Fanconi Anemia Fibroblasts Research Articles

Cellular sensitivity and low dose-rate recovery in Fanconi anaemia fibroblasts.

Fanconi anaemia (FA) is a rare inherited condition characterized by developmental abnormalities and progressive bone marrow failure, which requires bone marrow transplantation for successful treatment. This involves the use of alkylating agents and total body or thoraco-abdominal irradiation. Both chemical clastogens and irradiation cause increased chromosome damage in FA cells compared with controls. In some studies FA fibroblasts have been found to be more radiosensitive than normal. From these data it has been inferred that patients with FA might be more sensitive than normal to radiotherapy. However, increased radiosensitivity of FA fibroblasts has not been a uniform finding. The radiosensitivity of fibroblasts from two FA patients was studied at high and low dose-rate (LDR), and their sensitivity compared with normal strains. Both FA strains fell at the sensitive end of the range, but both demonstrated marked dose-rate sparing, with D0.01 recovery factors of 1.23 and 1.27, similar to the normal strains. These recovery factors are inconsistent with the suggestion that FA patients are recovery deficient. The data indicate that at least some FA strains are capable of LDR recovery, and imply that these patients would probably have a clinical benefit from fractionated or low dose-rate total body irradiation.

Hypersensitivity to oxygen is a uniform and secondary defect in Fanconi anemia cells

Cells from patients with Fanconi anemia (FA) frequently show an increased sensitivity to DNA crosslinking agents such as mitomycin C (MMC). FA cells also show abnormal sensitivity to oxygen tension. In order to examine the correlation between the two cellular defects in FA, several FA fibroblast lines were tested for their sensitivity to MMC and oxygen by colony-formation frequency. The sensitivity to MMC in different FA lines varied in a broad range from normal level to extreme hypersensitivity, whereas all of the FA lines showed similar hypersensitivity to oxygen. When FA fibroblasts were transformed by SV40 large T-antigen, the hypersensitivity to oxygen was normalized while the MMC sensitivity still remained. These results suggest that the cellular sensitivity to oxygen is a secondary defect rather than a primary effect of mutations in FA. However, it is a more uniform phenotype that the MMC sensitivity, and therefore, it may be closely related to the common clinical symptoms of FA. Since 1% oxygen showed the highest colony-formation frequency for FA cells, establishment of FA primary fibroblasts was attempted at the low oxygen condition. FA fibroblast cells showed greatly enhanced growth and migration at 1% oxygen resulting in fast establishment of FA primary fibroblasts.

Immunological probing of induction and repair of 8-methoxypsoralen photoadducts in DNA from Fanconi anemia and normal human fibroblasts: Quantitative analysis by electron microscopy

A direct method of immuno-electron microscopy has been employed to simultaneously determine 8-methoxypsoralen (8-MOP) photoinduced monoadducts (MA) and interstrand cross-links (CL), their relative localization along the DNA molecule, and their removal. It has been applied to DNA from cultures of Fanconi anemia (FA) fibroblasts (complementation groups A and D), and of normal human fibroblasts, following treatment by 8-MOP and 365 nm radiation. The immuno-reaction with monoclonal antibody 8G1 was performed on DNA extracted from the cells just after photoreaction, or after a 24 h repair period, and then denatured. Furan-side MA and also a significant proportion of pyrone-side MA were very efficiently immuno-detected. Only 1–2% CL were IgG-labeled. This is why CL were directly visualized and quantified on denatured DNA from the same cellular samples as used for immuno-detection. Results demonstrate that FA group A cells are not only impaired in the incision of CL, but also of MA. The response of FA group D cells is intermediate between normal and FA group A cells.

