Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bcl-2 family proteins and interleukin-1-beta converting enzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basic regulators of apoptosis. However, the precise mechanism by which they interact is unclear. In this study, we found that gamma-radiation-induced apoptosis of leukemia cells was associated with activation of multiple caspases and bax up-regulation. Membrane changes and caspase activities were suppressed by specific caspase inhibitors. Similarly, the serine protease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also prevented caspase activation and poly(ADP-ribose) polymerase cleavage in vivo but had no effect on caspase activity in vitro. TLCK also prevented bax up-regulation as a result of its inhibitory effect on p53 function. Inhibitors of caspases and serine proteases partially prevented cell death, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signaling events in Bax-mediated cell death, including steps upstream and downstream of p53 and bax up-regulation.
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PMID:Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. 1043 17

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.
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PMID:Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species. 1045 54

The hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, and its two analogues, EB 1089 and CB 1093, are novel putative anticancer agents with an interesting profile of induction of growth inhibition, differentiation, and apoptosis in tumor cells. To study the signaling pathways mediating these events, we used two human breast cancer cell lines: MCF-7 cells, expressing a wild-type p53 tumor suppressor protein, and T47D cells, lacking a functional p53. Vitamin D compounds induced a growth arrest followed by apoptosis in both cell lines at concentrations ranging from 1 to 100 nM, indicating that p53 is not necessary for growth-inhibitory effects induced by vitamin D compounds. Surprisingly, apoptosis induced by these compounds occurred also independently of known caspases. Inhibition of caspase activation by overexpression of a cowpox-derived caspase inhibitor CrmA or by addition of inhibitory peptides acetyl-Asp-Glu-Val-Asp-aldehyde (200 microM), acetyl-Ile-Glu-Thr-Asp-aldehyde (50 microM), and Z-Val-Ala-D,L-Asp-fluoromethylketone (1 microM) showed no effect on the induction of growth arrest or apoptosis by vitamin D compounds under assay conditions in which apoptosis induced by TNF or staurosporine was effectively inhibited. Moreover, overexpression of caspase-3 in MCF-7 cells had no sensitizing effect to vitamin D compounds, and neither caspase-3-like protease activity nor cleavage of a caspase substrate poly(ADP)ribose polymerase was detected in lysates from apoptotic cells following the treatment with these compounds. Contrary to CrmA, overexpression of an antiapoptotic protein Bcl-2 in MCF-7 cells conferred a nearly complete protection from apoptosis induced by vitamin D compounds. Taken together, these data indicate that vitamin D compounds induce apoptosis via a novel caspase- and p53-independent pathway that can be inhibited by Bcl-2. This may prove useful in the treatment of tumors that are resistant to therapeutic agents that are dependent on the activation of p53 and/or caspases.
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PMID:Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53. 1051 95

This study deals with the apoptotic effect exerted on human retinoblastoma Y79 cells by both sodium butyrate and an inhibitor of 26S proteasome [z-Leu-Leu-Leu-CHO (MG132)] and their synergistic effect. Exposure to sodium butyrate (1-4 mM) induced an accumulation of cells in the G2-M phase that was already visible after 24 h of treatment, when morphological and biochemical signs of apoptosis appeared only in a small number of cells (5-10%). Thereafter, the apoptotic effects increased progressively with slow kinetics, reaching a maximum after 72 h of exposure, when they concerned a large fraction of cells (>75% with 4 mM sodium butyrate). Sodium butyrate stimulated the conversion of procaspase-3 into caspase-3 and also induced the cleavage of poly-(ADP-ribose) polymerase and lamin B, two hallmarks of apoptosis. All of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone (a general inhibitor of caspase activities), whereas acetyl-Asp-Glu-Val-Asp aldehyde, a specific inhibitor of caspase-3 activity, only induced a partial reversion of the apoptotic effects. Sodium butyrate also decreased the Bcl-2 level, whereas it increased the Bax level and stimulated the release of cytochrome c from the mitochondria, an event that was most likely responsible for the activation of caspase-3. Finally, sodium butyrate activated 26S proteasome, the major extralysosomal degradative machinery, which is responsible for the degradation of short-lived proteins. Consequently, the levels of p53, N-myc, and IkappaBalpha (factors that play regulatory roles in apoptosis) diminished, whereas the nuclear level of nuclear factor kappaB concomitantly increased. Treatment of Y79 cells with MG132 induced apoptosis with more rapid kinetics than with sodium butyrate. The effects appeared after 8 h of incubation, reaching a maximum at 24 h, and they were accompanied by increased levels of N-myc, p53, and IkappaBalpha. MG132 also favored the release of cytochrome c from the mitochondria and increased the activity of caspase-3. When Y79 cells were exposed to combinations of sodium butyrate and MG132, the latter compound suppressed the decreasing effect induced by sodium butyrate on the levels of p53, N-myc, and IkappaBalpha and the increasing effect on the nuclear level of nuclear factor kappaB. Moreover, an increase in the level of Bax and an enhancement in the release of cytochrome c from the mitochondria were observed. Clear synergistic effects concerning the activation of both caspase-3 and apoptosis were induced by a combination of suboptimal doses of sodium butyrate and MG132. The results support the conclusion that MG132 potentiates the apoptotic effect of sodium butyrate by suppressing its stimulatory effect on 26S proteasome activity. Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in particular, the treatment of retinoblastoma.
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PMID:The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells. 1055 39

