol (EtOH-diet) or an isocaloric control diet for 4 weeks and were then pairwise subjected to H/R. Liver tissues and peripheral blood were sampled at 2 or 24 h after H/R. Cytokines in blood and tissue and leukocyte activation (as CD11b expression) were measured, along with EGFP as a marker of NF-κB activation. KEY RESULTS: The EtOH-diet increased mortality at 24 h after H/R and elevated liver injury, associated with an up-regulation of NF-κB-dependent genes and IL-6 release; it also increased production of NF-κB-driven intercellular adhesion molecule 1 (ICAM-1) and EGFP in liver tissue. At 2h after the H/R procedure in ethanol-fed mice we observed the highest proportion of NF-κB activated non-parenchymal cells and an NF-κB-dependent increase in polymorphonuclear leukocyte CD11b expression. CONCLUSIONS AND IMPLICATIONS: The EtOH-diet exacerbated liver injury after H/R, accompanying an overwhelming hepatic and systemic immune response. Our findings contribute to evidence implicating NF-κB as a key player in the orchestration of the immune response in haemorrhagic shock patients with a history of chronic ethanol abuse.

Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications.

Damage to the genetic material can affect cellular function in many ways. Therefore, maintenance of the genetic integrity is of primary importance for all cells. Upon DNA damage, cells respond immediately with proliferation arrest and repair of the lesion or apoptosis. All these consequences require recognition of the lesion and transduction of the information to effector systems. The accomplishment of DNA repair, but also of cell cycle arrest and apoptosis furthermore requires protein-protein interactions and the formation of larger protein complexes. More recent research shows that the formation of many of these aggregates depends on post-translational modifications. In this article, we have summarized the different cellular events in response to a DNA double strand break, the most severe lesion of the DNA.

Activation of p53 in cervical carcinoma cells by small molecules.

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21(WAF1/CIP1) and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

UV-Induced stabilization of c-fos and other short-lived mRNAs.

Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

Radiation-induced signal transduction. Mechanisms and consequences.

Over a dose range up to 50 Gy of low-LET (linear energy transfer) ionizing radiation and up to 5 kJ/m2 UVB, mammalian cells convert molecular damage into productive response (mostly gain of function). By inactivation of negative regulatory components, such as protein tyrosine phosphatases as one mechanism discovered, the balance between restraining and stimulating influences is disturbed and an increase in signal flow results. Also DNA damage causing transcriptional arrest produces a signalling cascade of as yet unknown details. Such stimulation of the intracellular communication network can lead to apoptosis, elevated cell cycling and differentiation processes possibly including repair and recombination. The outcome likely depends on integration of all signals received which is as yet ill-understood. Although accurate determinations of low-dose inductions have not been achieved for technical reasons, the dose-response curves of induced signal transduction likely show threshold characteristics, in contrast to the direct consequences of DNA damage.

DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation.

Abundance and activity of p53 are predominantly regulated posttranslationally. Structural disturbance in transcribed genes induced by radiation, e.g. DNA damage, or by transcriptional inhibitors cause p53 protein stabilization by a yet unknown mechanism. Using stable and transient transfections for the analysis of p53 mutant proteins, we have ruled out a role in stabilization by UV, gamma irradiation or actinomycin C for the following putative phosphorylation sites in the p53 protein: serines 6, 9, 15, 33, 315 and 392, and threonine 18. By double mutation combinations of phosphorylations were also ruled out; 6,9; 15,18; 15,37. These mutations eliminate modifications by casein kinases I and II, DNA-PK, ATM, CDK and JNK. Also the 30 carboxyterminal amino acids are not required for induced p53 stabilization. Thus neither phosphorylations of individual amino acids nor interactions of the carboxyterminus of p53 with cellular macromolecules appear to play a role in the stabilization process. The only single prerequisite for induced stabilization of p53 is its prior destabilization by Mdm2. However, the level of active Mdm2 must be controlled carefully: overexpression of Mdm2 inhibits UV induced p53 stabilization.

Transcription factor E2F-1 is upregulated in response to DNA damage in a manner analogous to that of p53.

