Photobiological studies of human cells in culture

Abstract

This thesis describes a photobiological study of human cells in culture. Experiments were conducted upon cells of two established tumour cell lines: HeLa, of normal uv sensitivity and MM96, a. melanoma line of high uv resistance. Further investigations made use of the technique of vacuum blistering to obtain fragments of viable human epidermis for experimentation.A direct comparison was made, between the two tumour cell lines, of three ultraviolet induced phenomena (1) Inhibition of DNA synthesis by uv;(2) The production and repair of protein-DNA crosslinks;(3) Inhibition of'the predominant DNA polymerase activities by uv lesions in the template molecule.In addition, DNA replication after uv irradiation was assessed autoradiographically in human epidermis and compared with equivalent measurements upon MM96 and HeLa cells. The purpose of this comparison was to determine whether the uv resistant properties of MM96, an epidermally derived tumour, were peculiar to that cell line or represented the continued expression of features of the untransformed melanocyte. Comparisons were also made of epidermal structure and replication frequency of basal cells, between donor groups of high or low susceptibility to sunlight induced cancer.Previous work had indicated that MM96 performed significantly higher levels of DNA synthesis than HeLa cells, after uv irradiation. More detailed examination revealed that, above a dose of 5 J/m2 the dose-dependent decrease in DNA synthesis was similar for both cell lines (=3% decrease/J/m2). However, an additional inhibitory factor was observed in HeLa cells. This second factor was saturated at a dose below 5 J/m2 and at such doses accounted for most of the inhibition. MM96 did not show this additional factor which was thought to be caused by the prevention of initiation of new replicons. In HeLa cells this phenomenon appeared to turn off DMA synthesis in approximately 50% of the genome. Irradiation of cells with a UVB source (290-320 nm) showed that differences in sensitivity between HeLa and MM96 were also evident with this form of uv. However, the inhibition of DNA synthesis by UVB differed in some respects from that of UVC. The major difference was that the phenomenon thought to be inhibition of replicon initiation was less severe in UVB irradiated HeLa cells, corresponding to the inactivation of approximately 20% of the genome. Measurement of protein-DNA crosslinking indicated that similar levels were sustained by both cell types i.e. 60 + 25 crosslinks/cell/J/m2. Repair of crosslinks could not be detected in either case. Two distinct methods were used for these measurements and the results suggested that previous values obtained for crosslinking in human cells (as % of DNA bound to protein) did not reflect true crosslinking levels but included apparently loose, non-covalent binding.Examination of DNA polymerizing enzymes showed the presence of a terminal transferase-like activity in one subline of MM96. However, this enzyme was absent from other sublines and could not be induced in them by uv. Since all sublines shared the uv resistant characteristics it was concluded that the terminal transferase-like activity was not a significant factor in uv resistance. ........