Gerhard Brandner

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 p21waf1/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 p21waf1/cip1 induction, and apoptosis, may explain anti-neoplastic effects of butyrate. Co-carcinogenic features of butyrate may result from inhibition of p53-mediated DNA damage response.

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 p53s 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 p21WAF1,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.

Influenza A virus infection of mice induces nuclear accumulation of the tumorsuppressor protein p53 in the lung

Summary. To investigate whether the tumor suppressor p53 protein, an indicator of DNA damage and cell stress, accumulates in the course of influenza-virus-induced murine pneumonia at the site of inflammation, female BALB/c mice were infected each with 5 × 104 infectious units of influenza virus A, strain Puerto Rico (PR) 8, by instillation into the nose and the pharynx. Two days later the mice became sick. Three and 6 days after infection the lungs of sacrificed infected and uninfected mice were examined. We assessed the presence and localisation of inflammation, the expression of influenza viral and p53 protein, as well as of the WAF1/Cip1/SDI gene product p21. Further, the appearance of nitrotyrosine, as an indicator of the formation of peroxynitrite, and eventually of apoptotic cells was examined. No significant nuclear p53 accumulation was found in influenza virus-infected murine cells in vitro. The results show, that in the course of influenza A virus-mediated murine pneumonia inflammatory bystander cells may cause activation of the tumor suppressor protein p53, due to oxidative stress and DNA damage, with ensuing p53-dependent upregulation of p21. Apoptosis is then mainly due to these indirect processes, with possible involvement of p53.

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.

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.

Inhibition of herpes simplex virus type 1 specific translation in cells treated with poly(rI).POLY(RC).

Summary Primary African green monkey kidney cells (GMK) treated with poly(rI).poly(rC) in the presence of DEAE-dextran (‘treated cells’) developed antiviral resistance and concomitantly released interferon into the medium. Treated and untreated cells were infected with herpes simplex virus type 1 (HSV1) in the presence of cytosine arabinoside (araC), and total RNA was isolated and hybridized with purified radio-labelled HSV1 DNA. The intracellular concentration of virus-specific transcripts was not significantly altered in treated cells, but a smaller proportion of the genome of HSV1 hybridized with the extracted RNA. Transcription was similarly restricted when protein synthesis was inhibited by cycloheximide. To analyse virus translation, proteins were radiolabelled between 6 and 10 h after infection and were immunoprecipitated with a pool of human sera and run on SDS-polyacrylamide gels. No virus-specific proteins could be detected in treated cells. In contrast about 25 HSV1-induced proteins were found in infected cells and about 22 proteins in cells infected in the presence of araC. In particular, two virus proteins with apparent mol. wt. of 128000 and 42500 were immunoprecipitated. Since these two were also detected in cells under conditions where elongation of polypeptide chains was non-specifically retarded, it is unlikely that a similar mechanism was responsible for the impaired growth of HSV1 in our treated cells. We conclude that this impairment probably resulted from regulation at the level of virus translation, probably mediated through interferon.

Zur biogenese der isoflavone V. Über die bildung von 4,4′,6′-trihydroxy[14C]chalkon aus [3-14C]zimtsäure in der kichererbse und die umwandlung des 4,4′,6′-trihydroxychalkon-4∼'-[β-14C]glucosid in zellfreien extrakten

Abstract 1. 1. When [3-14C]cinnamic acid was administered to shoots of chana germ (Cicer arietinum L.) radioactive 4,4′,6′-trihydroxychalcone could be isolated by dilution analysis. 2. 2. Using cell-free extracts from chana germ or acetone powder from red clover, the 4,4′,6′-trihydroxychalcone-4′-[β-14C]glucoside is converted to 7-hydroxy-4′-methoxy[2-14C]isoflavone and other not-identified radioactive products. From the results it can be concluded that the 4,4′,6′-trihydroxychalcone or its glucoside is a normal intermediate in the biogenesis of 7-hydroxy-4′-methoxyisoflavone (formononetin).

Protection of mice against SV40 tumours by Pam3Cys, MTP-PE and Pam3Cys conjugated with the SV40 T antigen-derived peptide, K(698)-T(708).

The intraperitoneal injection of Balb/c mice with synthetic analogues of adjuvants S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-cysteine (Pam3Cys) or muramyltripeptide phosphatidylethanolamine (MTP-PE) inhibited the tumourigenic growth of subcutaneously injected VLM cells, a syngeneic simian virus 40 (SV40)-transformed cell line. Furthermore, the Pam3Cys conjugate of K698-T708 (KT), which represents the C-terminal undecapeptide of the SV40 large tumour (T) antigen, was tumour-protective. Also syngeneic spleen cells, preincubated in vitro with this Pam3Cys-KT derivative, which anchores spontaneously at the cell membrane, were, through SV40 tumour mimicry, tumour-protective. The protection was impaired by treatment of the mice with either anti-CD4, anti-CD8 IgG, anti asialo GM1 antiserum or dextrane sulfate, which deplete the CD4+, CD8+ and NK cells or the macrophages, respectively. In summary, SV40 tumour transplantation resistance can be experimentally elicited by a tumour-epitope-specific vaccine. In the absence of an immunogenic epitope protection was obtained by administration of biological response modifiers. Protection is effected by SV40-T-antigen-specific cytotoxic lymphocytes in cooperation with NK cells and macrophages.

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.

Simulation and prevention of retrovirus--specific reactions by mycoplasmas.

From human mycosis fungoides tumor-derived cell lines,Mycoplasma hyorhinis was isolated. This mycoplasma shared the following characteristics with retroviruses: uptake of3H-uridine, but not of3H-thymidine in cell culture; banding at 1.16 g/ml sucrose density and partial shift to retrovirus core density position (≅1.24 g/ml) after detergent treatment; incorporation of3H-TMP into high molecular weight material in standard reverse transcriptase assays with the template-primer poly (A) · (dT)12. On the other hand, the specific reverse transcriptase reaction of retroviruses with poly(A) · (dT)12 and poly(C)·(dG)∼16 was almost completely abolished in the presence of the mycoplasma. Thus,M. hyorhinis may interfere with identification and isolation procedures for retroviruses.