Low-dose radiation hypersensitivity is associated with p53-dependent apoptosis

Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses <50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times postirradiation, we examined the response of human A549 lung carcinoma, T98G glioma, and MCF7 breast carcinoma cell lines exposed to gamma radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (Annexin V binding). We observed that caspase-3 activation and Annexin V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and Annexin V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no hyperradiosensitivity and apoptosis was not detectable by Annexin V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.     

Curcumin induced apoptosis is mediated through oxidative stress in mutated p53 and wild type p53 colon adenocarcinoma cell lines

Curcumin has anti‐oxidant, anti‐cancer and anti‐carcinogen property. Our laboratory had previously reported that, curcumin treatment induces reactive oxygen species (ROS) generation in HT‐29 cell line, an effect contradictory to its anti‐oxidant property. This study evaluates the role of p53 in curcumin mediated ROS generation and cell death. Curcumin induced ROS was determined by 2’,7’‐dichlorofluorescein and apoptosis by Hoechst33342/PI staining in HT‐29 and HCT‐116 cell lines. ROS generation occurs within 1 hour of 40 µM curcumin treatment and a reduction was observed by third hour in HCT‐116 insinuating p53 involvement. N‐acetyl cysteine (NAC) pre‐treatment effectively quenched ROS and inhibited membrane potential loss in HT‐29, but less effective in HCT‐116. Mitochondrial membrane potential loss is evident with 10 and 40 µM curcumin in HCT‐116 and at 40 µM curcumin in HT‐29. Total p53 protein level increase was observed by 24 hours in HCT‐116 upon NAC pre‐treatment. Our results indicate that curcumin induces ROS mediated cell death in colon adenocarcinoma cell lines and may be mediated via p53.     

Inactivation of Myc-induced p53-dependent apoptosis in human tumors

The Myc family of oncoproteins promote cell growth and are frequently overexpressed in human tumors. However, Myc can also trigger cell death by apoptosis. This is at least in part mediated via the ARF-p53 pathway. Myc activation leads to a selection for inactivation of ARF or p53, allowing cell survival and tumor progression. Restoration of p53-dependent apoptosis by various means is an attractive approach for new cancer therapy.     

p53 is frequently mutated in Burkitt''s lymphoma cell lines.

A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53.     

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

Calpain inhibitor 1 activates p53-dependent apoptosis in tumor cell lines.

Reports suggest a role of calpains in degradation of wild-type p53, which may regulate p53 induction of apoptosis. A calpain inhibitor, n-acetyl-leu-leu-norleucinal (calpain inhibitor 1), was assessed for ability to enhance p53-dependent apoptosis in human tumor cell lines with endogenous wild-type p53 and in altered p53 cell lines with the replacement of wild-type p53 by a recombinant adenovirus (rAd-p53). Calpain inhibitor 1 treatment resulted in increased levels of activated p53, increased p21 protein, and activation of caspases. Cell lines with wild-type, but not mutated or null, p53 status arrested in G0/G1 and were sensitive to calpain inhibitor-induced apoptosis. Regardless of endogenous p53 status, calpain inhibitor treatment combined with rAd-p53, but not empty vector virus, enhanced apoptosis in tumor cell lines. These results demonstrate p53-dependent apoptosis induced by a calpain inhibitor and further suggest a role for calpains in the regulation of p53 activity and induction of apoptotic pathways.     

Variation in apoptosis profiles in radiation-induced genomically unstable cell lines

Delayed reproductive cell death or lethal mutations in the survivors of irradiated cells is a well-characterized end point associated with radiation-induced genomic instability. Although the mechanism for this delayed lethality has not been identified, it is thought to be a means of eliminating cells that have sustained extensive damage, thus preventing tissue disruption after radiation exposure. In this study we have tested the hypothesis that delayed reproductive cell death in chromosomally unstable GM10115 clones is due to persistently increased levels of apoptosis. Evidence for differences in apoptosis in two representative genomically unstable clones after irradiation is presented. In addition, one of the unstable clones was found to have abnormal levels of apoptosis after radiation exposure. An understanding of apoptosis in genomically unstable clones may provide insight into the maintenance of genomic instability and the mechanism by which genomically unstable cells evade cell death, potentially contributing to carcinogenesis.     

Induction of apoptosis by transiently transfected metabolically stable wt p53 in transformed cell lines

Biochemical and functional properties of wild-type (wt) and mutant p53 were studied under the same cellular environment by transient transfection. Exogenous wt p53 expressed in transformed cell lines was found to be as metabolically stable as mutant p53. Yet only mutant p53 bound to hsp70 whereas wt p53 did not, suggesting that the metabolic stability of p53 does not depend on its ability to form complexes with hsp70. The wt protein was expressed essentially in the nucleus, while mutant p53 showed both nuclear and cytoplasmic expression, as determined by immunofluorescence staining with PAb122. In addition, staining with PAb1801 revealed a number of strongly fluorescent cell fragments in cultures transfected by wt p53. Morphological features of apoptosis were observed in these cultures. Quantitative analysis by flow cytometry confirmed that only the cell population expressing wt p53 had a significant amount of cell debris. Thus, transient expression of a metabolically stable wt, but not mutant, p53 induces cell death by apoptosis. The present study demonstrates a model system to investigate the functional domains of p53 in the induction of apoptosis.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Cytoplasmic p53 polypeptide is associated with ribosomes.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

p53 is associated with p34cdc2 in transformed cells.

The normal functioning of p53 is thought to involve p53 target proteins. We have previously identified a cellular 35 kd protein associated with p53 and now report evidence identifying this 35 kd protein as p34cdc2, product of the cell cycle control cdc2 gene. The association between p53 and p34cdc2 was detected in SV3T3 and T3T3 cell lines, both expressing the wild-type p53 phenotype, and in 3T3tx cells, expressing 'mutant' p53 phenotype. Binding of the mutant p53 phenotype with p34cdc2 was greatly reduced relative to wild-type. Complexes of p53-p34cdc2 may represent inactivation or activation of either component. The p34cdc2 kinase functions at cell cycle control points and is necessary for entry and passage through mitosis. It also operates in G1 and is involved in the commitment of cells into the proliferative cycle. Since we were unable to detect p53-p34cdc2 complexes in mitotic cells we propose that the interaction between p53 and p34cdc2 may be functional in cell growth control, possibly to promote or to suppress cell proliferation.