Photosensitivity and heat resistance conferred by brdu incorporation upon a thymidine kinase-deficient mouse cell line with persistent mitochondrial enzyme activity
Authors: Attardi G. , Keeley Benneta , Tu Catherina 
Published: April 2021

The LM(TK−) Cl 1D cell line, a thymidine kinase-deficient derivative of mouse L fibro-blasts with persistent mitochondrial enzyme activity, can incorporate thymidine and 5-bromo-deoxyuridine (BrdU) into mitochondrial DNA (mit-DNA) at a substantial rate, in the absence of any significant incorporation of these precursors into nuclear DNA. Furthermore, these cells can be grown indefinitely in the dark in the presence of 30 μg/ml BrdU. In the present work, advantage was taken of these properties to investigate the possible photosensitizing effect of BrdU which would depend on its incorporation into the organelle DNA, in the absence of nuclear effects.

LM(TK−) Cl 1D cells, which had been grown in medium containing 30 μg/ml BrdU or in BrdU-free medium, were irradiated for different lengths of time with ‘visible’ light, in the presence or absence of the halogenated precursor, respectively. ‘Visible light’ indicates the source of light used, although the components most active photochemically on BrdU-substituted DNA are in the near-visible range (300–340 nm) (Szybalski & Opara-Kubinska, 1965).

A two-to several-fold decrease in growth rate of mass cultures and an exponential decline in cloning forming capacity with a half-life of 2-3 h, after a lag of a few hours, were observed during irradiation both of cells grown in the presence of BrdU and of cells grown in its absence, the effects being somewhat more pronounced in BrdU-maintained cells. In cells maintained in BrdU-free medium, this loss of cell viability could be almost completely reversed if the irradiated cultures were trypsinized and replated either for mass growth or for cloning experiments. On the contrary, the reversal of the irradiation effects by trypsinization and replating was only partial in the case of BrdU-grown cells: these exhibited, in fact, after increasing periods or irradiation, without any significant lag, an exponential loss of cloning efficiency with a half-life of about 8 h down at least to 01 % survival. Control experiments showed that the photosensitivity of Cl 1D cells required their previous exposure to the halogenated precursor, and did not depend on the presence of BrdU in the medium during irradiation.

An estimate of the possible residual extramitochondrial thymidine kinase activity in Cl iD cells, as compared to that of A9 cells, another L cell derivative with normal enzyme activity, was obtained from the relative incorporation of [Me-3H]thymidine into total cell DNA in the 2 cell lines. From this estimate and from the photosensitivity of A9 cells grown for 2 or 3 generations in the presence of 30 μg/ml BrdU, it can be calculated that the photosensitivity of Cl 1D cells is about an order of magnitude higher than expected from any possible residual incorporation of BrdU into nuclear DNA, if this were random. The present results are, on the contrary, compatible with the idea that mit-DNA is the killing target of ‘visible’ light irradiation.

An increased thermoresistance, as revealed by measurements of growth capacity of mass cultures and by cloning efficiency determinations at 40 °C, was observed in BrdU-grown Cl 1D cells in comparison with cells maintained in the absence of the halogenated precursor.