A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, in spite of several morphological alterations, BrdU labelling was present even in cells displaying early and late apoptotic features.
The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also described both with terminal deoxynucleotidyl transferase and DNA polymerase I, aimed at the study of the distribution of DNA cleavage areas. Flow cytometry is also proposed and different technical approaches, based on different laser utilizations, are discussed. Ultrastructural analysis, allowing the study of apoptotic cell details, is finally considered.
Abstract. A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G 1 /S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.