The transcription factor E2F-1 directs the expression of genes that induce or regulate cell division, and a role for E2F-1 in driving cells into apoptosis is the subject of intense discussion. Recently it has been shown that E2F-1 binds and coprecipitates with the mouse double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino acids 390 to 406) shows striking similarity to the Mdm2 binding domain of the tumor suppressor protein p53. It is known that interaction of Mdm2 with p53 through this domain is required for Mdm2-dependent degradation of p53. We show here that E2F-1 protein is upregulated in response to DNA damage. The kinetics of induction are dependent upon the source of DNA damage, i.e., fast and transient after irradiation with X rays and delayed and stable after irradiation with UVC, and thus match the kinetics of p53 induction in response to DNA damage. We show further that E2F-1 is also upregulated by treatment with the transcription inhibitor actinomycin D and with the kinase inhibitor DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not able to see an increase in E2F-1 RNA production but did find an increase in protein stability in UVC-irradiated cells. Upregulation of E2F-1 in response to DNA damage seems to require the presence of wild-type p53, since we did not observe an increase in the level of E2F-1 protein in several cell lines which possess mutated p53. Previous experiments showed that p53 is upregulated after microinjection of an antibody which binds to a domain of Mdm2 that is required for the interaction of Mdm2 with p53. Microinjection of the same antibody also increases the expression of E2F-1 protein, while microinjection of a control antibody does not. Furthermore, microinjection of Mdm2 antisense oligonucleotides upregulates E2F-1 protein, while microinjection of an unrelated oligonucleotide does not. These data suggest that E2F-1 is upregulated in a similar way to p53 in response to DNA damage and that Mdm2 appears to play a major role in this pathway.

Photoproducts in transcriptionally active DNA induce signal transduction to the delayed U.V.-responsive genes for collagenase and metallothionein.

Mammalian cells in culture react to ultraviolet irradiation with the massive transcriptional activation of several genes and with the stabilization of the p53 protein. While U.V.-induced transcription of several immediate-response genes depends on U.V.-induced activation of signal transduction generated by non-nuclear mechanisms, stabilization of p53 and the transcription of several delayed-response genes are triggered by U.V.-induced DNA damage. By comparing dose responses for the activation by U.V. of delayed-responsive genes (collagenase 1, metallothionein IIA) in cells from patients with different DNA repair deficiencies (complementation groups of Xeroderma pigmentosum, Cockayne's syndrome and Trichothiodystrophy), we show here that U.V.-induced transcription of these genes does depend on pyrimidine dimers in transcribed regions of the genome (but not on damage in its silent part). Since all cells with defects in DNA repair that had been tested and which lack different enzymes, respond to U.V. with expression of these same genes, functional repair does not appear to be required for the induction of expression, and repair intermediates (which would not be identical in cells of different repair deficiency) cannot be responsible for signal generation.

UV-induced signal transduction.

Irradiation of cells with wavelength ultraviolet (UVA, B and C) induces the transcription of many genes. The program overlaps with that induced by oxidants and alkylating agents and has both protective and other functions. Genes transcribed in response to UV irradiation include genes encoding transcription factors, proteases and viral proteins. While the transcription factor encoding genes is initiated in minutes after UV irradiation (immediate response genes) and depends exclusively on performed proteins, the transcription of protease encoding occurs only many hours after UV irradiation. Transcription factors controlling the activity of immediate response genes are activated by protein kinases belonging to the group of proline directed protein kinases immediately after UV irradiation. Experimental evidence suggests that these kinases are activated in UV irradiated cells through pathways which are used by growth factors. In fact, the first cellular reaction detectable in UV irradiated cells is the phosphorylation of several growth factor receptors at tyrosine residues. This phosphorylation does not depend on UV induced DNA damage, but is due to an inhibition of the activity of tyrosine phosphatases. In contrast, for late cellular reactions to UV, an obligatory role of DNA damage in transcribed regions of the genome can be demonstrated. Thus, UV is absorbed by several target molecules relevant for cellular signaling, and it appears that numerous signal transduction pathways are stimulated. The combined action of these pathways establishes the genetic program that determines the fate of UV irradiated cells.

Nuclear and non-nuclear targets of genotoxic agents in the induction of gene expression. Shared principles in yeast, rodents, man and plants.

The interplay between environmental cues and the genetic response is decisive for the development, health and well-being of an organism. For some environmental factors a narrow margin separates beneficial and toxic impacts. With the increasing exposure to UV-B this dichotomy has reached public attention. This review will be concerned with the mechanisms that mediate a cellular genetic response to noxious agents. The toxic stimuli find access to the regulatory network inside cells by interacting at several points with cellular molecules - a process that converts the 'outside information' into 'cellular language'. As a consequence of such interactions, many adverse agents cause massive signal transduction and changes of gene expression. There is an interesting conservation of the mechanisms from yeast to man. An understanding of the genetic programs and of their phenotypic consequences is lagging behind.

The mammalian UV response: mechanism of DNA damage induced gene expression.