Increased amount and contour length distribution of small polydisperse circular DNA (spcDNA) in Fanconi anemia

Small polydisperse circular DNA (spcDNA) in Fanconi anemia (FA) was analyzed from cultured fibroblast-like cells by electron microscopy. Application of the mica-press adsorption technique for the semi-quantitative determination of spcDNA amounts to three FA and three normal control skin-derived fibroblast strains revealed 85-fold increased levels of spcDNA in the FA cells. An even higher excess over controls was suggested when the FA fibroblasts were propagated for up to 11 serial in vitro passages, consistent with the short replicative life-span of primary FA cells and their rapid transition into a poorly dividing state, in which spcDNA reportedly further increases. In addition, contour length distributions of gradient-purified spcDNA preparations from five FA fibroblast strains were compared with those from five normal control strains. Mean spcDNA contour lengths were significantly greater in the FA than in the control cells. The reported findings of increased spcDNA amounts and sizes in FA coincide with a similar association of chromosome instability and abnormal spcDNA formation previously observed in cultured cells derived from angiofibromas in tuberous sclerosis. Circumstantial evidence from the present study in the paradigmatic chromosome breakage syndrome FA further supports the suggestion that a common mechanism underlies chromosome instability and the surplus generation of spcDNA. Notably, this apparent mechanism is functional in homonuclear primary cell strains with a distinct inherited basis of their chromosome instability, and is not restricted to heteroploid and neoplastoid cell lines.

Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.

Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells

The basal levels of superoxide dismutase (SOD) activity and chromosome aberration (CA) and sister-chromatid exchange (SCE) frequencies were examined in cultured fibroblasts or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs). These cells were derived from patients with chromosome instability syndromes (CISs) including Bloom's syndrome (BS), Fanconi's anemia (FA) and ataxia telegiectasia (AT). Embryonal fibroblasts and LCLs from normal subjects served as controls. Although LCLs tended to exhibit a higher SOD level than fibroblasts due to an elevation of Cu/ Zn-SOD activity, BS and FA fibroblasts with increased frequencies of CAs and/or SCEs showed abnormally elevated SOD activity due to the manifold increase of Mn-SOD levels compared with control cells. However, BS and AT LCLs with almost control levels of CA and SCE frequencies showed no, or a slightly elevated, SOD activity, suggesting a possible selection of such cells during EBV transformation. The observed parallelism between the SOD activity and the cytogenetic manifestation may imply an involvement of active oxygen species, especially superoxide radicals, in the increased chromosome damage of CIS cells.

DNA polymerase alpha defect in the N syndrome

The N syndrome is characterized by mental retardation, malformations, chromosome breakage, and development of T-cell leukemia (Opitz et al.: Proceedings of the II International Congress IASSMD pp 115-119, 1971; Hess et al.: Clinical Genetics 6:237-246, 1974b, American Journal of Medical Genetics [supplement] 3:383-388, 1987). N syndrome fibroblasts were studied to see if the high chromosome breakage rate associated with this apparently X-linked syndrome could be related to a deficiency of DNA polymerase alpha, a product of a gene localized to the X chromosome. Bleomycin, which is known to break double-stranded DNA, produced increased chromosome breakage in normal control, Fanconi anemia, and N syndrome fibroblasts. When aphidicolin was used to inhibit repair mediated by DNA polymerase alpha, both normal control and Fanconi anemia fibroblasts showed significantly more chromosome breakage than was produced by bleomycin alone, but there was no increase in the amount of breakage seen in the N syndrome fibroblasts over that seen with bleomycin alone. This suggests that the N syndrome is due to a mutation affecting the region of the X chromosome on which the gene for DNA polymerase alpha is located, and that the high risk of T-cell leukemia observed in the hemizygote is due to this DNA repair defect.

Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.

Ultrastructure of Fanconi anemia fibroblasts.

