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

Abstract

Primary skin fibroblast cell lines from patients with Fanconi anemia were cotransfected with UVirradiated 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-. 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.Fanconi anemia (FA) is an autosomal recessive disorder in which patients show pancytopenia and predisposition to leukemia and carcinoma (1, 2). These patients show a high level of chromatid-type damage in their lymphocytes and fibroblasts (2-4). Cultured FA cells are unusually sensitive to DNA crosslinking agents by chromosomal criteria (5-9) and by clonogenic cell survival (10-14) whereas their sensitivity to monofunctional agents or to radiation is close to normal (12, 15).A number of antitumor drugs and environmental pollutants produce interstrand crosslinks in DNA. Hence, it is important to understand the consequences of such lesions. Since FA is characterized by an almost specific sensitivity to DNA crosslinking agents it may serve as a model system for research in this area. The nature of the repair defect in FA cells is, however, still debated (11,14,(16)(17)(18) (20) has been shown. However, almost nothing is known about the chromosomal location, expression, and regulation of the genes controlling sensitivity of human cells to DNA crosslinking agents. To investigate these aspects, a project aimed at the isolation of the genes involved was initiated, and we established conditions for the DNA-mediated transfer and expression of wild-type genes in FA cell lines. The isolation and characterization of the transformed population obtained are described here. MATERIALS AND METHODSCell Lines and Cell Cultures. Three...

Insulin delivery by somatic cell gene therapy

“…Previous studies have inserted and expressed cDNA encoding human preproinsulin in fibroblasts (Moore et al 1983;Diatloff-Zito et al 1986;Seiden et al 1987e;Kawakami et al 1992) and pituitary AtT20 cells (Moore et al 1983;. Unlike fibroblasts, which release proinsulin almost exclusively, AtT20 cells can process pro¬ insulin to insulin.…”

“…AtT20 cells have been transfected with genes en¬ coding rat and human insulins (Lomedico, 1982;Laub & Rutter, 1983;Moore et al 1983;Diatloff-Zito et al 1986;Seiden et al 19876;Gross et al 1989;Kawakami et al 1992;. Since proinsulin exerts only weak insulin-like activity in vivo (<10% of the potency of insulin) (Revers et al 1984), it is important that proinsulin is processed to insulin.…”

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA

“…1986) was applied to FA 150 and FA 145 primary fibroblasts. For this reason, selection of the corrected population was based on differential growth rates (for a detailed description of this point, see Diatloff-Zito et al 1986). However, the classical cloning of MMC resistant cells selected from transfected FA primary fibroblasts could not be carried out since the number of divisions in vitro is limited.…”

Insulin-releasing pituitary cells as a model for somatic cell gene therapy in diabetes mellitus

“…Several studies have demonstrated the introduction and expression of an exogenous copy of a preproinsulin gene in somatic cells such as fibroblasts and murine pituitary AtT20 cells (Lomedico 1982, Laub & Rutter 1983, Moore et al 1983, Diatloff-Zito et al 1986, Gross et al 1989, Seiden et al 1987a, Kawakami et al 1992, Stewart et al 1993). Fibroblasts do not process proinsulin to insulin, whereas AtT20 cells operate constitutive and regulated pathways of hormone secretion, and express endopeptidases which convert proinsulin to insulin (Moore et al 1983, Orci et al 1987, Powell et al 1988).…”