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.
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PMID:p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release. 1070 5

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.
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PMID:A new approach to gene mutation analysis using "GFP-Display". 1073 55

Ligation of CD95 on T lymphocytes resulted in the up-regulation of a cell cycle control protein, p21cip-1/WAF-1, an inhibitor of cyclin-dependent kinases. This up-regulation was completely blocked by the cysteine protease inhibitor Z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), whereas DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde), a caspase 3 inhibitor, had no effect. In Faslpr-cg mice, a point mutation in the death domain of CD95 results in failure to recruit FADD (Fas-associated death domain), and in the present study this mutation prevented both CD95-mediated apoptosis and p21cip-1/WAF-1 induction. During apoptotic cell death due to irradiation, p21cip-1/WAF-1 is up-regulated by a p53-dependent pathway that responds to DNA damage. However, CD95-induced up-regulation of p21cip-1/WAF-1 in T cells was p53-independent. T cells deficient in p21cip-1/WAF-1 were less susceptible to CD95-induced apoptosis. We conclude that in T cells, ligation of CD95 and activation of caspases cause the induction of p21cip-1/WAF-1, which acts to promote cell death.
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PMID:CD95/Fas signaling in T lymphocytes induces the cell cycle control protein p21cip-1/WAF-1, which promotes apoptosis. 1075 95

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.
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PMID:Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis. 1076 89

The dietary isothiocyanates and cancer chemopreventive agents phenethyl isothiocyanate and allyl isothiocyanate and their cysteine conjugates inhibited the growth and induced apoptosis of human leukaemia HL60 (p53-) and human myeloblastic leukaemia-1 cells (p53+) in vitro. The median growth inhibitory concentration (GC(50)) values were in the range 1.49-3.22 microM in cultures with 10% serum. Isothiocyanates and cysteine conjugates had increased potency against HL60 cells in serum-free medium, with GC(50) values of 0.8-0. 9 microM. The potency of the compounds decreased with increased serum content of the medium, but that of the cysteine conjugates decreased more markedly. Growth inhibition and toxicity was characterised by either a rapid interaction of the isothiocyanate with the cells in the first hour of culture or exposure to isothiocyanate liberated from the cysteine conjugate in the initial 3 hr of culture, inhibition of macromolecule synthesis, and a commitment to apoptosis which developed in the initial 24 hr. Activities of caspase-3 and caspase-8 were increased during isothiocyanate-induced apoptosis, but caspase-1 activity was not. The general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and the specific caspase-8 inhibitor N-benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone inhibited apoptosis, but specific caspase-1 and caspase-3 inhibitors did not. The antiproliferative activities were limited by hydrolysis of the isothiocyanate. This suggests that caspase-8 has a critical role, and caspase-3 a supporting role, in isothiocyanate-induced apoptosis in which p53 is not an obligatory participant. Isothiocyanate-induced apoptosis may suppress the growth of preclinical tumours and contribute to the well-established decreased cancer incidence associated with a vegetable-rich diet.
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PMID:Studies on the mechanism of the inhibition of human leukaemia cell growth by dietary isothiocyanates and their cysteine adducts in vitro. 1082 67

DNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound full-length p53 transactivation domain, although similar to the recently discovered group of loosely folded proteins in that it does not have tertiary structure, is nevertheless populated by an amphipathic helix and two nascent turns. The helix is formed by residues Thr(18)-Leu(26) (Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu), whereas the two turns are formed by residues Met(40)-Met(44) and Asp(48)-Trp(53), respectively. It is remarkable that these local secondary structures are selectively formed by functionally critical and positionally conserved hydrophobic residues present in several acidic transactivation domains. This observation suggests that such local structures are general features of acidic transactivation domains and may represent "specificity determinants" (Ptashne, M., and Gann, A. A. F. (1997), Nature 386, 569-577) that are important for transcriptional activity.
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PMID:Local structural elements in the mostly unstructured transcriptional activation domain of human p53. 1088 88


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