Employing indirect immunofluorescence and conventional electron microscopy, gross nuclear aberrations were observed in cultured interphase fibroblasts derived from a patient suffering from Fanconi's anemia (FA). Such aberrations were predominantly expressed in cells at high passages between 28 and 34. The structure of the nuclei appeared compound in nature, often consisting of two to three nuclear fragments connected to each other by thin nuclear bridges containing chromatin and nuclear lamin material. In other cases, the nuclei appeared lobed or budded but the cells did not contain distinct nuclear fragments. Chromatin was conspicuously absent from some nuclear lobes, revealing empty, cage-like structures comprising nuclear lamin material. Micronuclei were often abundant in the perinuclear cytoplasm but in some instances they appeared to be composed of chromatin lacking a delineating nuclear lamin matrix. Residual cytoskeletons examined by whole-mount electron microscopy revealed a network of intermediate filaments (IFs) within FA fibroblasts forming a bridge between the plasma membrane and the nucleus or its major fragments. In addition, there were thinner, 3-4 nm filaments connecting individual IFs with the surface of the nucleus. Micronuclei that were not connected to the main nuclear body, but which were delineated by a distinct lamina and possessed nuclear pores, did not appear to be anchored to the IF network. Multinuclearity, nuclear fragmentation, irregular chromatin distribution and inter-nuclear chromatin/lamin bridges might result from a failure in the redistribution of chromatin to sister nuclei, incomplete cytokinesis and proliferation of nuclear envelope material. These phenomena point to precocious aging of FA fibroblasts and may occur as a consequence of spontaneous damage to the sister chromatids or through the action of DNA-toxic agents.

RecA-like activity in mammalian cell extracts of different origin

The occurence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from ΦX174 RFI DNA and fragments of ΦX174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg 2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4–7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus-(Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.

Antioxidant status of Fanconi anemia fibroblasts.

Several observations in the recent literature have indicated that Fanconi anemia (FA) cells may be primarily deficient in the detoxification of activated oxygen species. To evaluate the antioxidant status of FA fibroblasts, we measured Mn-containing superoxide dismutase (Mn-SOD), CuZn-containing superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase activities, as well as cellular glutathione contents and total nonenzymatic antioxidant potential in FA and control fibroblasts at multiple time points during a single passage. All parameters exhibited a characteristic pattern of changes during a period of 19 days following trypsinization. Unlike FA erythrocytes, which are known to be deficient in CuZn-SOD, FA fibroblasts exhibited normal CuZn-SOD activities. Also, the nonenzymatic "antioxidant potential" as well as glutathione levels were similar in FA and control fibroblasts. However, Mn-SOD, catalase, and glutathione peroxidase activities were consistently higher in FA fibroblasts. We hypothesize that the elevation of these enzyme activities might reflect a cellular "prooxidant" state in FA resulting from an increased formation of endogenous oxidizing molecular species that trigger enhanced synthesis of certain enzymatic antioxidant defenses.

Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts.

Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). We report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental or Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 micrograms/ml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEBr) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEBr colonies independently derived from transfection of human DNA and one of three DEBr colonies independently derived from transfection of Chinese hamster DNA. In addition, Alu-equivalent hamster sequences were detected in three of seven additional independently derived colonies from transfection of Chinese hamster DNA. The DEBr phenotype of these colonies was stably maintained over several subcultures. Our results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA.

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

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.

Characterization of a Simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer.. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D 10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 × 10 −4, values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.

Sensitivity of Hyperthermia-Treated Human Cells to Killing by Ultraviolet or γ Radiation

