Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents.

Cancer therapy drugs, such as diamminedichloroplatinum (cisplatin), mitomycin C, etoposide and a number of other compounds, as well as energy-rich radiation, are known to act on cellular DNA. These agents are shown to induce nuclear accumulation of the so-called tumor-suppressor protein p53 in fibroblastoid cells, as well as in epithelioid normal and immortalized cells of murine, simian, and human origin. p53 accumulation starts a few hours after treatment and can remain detectable in surviving cells for at least 20 days. Accumulation occurs because of increased p53 protein stability and depends on ongoing translation. It is not the result of enhanced gene expression. A number of cell cycle inhibitors do not affect p53 protein accumulation, suggesting that the process may start from several points in the cell cycle. Since the increase in the nuclear p53 protein levels occurs within a few hours in most of the treated normal diploid cells, it is unlikely that the accumulated p53 protein is derived from a mutated p53 gene. The results obtained are in accordance with the view that the DNA damage-induced p53 accumulation may either inhibit cell growth, allowing DNA repair processes, or, in the case of severe damage, initiate apoptosis.

Butyrate modulates DNA-damage-induced p53 response by induction of p53-independent differentiation and apoptosis.

Butyrate, a physiologically occurring agent, has been reported to decrease constitutively high expressed p53 levels in transformed cells. To elucidate whether butyrate also inhibits DNA-damage-induced p53 response we investigated the effects of butyrate and the anticancer drug mitomycin C in normal C3H10T1/2 cells harbouring wild-type p53. In comparison with p53-deficient fibroblasts we examined p53 protein level, cell cycle arrest, differentiation, and apoptosis. Butyrate induced G1 phase arrest, differentiation, and p53-independent increase in p21(waf1/cip1) protein. Moreover, butyrate induced p53-independent apoptosis, which was, as well as p53-mediated apoptosis, associated with a dose-dependent increase in Bax and c-Myc protein. Pretreatment with butyrate repressed dose-dependently mitomycin-C-induced p53 accumulation and interfered with p53-dependent cell cycle arrest. Butyrate further partially inhibited p53-mediated apoptosis, but low doses of butyrate were more effective than higher concentrations. This was reflected in an enhanced decrease in c-Myc and Bax protein in response to mitomycin C with low concentrations of butyrate. Our data indicate that the differentiation stimulus of butyrate, in association with p21(waf1/cip1) induction, and apoptosis, may explain antineoplastic effects of butyrate. Co-carcinogenic features of butyrate may result from inhibition of p53-mediated DNA damage response.

DNA-damage-inducible p53 activity in SV40-transformed cells.

The biological state of the tumour suppressor proteins Rb and p53 is altered in papillomavirus- and SV40-transformed cells, due to interaction with the DNA tumour virus oncogene proteins E6/E7 and the tumour (T) antigen. Thus, the DNA damage response function of p53, a crucial feature of the tumour suppressor p53, might be considered as inactive. To investigate this subject, C57SV and VLM, two SV40-transformed murine cell lines enharboring constitutively high nuclear p53 and SV40 large T antigen levels, were treated with mitomycin C. Mitomycin C is known for its activity to elicit DNA damage, followed by nuclear accumulation of biologically active p53. Surprisingly, the nuclear p53 level significantly increased in mitomycin-C-treated C57SV cells and to a lesser degree in VLM cells. In addition, expression of p21WAF1 protein was induced in C57SV and VLM cells. This indicates a possible DNA-damage-elicited p53 activation. Finally, nuclear extracts of mitomycin-C-treated C57SV and VLM cells, but not of untreated cells, exhibited PAb421-enhanced specific DNA-binding activity of p53, as proven by gel shift analysis. Thus, DNA damage induced essential biological functions typical for wild-type p53 in the SV40-transformed cell lines examined so far.

Enhanced p53 activity and accumulation in response to DNA damage upon DNA transfection.

In response to DNA damage the wild-type tumor suppressor protein p53 accumulates in the nucleus of rodent and primate cells. To investigate the minimal requirement for this reaction the cellular DNA was restricted by two alternative ways: (i) by calicheamicin gamma 1, an enediyne, which causes direct, sequence-specific DNA damage, as shown by fluorimetric analysis of DNA unwinding and by poly(ADP-ribose) polymerase activation. The dose-dependent DNA damage correlated with the nuclear p53 accumulation. In addition, restriction was generated (ii) by the intracellular introduction of the restriction enzyme PvuII, which generates blunt-ended DNA breaks, applying a mild hypotonic shock (pellet method). Previous transfection of linear or circular, single- or ds, DNA, followed by mitomycin C-treatment, lead to a dramatic increase in nuclear p53 accumulation and p53 activity according to electrophoretic mobility shift analysis. The nature of transfected DNA was irrelevant for enhanced accumulation. The data suggest, that the cellular p53 response to DNA damage is sensitized by uptake of exogenous DNA.

Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression.

Many tumor cells produce vascular endothelial growth factor (VEGF), which is thought to be a pivotal mediator of tumor neoangiogenesis. Expression of the VEGF gene can be induced by tumor promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate protein kinase C (PKC). Here we show that in transient transfection assays a mutated form of the murine p53 tumor suppressor gene (ala135-->val) induces expression of VEGF mRNA and potentiates TPA stimulated VEGF mRNA expression. In NIH 3T3 cells which stably overexpress the temperature sensitive p53 (ala135-->val), displaying mutant phenotype at 37 degrees C and wildtype phenotype at 32.5 degrees C, induction of VEGF mRNA and protein by activated PKC is strongly synergistic with mutant, but not wildtype p53. Mutant p53 specifically increases TPA induction of VEGF without affecting the expression of other TPA inducible genes. TPA dependent VEGF expression is also enhanced by human p53 mutated at amino acid 175. Thus, our data link PKC and p53, the gene most frequently altered in human tumors, with the regulation of tumor angiogenesis.

Flavonoids activate wild-type p53.

Flavonoids are diphenyl propanoids widely distributed in edible plants. They play a dual role in mutagenesis and carcinogenesis. Some of them act as anticarcinogens or inhibit the growth of tumour cells, whereas others act as cocarcinogens, are mutagenic or able to induce DNA damage. To further elucidate this dual role, we investigated the influence of apigenin, luteolin and quercetin on the tumour suppressor protein p53, regarding p53 accumulation, cell cycle arrest, apoptosis, and biological activity. We found that incubation of the non-tumour cell line C3H10T1/2CL8 with these flavonoids resulted in induction of p53 accumulation and apoptosis. Apoptosis occurred out of the G2/M phase of the cell cycle. The G2/M arrest seems to be p53-dependent as it did not occur in p53 knockout fibroblasts which further supports the recent finding that p53 is involved in the G2/M checkpoint control. Differences between the flavonoids tested concerned p53 accumulation kinetics as well as the biological activity of accumulated p53 and might be due to different modes of flavonoid action. These data suggest that both aspects of flavonoid effects, i.e. inhibition of tumour growth through cell cycle arrest and induction of apoptosis, are functionally related to p53.

Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex.

Neuroblastoma (NB) cells reportedly accumulate wild-type p53 exclusively in the cytoplasm. However, immunofluorescence assays with five different antibodies showed that p53 accumulates in the nucleus of up to 10% of NB cells. PAb1801 detected cytoplasmic 'punctate structures' which were also found in p53-null cells, rendering this antibody unsuitable for p53 detection. A comparison of DO-1 and PAb1801 staining in NB tissue sections confirmed the results obtained with NB cells. Nuclear accumulation of p53 was induced in NB cells using substances which disturb p53's tertiary structure at its zinc finger motif, or by treatment with mitomycin C. Constitutive nuclear accumulation was observed in an SK-N-SH variant, AW-1, which has a point mutation in p53 at Cys176>Ser, disturbing the same motif. Even though p53 showed DNA-binding capability after mitomycin C treatment of NB cells, the target gene products MDM2 and p21(WAF1,CIP1,SDI1) were not synthesized and no p53 transactivating activity measured in a reporter gene assay. Therefore we suggest that p53 in NB cells might be predominantly in a conformation refractory to integration into the transcriptional complex, resulting in at least partial transcriptional inactivity, hyperactive nuclear export and resistance to degradation by exogenously expressed MDM2.

Incidence of radiation lymphomas in C57BL/6 mice is not promoted after intraperitoneal treatment with the phorbol ester tetradecanoylphorbol acetate.

Female C57BL/6 mice, given 4 X-ray irradiations each with 1.7 Gy, according to H.S. Kaplan and M.S. Brown (J. Natl. Cancer Inst., 13 (1952) 185-192) developed lethal lymphomas in more than 90%, 270 days after irradiation. Intraperitoneal application of tetradecanoylphorbol acetate (TPA), 30 ng/g, twice weekly for 240 days had no influence on survival of the animals and incidence of the malignant lymphomas.

