Effects of trypsin on cellular, chromosomal and DNA damage induced by X-rays
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When cells are trypsinized before irradiation, potentiation of cell killing is seen; this is known as the 'trypsin effect'. The trypsin effect is re-examined here in the light of experiments in which enzymatic modifications of DNA in permeabilized cells has become a powerful experimental tool (Bryant et al, 1978, Ahnstrom and Bryant,1982; Natarajan et al, 1980; Bryant, 1984, 1985; Natarajan and Obe, 1984) and where in some cases it is suspected that trypsinization as part of the technique could significantly alter cell membrane permeability and chromatin structure (Obe et al, 1985; Obe and Winkel, 1985; Bryant and Christie, 1989). The trypsin effect was investigated at various cellular levels, assaying for cell survival (to verify the potentiation), anaphase chromosomal aberrations, DNA damage and repair and lastly using a nucleoid assay to investigate the effect of trypsin on DNA-nuclear matrix interactions. Each of these are considered in separate chapters as individual studies, then all compared in the final discussion. A small potentiation effect of X-ray damage on cell killing was seen when using Chinese Hamster Ovary (CHO) cells but no potentiating effect was found in the murine Ehrlich ascites tumour (EAT) cell line. Trypsinization was found to increase the number of X-ray induced chromosomal anaphase abnormalities in EAT cells. To investigate the possibility that the basis of the trypsin effect lies in its action at the DNA level, further experiments were performed to monitor DNA damage and repair using the DNA unwinding and neutral elution techniques. No difference was seen in the unwinding kinetics or in the DNA unwinding dose-effect curves for induction of DNA single strand breakage (ssb); when using neutral elution however, treatment of cells with trypsin or buffer alone increased the incidence of X-ray induced double strand breaks (dsb) at higher doses. Trypsinized EAT cells were found to repair ssb after 12 Gy less rapidly than those treated with buffer (EDTA) indicating an inhibitory effect of trypsin on repair. A progressive decrease in repair capacity with increase in time of trypsin treatment was seen. The dsb repair kinetics as measured by the DNA unwinding technique after 50 Gy showed that either trypsin of buffer (EDTA) alone reduced the dsb repair rate, no difference between their repair kinetics being evident (this was also seen with neutral elution repair after 40 Gy). This indicates that the EDTA/buffer solution in which the trypsin is dissolved may also be contributing to the trypsin effect. A new nucleoid assay was developed and used to investigate the effect of prolonged trypsinization and electroporation on nucleoid morphology. The results confirm that routine trypsinization of cells enhances X-ray induced cell killing in some cell lines. It is postulated that this may occur by reducing the repair capacity of the cells rather than by increasing the amount of damage initially caused.
Thesis, PhD Doctor of Philosophy
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