DNA damage inducing treatment of cultured mammalian cells triggers the activation of transcription factors and the prolongation of the half life of p53. As the earliest event detectable in the nucleus (5 min), AP-1 (c-Jun/c-Fos) is post-translationally modified. Triggering this early event and triggering subsequent transcription factor dependent processes requires extra-nuclear components of signal transduction such as Src, Ras, Raf-1 and MAP-2 kinase. Recent efforts have concentrated on examining whether DNA damage or other secondary effects of the damaging agent generate the signal then passed on to transcription factors. Further, it has been studied whether a pathway of reverse signalling exists that originates in the nucleus and reaches the cell surface. At the cell surface the UV induced signalling chain can be interrupted experimentally. Beyond this step DNA damage and signal transduction induced by phorbol esters and growth factors merge and reach the nuclear proteins through common components.

Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases.

In chicken myeloid cells but not in erythroid cells, kinase-type oncogenes activate expression of the chicken myelomonocytic growth factor (cMGF). The autocrine loop established this way plays a key role in lineage-specific cooperation of nuclear and kinase-type oncogenes in retrovirally induced myeloid leukemia. In this report, we describe the cloning of the cMGF gene, including its promoter. The structure of the cMGF gene is homologous to those of the granulocyte colony-stimulating factor and interleukin-6 genes. Expression from reporter constructs containing the cMGF promoter is specific to myelomonocytic cells. Kinases activate cMGF at the transcriptional level in macrophages and strongly induce reporter expression in myelomonocytic cells.

A new role for the low density lipoprotein receptor.

It is well established that the low density lipoprotein (LDL) pathway functions to maintain a constant concentration of cellular cholesterol, but LDL effects that are unrelated to cholesterol metabolism have not been studied in great detail. In the present investigation we demonstrate that the LDL receptor pathway regulates cellular levels of free arachidonic acid (AA) and hence prostaglandin (PG) synthesis. We used platelet-derived growth factor (PDGF)-stimulated fibroblasts as a model system to investigate mechanism of LDL-dependent PG synthesis. PDGF-stimulated but not quiescent cells formed radiolabelled prostacyclin (PGI2) and PGE2 upon incubation with LDL that had been reconstituted with cholesteryl-(1-14C)-arachidonate (rec-LDL), while fibroblasts from patients that are afflicted with the LDL receptor negative phenotype of familial hypercholesterolaemia (FH) failed to synthesize significant amounts of PGs. Furthermore cells that had been preincubated with chloroquine or an anti LDL receptor antibody, that prevents binding of LDL to its receptor, did not produce significant amounts of PGs upon incubation with rec-LDL. Moreover incubation of PDGF-stimulated cells with LDL or AA led to a time and concentration-dependent inactivation of PGH synthase, the rate limiting enzyme of PG synthesis. When taken together our results establish a new role of the classical LDL receptor pathway of Brown and Goldstein by demonstrating that LDL provides AA to fibroblasts for eicosanoid formation and that LDL has a profound inhibitory effect on the key enzyme of PG synthesis, the PGH synthase.

The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor.

Animal cells can convert 20-carbon polyunsaturated fatty acids into prostaglandins (PGs) and leukotrienes. These locally produced mediators of inflammatory and immunological reactions act in an autocrine or paracrine fashion. Arachidonic acid (AA), the precursor of most PGs and leukotrienes, is present in the form of lipid esters within plasma lipoproteins and cannot be synthesised de novo by animal cells. Therefore, AA or its plant-derived precursor, linoleic acid, must be provided to cells if PGs or leukotrienes are to be formed. Because several classes of lipoproteins, including low-density lipoproteins (LDL), very-low-density lipoproteins, and chylomicron remnants, are taken up by means of the LDL receptor, and because LDL and very-low-density lipoproteins, but not high-density lipoproteins, stimulate PG synthesis, we have suggested previously that PG formation is directly linked to the LDL pathway. Using fibroblasts with the receptor-negative phenotype of familial hypercholesterolaemia and anti-LDL receptor antibodies, we show here that LDL deliver AA for PG production and that an LDL receptor-dependent feedback mechanism inhibits the activity of PGH synthase, the rate-limiting enzyme of PG synthesis. These results indicate that the LDL pathway has a regulatory role in PG synthesis, in addition to its well-known role in the maintenance of cellular cholesterol homeostasis.

Platelet-derived growth factor--a growth factor with an expanding role in health and disease.

Platelet-derived growth factor (PDGF) is the principal mitogen for connective tissue-derived cells such as fibroblasts, smooth muscle cells, and glial cells. It is synthesized by a variety of cell types and the synthesis of PDGF and its receptors is tightly controlled. Accumulating evidence obtained in vitro and in vivo suggests that PDGF plays important roles in the pathogenesis of clinically important diseases such as atherogenesis and cancer. Moreover, PDGF is an important research tool to study the signal transmission pathway of growth factors and other hormones.