Activation of CPP32 and Mch3 alpha in wild-type p53-induced apoptosis.
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ABSTRACT:
SUBMITTER: Chandler JM
PROVIDER: S-EPMC1218152 | biostudies-other | 1997 Feb
REPOSITORIES: biostudies-other
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Project description:Resveratrol is a promising chemopreventive agent that mediates many cellular targets involved in cancer signaling pathways. p53 has been suggested to play a role in the anticancer properties of resveratrol. We investigated resveratrol-induced cytotoxicity in H1299 cells, which are non-small lung cancer cells that have a partial deletion of the gene that encodes the p53 protein. The results for H1299 cells were compared with those for three cell lines that constitutively express wild-type p53: breast cancer MCF-7, adenocarcinomic alveolar basal epithelia A549 and non-small lung cancer H460. Cell viability assays revealed that resveratrol reduced the viability of all four of these cell lines in a dose- and time-dependent manner. MCF-7, A549 and H460 cells were more sensitive to resveratrol than were H1299 cells when exposed to the drug for 24 h at concentrations above 100 µM. Resveratrol also increased the p53 protein levels in MCF-7 cells without altering the p53 mRNA levels, suggesting a post-translational modulation of the protein. The resveratrol-induced cytotoxicity in these cells was partially mediated by p53 and involved the activation of caspases 9 and 7 and the cleavage of PARP. In H1299 cells, resveratrol-induced cytotoxicity was less pronounced and (in contrast to MCF-7 cells) cell death was not accompanied by caspase activation. These findings are consistent with the observation that MCF-7 cells were positively labeled by TUNEL following exposure to 100 µM resveratrol whereas H1299 cells under similar conditions were not labeled by TUNEL. The transient transfection of a wild-type p53-GFP gene caused H1299 cells to become more responsive to the pro-apoptotic properties of resveratrol, similarly to findings in the p53-positive MCF-7 cells. Our results suggest a possible therapeutic strategy based on the use of resveratrol for the treatment of tumors that are typically unresponsive to conventional therapies because of the loss of normal p53 function.
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Project description:We examined the temporal profile of apoptosis after fluid percussion-induced traumatic brain injury (TBI) in rats and investigated the potential pathophysiological role of caspase-3-like proteases in this process. DNA fragmentation was observed in samples from injured cortex and hippocampus, but not from contralateral tissue, beginning 4 hr after TBI and continuing for at least 3 d. Double labeling of brain with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and an antibody directed to neuronal nuclear protein identified apoptotic neurons with high frequency in both traumatized rat cortex and hippocampus. Cytosolic extracts from injured cortex and hippocampus, but not from contralateral or control tissue, induced internucleosomal DNA fragmentation in isolated nuclei with temporal profiles consistent with those of DNA fragmentation observed in vivo. Caspase-3 mRNA levels, estimated by semiquantitative RT-PCR, were elevated fivefold in ipsilateral cortex and twofold in hippocampus by 24 hr after TBI. Caspase-1 mRNA content also was increased after trauma, but to a lesser extent in cortex. Increased caspase-3-like, but not caspase-1-like, enzymatic activity was found in cytosolic extracts from injured cortex. Intracerebroventricular administration of z-DEVD-fmk-a specific tetrapeptide inhibitor of caspase-3-before and after injury markedly reduced post-traumatic apoptosis, as demonstrated by DNA electrophoresis and TUNEL staining, and significantly improved neurological recovery. Together, these results implicate caspase-3-like proteases in neuronal apoptosis induced by TBI and suggest that the blockade of such caspases can reduce post-traumatic apoptosis and associated neurological dysfunction.
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Project description:The tumor suppressor p53 is inactivated by mutations in the majority of human solid tumors. Conversely, p53 mutations are rare in leukemias and are only observed in a small fraction of the patient population, predominately in patients with complex karyotype acute myeloid leukemia or hypodiploid acute lymphoblastic leukemia. However, the loss of p53 function in leukemic cells is often caused by abnormalities in p53-regulatory proteins, including overexpression of MDM2/MDMX, deletion of CDKN2A/ARF, and alterations in ATM. For example, MDM2 inhibits p53-mediated transcription, promotes its nuclear export, and induces proteasome-dependent degradation. The MDM2 homolog MDMX is another direct regulator of p53 that inhibits p53-mediated transcription. Several small-molecule inhibitors and stapled peptides targeting MDM2 and MDMX have been developed and have recently entered clinical trials. The clinical trial results of the first clinically used MDM2 inhibitor, RG7112, illustrated promising p53 activation and apoptosis induction in leukemia cells as proof of concept. Side effects of RG7112 were most prominent in suppression of thrombopoiesis and gastrointestinal symptoms in leukemia patients. Predictive biomarkers for response to MDM2 inhibitors have been proposed, but they require further validation both in vitro and in vivo so that the accumulated knowledge concerning pathological p53 dysregulation in leukemia and novel molecular-targeted strategies to overcome this dysregulation can be translated safely and efficiently into novel clinical therapeutics.
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Project description:Classic but also novel roles of p53 are becoming increasingly well characterized. We previously showed that ex vivo retroviral transfer of mitochondrially targeted wild type p53 (mitop53) in the Emu-myc mouse lymphoma model efficiently induces tumor cell killing in vivo. In an effort to further explore the therapeutic potential of mitop53 for its pro-apoptotic effect in solid tumors, we generated replication-deficient recombinant human Adenovirus type 5 vectors. We show here that adenoviral delivery of mitop53 by intratumoral injection into HCT116 human colon carcinoma xenograft tumors in nude mice is surprisingly effective, resulting in tumor cell death of comparable potency to conventional p53. These apoptotic effects in vivo were confirmed by Ad5-mitop53 mediated cell death of HCT116 cells in culture. Together, these data provide encouragement to further explore the potential for novel mitop53 proteins in cancer therapy to execute the shortest known circuitry of p53 death signaling.
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Project description:The estrogen receptor alpha (ERα) plays a central role in the etiology, progression, and treatment of breast cancers. Constitutively activating somatic mutations Y537S and D538G, in the ligand binding domain (LBD) of ESR1, are associated with acquired resistance to endocrine therapies. We have previously shown that the metalloestrogen calcium activates ERα through an interaction with the LBD of the receptor. This study shows that cadmium activates ERα through a mechanism similar to calcium and contributes to, and further increases, the constitutive activity of the ERα mutants Y537S and D538G. Mutational analysis identified C381, N532A, H516A/N519A/E523A, and E542/D545A on the solvent accessible surface of the LBD as possible calcium/metal interaction sites. In contrast to estradiol, which did not increase the activity of the Y537S and D538G mutants, cadmium increased the activity of the constitutive mutants. Mutation of the calcium/metal interaction sites in Y537S and D538G mutants resulted in a significant decrease in constitutive activity and cadmium induced activity. Mutation of calcium/metal interaction sites in wtERα diminished binding of the receptor to the enhancer of estrogen responsive genes and the binding of nuclear receptor coactivator 1 and RNA polymerase II. In contrast to wtERα, mutation of the calcium/metal interaction sites in the Y537S and D538G mutants did not diminish binding to DNA but prevented a stable interaction with the coactivator and polymerase. Growth assays further revealed that calcium channel blockers and chelators significantly decreased the growth of MCF7 cells expressing these constitutively active mutants. Taken together, the results suggest that exposure to cadmium plays a role in the etiology, progression, and response to treatment of breast cancer due, in part, to its ability to activate ERα.
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Project description:BackgroundNaturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored.MethodsHuman cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit.ResultsWe show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors.ConclusionNutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.
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Project description:Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.
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Project description:Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.
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