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.

Nitric oxide production by cells isolated from regenerating rat liver.

Nitric oxide (NO) production by cells of the regenerating liver was estimated from the amount of nitrite accumulated during 24 h in the culture media of hepatocytes, Kupffer cells and sinusoidal endothelial cells isolated at different times after partial hepatectomy (PHE). The time course of NO production was compared with the course of the proliferating activity of the same cells. During the time when liver cells pass through their first cell cycles, hepatocytes were the main producers of NO in the liver. The time-dependent changes of their NO production corresponded to those obtained with the whole liver and were inversely correlated with the DNA-synthesizing activity. The NO production by Kupffer and endothelial cells followed that by hepatocytes in this order; the time displacement between them corresponded to the schedule of their proliferating activity. The NO synthesis in non-parenchymal cells fluctuated in a similar way as in parenchymal cells and was minimal when DNA synthesis was manifest.

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.

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.