Epothilone A induces apoptosis in neuroblastoma cells with multiple mechanisms of drug resistance.

Epothilone A, a novel macrolide antibiotic, is produced by the myxobacterium Sorangium cellulosum. Similarly to paclitaxel (Taxol), epothilone A inhibits cell proliferation and induces apoptosis by binding to tubulin and stabilizing of microtubuli. Like paclitaxel, epothilone A induced apoptosis in neuroblastoma cells which exhibit constitutive cytoplasmic sequestration of p53 and, hence, an impaired DNA-damage-dependent apoptosis. However, in contrast to paclitaxel, epothilone A was also effective against a constitutively Pgp-expressing, multidrug resistant neuroblastoma cell line (SK-N-SH). Moreover, the efficacy of epothilone A was not impaired even though the Pgp level was further increased during treatment with the drug.

Cross-reactivity of the monoclonal antibody 9E10 with murine c-MYC.

The C-terminal regions of the human and the murine c-MYC consist of a common conserved sequence, the amino acids (a.a.) 418-439 with one terminal exchange (C438G). The pre-C-terminal region of both proteins, a.a. 408-417, exhibit four exchanges. A commercially available monoclonal antibody, 9E10, raised against the C-terminal a. a. 408-439 of human c-MYC, is declared to recognize specifically and exclusively human c-MYC. However, in an immunofluorescence assay we observed, in addition to the reaction with a human cell line (SV80), reactivity with the murine cell line L929. In analogy, a rabbit polyclonal antiserum raised against a peptide which corresponds to the murine pre-C-terminus of c-MYC, a.a. 408-417, showed also cross-reactivity in immunofluorescence. The immunostaining with both anti-bodies in the human and the murine cell line was competed by the peptide, corresponding to the murine pre-C-terminal a.a. 408-417, whereas the staining of both cell lines with an antiserum raised against the conserved N-terminal region of c-MYC was not competed by this peptide. The cross-reactivity of 9E10 with murine c-MYC was confirmed by Western blot using two additional cell lines. In conclusion, our findings indicate that 9E10 which is generally regarded as specific for human c-MYC cross-reacts with denaturated murine c-MYC.

Cell cycle arrest in ovarian cancer cell lines does not depend on p53 status upon treatment with cytostatic drugs.

Human ovarian cancer cell lines with different p53 status were investigated for p53-dependency of cell cycle arrest upon treatment with cytostatic drugs. For this purpose commonly used anti-cancer drugs and a novel anti-cancer drug, gemcitabine, were applied. Cell cycle arrest was dependent on the drug dose used, as observed for all anti-cancer drugs applied, but not related to functional p53. With the exception of the etoposide-effected G2/M arrest at high concentrations, which seems to depend on functional p53, since it did not occur in cells with inactive p53. Only in cells with wt p53 and quasi-wild-type, p53 accumulated in the nucleus upon drug treatment with all anti-cancer agents applied. The level of accumulation was drug dose-dependent for each drug tested. The accumulated p53 was biochemically active, as measured in a transient transfection assay upon treatment with gemcitabine, cisplatin, etoposide, and Taxol. Activity was dependent on the drug dose applied and proportional to the level of accumulated p53, except for Taxol-induced p53 accumulation which correlated inversely with p53 biochemical activity. Apoptosis was estimated by in situ end labeling by biotinylated dUTP with the terminal deoxyribonucleotidyl transferase assay. Apoptosis occured after arrest at the various phases of the cell cycle in all cell lines tested, depending on the drug and the drug dose used. Nevertheless, cells with wt p53 exhibited the highest fraction of apoptotic cells.

p53 in SV40-transformed DNA-damaged human cells binds to its cognate sequence but fails to transactivate target genes.

