Decreased DNA repair capacity of UV-irradiated cells following interferon treatment.

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-alpha, IFN-beta and IFN-gamma on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-alpha had a stronger inhibitory effect than IFN-gamma. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.

Influence of interferons on the repair of UV-damaged DNA.

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-alpha and hrIFN-gamma) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-alpha were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-gamma was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent.

Spontaneous transfection of mammalian cells with plasmid DNA.

A simple method for spontaneous transfection into mammalian cells (both adherent and suspension in culture) with plasmid DNA is described. This method does not require any specific DNA carrier or technical device and can be applied for obtaining both transient and stably transfected cells. The efficiency of spontaneous transfection is slightly lower in comparison with that of the conventional calcium phosphate and lipofectin transfection methods and does not depend on the type of cell culture used.

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells.

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.