GENE-SPECIFIC DAMAGE PRODUCED BY CISPLATIN, ORMAPLATIN AND UV-LIGHT IN HUMAN-CELLS AS ASSAYED BY THE POLYMERASE CHAIN-REACTION

The level of DNA damage in the total genome of leukocytes from patient s receiving cisplatin therapy has been previously correlated with ther apeutic outcome. Based on the fact that unrepaired damage in transcrib ed genes has been well correlated with sensitivity to a number of DNA damaging agents, the correlations with therapeutic response might be i mproved if the level of DNA damage in specific genes was assessed. We previously established a polymerase chain reaction (PCR)-based assay t o measure cisplatin lesions in specific genes. The major advantages of this assay include its potential to quantify DNA lesions produced by any DNA damaging agent, and to measure it in minute samples of cells. This assay has been further improved to realize this potential. Specif ically, cells were damaged with cisplatin, a related analogue ormaplat in, and ultraviolet light, and PCR was performed on the DNA from 1000 cells after direct lysis with no purification. Human HL-60 cells were used to compare the efficacy of the various agents to damage the alpha -fetoprotein gene. This was then extrapolated to damage in the total g enome and compared to the cytotoxicity for each agent. At a dose that killed 50% of the cells, ormaplatin, cisplatin and ultraviolet light p roduced 19,200, 48,000, and 1,080,000 lesions per cell, demonstrating that ormaplatin lesions were the most effective at killing cells. Mono nuclear cells were obtained from freshly isolated blood from five indi viduals. These cells were damaged with the three agents, and PCR of th e alpha-fetoprotein gene was performed. No significant difference betw een the individuals was observed in the level of DNA damage produced b y any of the agents. The results show that this is a rapid and facile assay for quantitation of DNA damage in very small numbers of cells, a nd is particularly applicable to analysis of DNA damage in freshly iso lated human blood.