Human SV40-transformed cells contain high levels of stabilized p53 of which only a fraction is complexed with the SV40 large tumor antigen (T-antigen). This raises the question whether the p53 which is not complexed with T-antigen retains some biological activity. Two human SV40-transformed cell lines, BEAS and SV80, were investigated. A significant level of constitutive cognate-sequence-specific DNA-binding of p53 was detected by electrophoretic mobility shift assay (EMSA) of cell extracts. Upon DNA damage by treatment with mitomycin C the DNA-binding activity was increased, as known for cells with wild-type p53. However, in both cell lines, before and after DNA damage, p53 was not able to transactivate a target gene as shown by reporter gene assay. Hence, the capability of p53 to bind its cognate sequence is a prerequisite but no proof of p53 transactivating activity. Nuclear p53 levels were not further increased after mitomycin C treatment, occasionally rather slightly decreased, often accompanied by an even larger decrease in amount of T-antigen. In conclusion, SV40-transformation of human cells has caused a loss of essential features of wild-type p53 activity, even in that fraction of p53 not in physical complex with SV40 T-antigen.

Filtered, tar-free and aerosol-free cigarette-smoke causes accumulation of the tumor-suppressor protein p53 in rodent cells.

Cigarette smoke was filtered with a Cambridge glass fiber filter retaining 99.9% of the tar and aerosol fraction and diluted 1:5 with air. The murine cell line L929 was exposed to this smoke preparation for periods of up to 10 min. Thereafter the following parameters were determined at different times: Nuclear accumulation of the tumor suppressor protein p53 indicating chromatin injury (by immunostaining); apoptotic DNA fragmentation (by DNA end labelling with biotin-16-dUTP in the presence of terminal deoxyribonucleotidyl transferase); the intracellular level of reactive oxygen intermediates (ROI) (by cytofluorimetry with the fluorigenic stain 2',7'-dichlorofluorescin diacetate). After 1 min exposure to 1:5 air-diluted filtered cigarette smoke maximal p53 accumulation occured about 20 h later, whereas maximal DNA fragmentation and apoptosis and maximal ROI levels were found after 10 min of exposure. Obviously, even the diluted, tar- and aerosol-free fraction of cigarette smoke has the potency, after 1 min of exposure only, to exert severe DNA damage, a potential transformation risk for the surviving cell fraction, in murine cell cultures as indicated by stabilization and accumulation of the tumor suppressor protein p53.

DNA damage by filtered, tar- and aerosol-free cigarette smoke in rodent cells: a novel evaluation.

Nuclear accumulation of the tumorsuppressor protein p53 indicates the occurrence of chromatin injury (J. Cancer Res. Clin. Oncol. 1991, 117, 30; Oncogene 1993, 8, 307) and may be used as an analytical tool to detect genotoxic agents. This mechanism was used to evaluate the DNA-damaging potency (clastogenicity) of the tar- and aerosol-free, gaseous phase of cigarette smoke which is obtained by filtration through Cambridge glass fiber filters. This condensate-free gas phase was absorbed by phosphate-buffered saline and immediately thereafter poured onto monolayers of the murine cell line L929 for 10 min. Eighteen hours later the nuclear accumulation of p53, an indicator for DNA damage, was determined. The elicited level of p53 was similar to that obtained by direct incubation with the gas phase of filtered cigarette smoke for 2 min or with several micrograms of mitomycin C per ml. Previous exhaustive filtration obviously does not inhibit the clastogenic property of tobacco smoke to exert severe DNA damage.

Nuclear accumulation of p53 in response to treatment with DNA-damaging agents.

A number of agents which damage DNA also trigger the nuclear accumulation of the tumor suppressor protein p53. Here we show the correlation with different p53 detection methods. As an example we investigated the effects of the cancer therapy drug mitomycin C on different mammalian cell lines. Our findings demonstrate that either the immunofluorescence techniques (indirect immunofluorescence staining or flow cytometric analysis) or ELISA or immunoblot assays are useful methods in detecting p53 accumulation. Simultaneously we measured DNA damage with the terminal deoxynucleotidyl transferase assay. Compatible data were obtained. Thus p53 accumulation may be used as indicator of DNA injury.

Transformation-related cellular protein p53: increased level in untransformed rat cells following treatment with the tumorpromoter, tetradecanoylphorbol-acetate.

12-O-Tetradecanoylphorbol-13-acetate (TPA, 100 ng ml-1), a tumor promoting phorbol ester, is able to induce enhanced levels of the transformation-associated cellular antigen p53 in normal rat 2 cells which had not been previously initiated by a carcinogen. p53 was estimated in ethanol-fixed treated cells on microtiter plates with ELISA using the monoclonal antibody Pab 1620 [EMBO J. 7, 1485, (1984)]. Induction of p53 was confirmed by immunoblotting. This effect of TPA is an additional phenotypic characteristic of tumor cells which can be induced by TPA in untransformed rodent cells.