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.

Silencing of retroviral vector transduced LacZ reporter gene by frameshift mutation.

Moloney murine leukemia virus-based vector expressing Escherichia coli beta-galactosidase (lacZ) as reporter gene and the transposon Tn5 neomycin resistance (neo) gene was transduced at low-multiplicity of infections into NIH 3T3 cells. Geneticin (G418)-resistant cells were recloned and cell lines containing beta-galactosidase positive or beta-galactosidase negative cells were obtained. Both positive and negative cell lines contained a single proviral copy at distinct integration sites. RNA complementary to lacZ was detected in beta-galactosidase positive as well as in one of three investigated beta-galactosidase negative cell lines. DNA sequence analysis of proviral LacZ gene in beta-galactosidase negative cell line C6 showed a single nucleotide insertion at position 1567 resulting in reading frame shift and translational stop codon at position 1629. This mutation explains the enzyme inactivation. The absence of beta-galactosidase after retroviral transduction of LacZ reproter gene may be a consequence of definite mutation but not a consequence of ineffective transduction or transcriptional inactivation of transgene.