Specific cellular defects in patients with Fanconi anemia

Weksberg, Rosanna; Buchwald, Manuel; Sargent, P. A.; Thompson, Margaret; Siminovitch, Louis

doi:10.1002/jcp.1041010211

Cited by 106 publications

(22 citation statements)

References 29 publications

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“…1). TS fibroblasts thus probably do not bear resemblance to chromosomal instability syndromes such as AT, Fanconi anemia, or Bloom syndrome, with respect to the growth rate Swift, 1976a, 1976b;Weksberg et al, 1979) and to the colony-forming efficiency (Shiloh et al, 1982).…”

Section: Discussionmentioning

confidence: 99%

“…MNNG was dissolved in dimethylsulfoxide at a concentration of 10 mg/ml and kept at -20~ 4NQO was dissolved in ethanol at a concentration of 250 yg/ml and kept at -20~ These compounds were diluted into medium immediately before use. After cells were exposed to each chemical for 24 hours, the periods being determined according to Weksberg et al (1979), cells were rinsed three times with warmed medium without FBS. Then fresh DEM containing 10 FBS was added to and the cells were cultured.…”

Section: Methodsmentioning

confidence: 99%

“…In the case of several disorders, the in vitro behavior of cells from such patients is abnormal with respect to growth parameters and sensitivity to a variety of DNA damaging agents. Decreased growth was evident in fibroblast strains from patients with ataxia telangiectasia (AT) (Elmore and Swift, 1976a), Fanconi anemia Swift, 1976b, Weksberg et al, 1979) and yon Recklinghausen disease (Zelkowitz, 1981), and has been suggested in case of cells from patients with Cockayne syndrome and Bloom syndrome (Weksberg et al, 1979). These disorders, except for yon Recklinghausen disease, have al!…”

Section: Introductionmentioning

confidence: 99%

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Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylating agents

Ohno

Takeshita

1984

Jap J Human Genet

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SummaryTuberous sclerosis (TS) is an autosomal dominant neurocutaneous syndrome characterized by hamartomatous growth in multisystems. To determine the gene expression in cultured fibroblasts, we studied several in vitro properties of skin fibroblasts. Measurements of the attachment ratio, the colony-forming efficiency, the population doubling time, and the saturation density in 10~ fetal bovine serum show that fibroblasts from five TS patients proliferate in the same manner as do the age-matched controls. We also measured the survival of TS fibroblasts after treatment with a variety of chemical agents. TS fibroblasts are equal to controls in their sensitivity to four types of alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine to which TS fibroblasts have been reported to be hypersensitive. These results suggest that the in vitro properties of TS fibrobtasts do not resemble cells from patients with several autosomal recessive disorders predisposing to neoplasms and displaying hypersensitivity to DNA damages.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylating agents

Ohno

Takeshita

1984

Jap J Human Genet

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“…These patients show a high level of chromatid-type damage in their lymphocytes and fibroblasts (2)(3)(4). Cultured FA cells are unusually sensitive to DNA crosslinking agents by chromosomal criteria (5)(6)(7)(8)(9) and by clonogenic cell survival (10)(11)(12)(13)(14) whereas their sensitivity to monofunctional agents or to radiation is close to normal (12,15).…”

Section: Introductionmentioning

confidence: 99%

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

Diatloff-Zito¹,

Papadopoulo²,

Averbeck³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…In particular, their G 2 phase is unusually long, and many cells remain permanently arrested within this phase (44,47,60). G 2 arrest may reflect the accumulation of DNA damage and the need to repair this damage before mitosis.…”

mentioning

confidence: 99%

Irreversible Repression of DNA Synthesis in Fanconi Anemia Cells Is Alleviated by the Product of a Novel Cyclin-Related Gene

Digweed

Günthert

Schneider

et al. 1995

Molecular and Cellular Biology

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Primary fibroblasts from patients with the genetic disease Fanconi anemia, which are hypersensitive to cross-linking agents, were used to screen a cDNA library for sequences involved in their abnormal cellular response to a cross-linking challenge. By using library partition and microinjection of in vitro-transcribed RNA, a cDNA clone, pSPHAR (S-phase response), which is able to correct the permanent repression of semiconservative DNA synthesis rates characteristic of these cells, was isolated. Wild-type SPHAR mRNA is expressed in all fibroblasts so far analyzed, including those of Fanconi anemia patients. Correction of the abnormal response in these cells appears therefore to be due to overexpression after cDNA transfer rather than to genetic complementation. The cDNA contains an open reading frame coding for a polypeptide of 7.5 kDa. Rabbit antiserum directed against a SPHAR peptide detects a protein of 7.9 kDa in Western blots (immunoblots) of whole-cell extracts from proliferating, but not resting, fibroblasts. The deduced amino acid sequence of SPHAR contains a motif found in the cyclins, and it is proposed that SPHAR acts within the injected cell by interfering with the cyclin-controlled maintenance of S phase. In agreement with this proposal, normal cells transfected with an antisense SPHAR expression vector have a significantly reduced rate of DNA synthesis during S phase and a prolonged G 2 phase, reflecting the need for postreplicative DNA processing before entry into mitosis.

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Specific cellular defects in patients with Fanconi anemia

Cited by 106 publications

References 29 publications

Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylating agents

Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylating agents

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

Irreversible Repression of DNA Synthesis in Fanconi Anemia Cells Is Alleviated by the Product of a Novel Cyclin-Related Gene

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