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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Sep;83(18):7034–7038. doi: 10.1073/pnas.83.18.7034

Abnormal response to DNA crosslinking agents of Fanconi anemia fibroblasts can be corrected by transfection with normal human DNA.

C Diatloff-Zito, D Papadopoulo, D Averbeck, E Moustacchi
PMCID: PMC386647  PMID: 3092225

Abstract

Primary skin fibroblast cell lines from patients with Fanconi anemia were cotransfected with UV-irradiated pSV2neo plasmids and high molecular weight DNA from normal human cells. Restoration of a normal cellular resistance to mitomycin C (MMC) was observed provided that a Fanconi anemia cell line is selected for DNA-mediated transformation (neo gene) and that at least two successive rounds of transfection are performed. Cells were selected by taking advantage of the higher proliferation rate and plating efficiency of the MMC resistant transformants. As estimated from reconstruction experiments, the frequency of transfer of MMC resistance lies between 1 and 30 X 10(-7). The MMC resistance phenotype was maintained for at least 10 generations following transfection. Evidence for DNA-mediated transformation also includes the recovery of a normal pattern of DNA semiconservative synthesis after treatment with 8-methoxypsoralen and 365-nm UV irradiation, and the presence of exogenous pSV2neo DNA sequences was shown by Southern blot analysis. The acquired MMC resistance is probably due to the presence of DNA from normal cells. Indeed, sensitivity to MMC was maintained when Fanconi anemia cells were cotransfected with the UV-irradiated pSV2neo plasmid mixed with their own DNA or with yeast or salmon sperm DNA. These negative results also render unlikely the selection of spontaneous MMC resistant revertants in transfection of Fanconi anemia cells with normal DNA. These experiments establish the prerequisites for the isolation of the gene(s) involved in the response to DNA crosslinking lesions in human cells.

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Selected References

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