Human xeroderma pigmentosum (XP) or Fanconi anemia (FA) fibroblasts displayed shouldered 45 degrees C heat survival curves not significantly different from normal fibroblasts, a result similar to that previously found for ataxia telangiectasia (AT) cells, indicating heat resistance is not linked to either uv or low-LET ionizing radiation resistance. Hyperthermia (45 degrees C) sensitized normal and XP fibroblasts to killing by gamma radiation but failed to sensitize the cells to the lethal effects of 254 nm uv radiation. Thermal inhibition of repair of ionizing radiation lesions but not uv-induced lesions appears to contribute synergistically to cell death. The thermal enhancement ratio (TER) for the synergistic interaction of hyperthermia (45 degrees C, 30 min) and gamma radiation was significantly lower in one FA and two strains (TER = 1.7-1.8) than that reported previously for three normal strains (TER = 2.5-3.0). These XP and FA strains may be more gamma sensitive than normal human fibroblasts. Since hyperthermia treatment only slightly increases the gamma-radiation sensitivity of ataxia telangiectasia (AT) fibroblasts compared to normal strains, it is possible that the degree of thermal enhancement attainable reflects the genetically inherent ionizing radiation repair capacity of the cells. The data indicate that both repair inhibition and particular lesion types are required for lethal synergism between heat and radiation. We therefore postulate that the transient thermal inhibition of repair results in the conversion of gamma-induced lesions to irrepairable lethal damage, while uv-type damage can remain unaltered during this period.

DNA semi-conservative synthesis in normal and Fanconi anemia fibroblasts following treatment with 8-methoxypsoralen and near ultraviolet light or with X-rays.

The effect of treatment with 8-methoxypsoralen (8-MOP) plus near-UV radiation (UVA) or with X-rays on the rate of DNA semi-conservative synthesis of fibroblasts from 10 Fanconi anemia (FA), two heterozygous, and three normal cell lines was studied. Following treatments with either X-rays or low doses of 8-MOP plus UVA leading to a majority of monoadducts over cross-links per genome, the FA and heterozygous cell lines were indistinguishable from normals: the transient inhibition of semi-conservative DNA synthesis was followed by the recovery of a normal rate of synthesis. In contrast treatment with higher (but not saturating) doses of 8-MOP plus UVA allowed us to distinguish two classes among the FA cell lines. One class demonstrated a pattern of recovery similar to that of heterozygous and normal cell lines. This indicates that in such cell lines, the predominant lesion in this condition, the cross-links, do not arrest DNA synthesis and are likely to be normally repaired. Another class of FA cell lines did not show a recovery of a normal rate of DNA synthesis even after prolonged post-treatment incubation and although the proportion of cells in S phase was similar to that of the strains of the first category. This indicates that in such cell lines the repair of cross-links is inhibited at some step which is not necessarily the incision one.

Inhibition of DNA synthesis by psoralen-induced lesions in xeroderma pigmentosum and Fanconi's anemia fibroblasts.

Abstract— Exposure of human cells to psoralens and near‐UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8‐Methoxypsoralen (8‐MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8‐MOP and 7500 J/m2 of near‐UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near‐UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8‐MOP plus near‐UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8‐MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.

Further studies on compartmentalisation of DNA-topoisomerase I in Fanconi anemia tissue.

Previously we found increased DNA-topoisomerase I activity in the cytoplasmic fraction of a mature placenta from a child homozygously affected with Fanconi anemia (FA). Now determination of this enzyme was extended to one heterozygous and four homozygous FA fibroblast cultures in confluent stage, and one mature and one immature placenta from homozygously affected children. In all these cases enzyme activity was found in the cytoplasmic fraction in the range between 0.2 and 1.3 U/micrograms protein. For comparison six normal fibroblast cultures, five normal mature and two immature placentae were studied. The cytoplasmic fractions from all but one fibroblast culture, which showed trace activity, showed no detectable activity. The normal mature placentae had no measurable cytoplasmic activity, while the immature trisomy 18 placentae contained 0.1 and 0.27 U/microgram protein. In a homozygous FA fibroblast culture the total cellular distribution was determined; the cytoplasmic fraction contained 6.5% of the whole cellular activity.

Investigation of the cell cycle response of normal and Fanconi's anaemia fibroblasts to nitrogen mustard using flow cytometry.

Cell survival has been measured in normal and Fanconi's anaemia (FA) human fibroblasts after treatment with the bifunctional alkylating agent, nitrogen mustard (HN2). Two FA cell lines exhibited 6- to 10-fold greater sensitivity than the normal cell line. Flow cytometry was used to investigate the effects of HN2 on cell cycle progression of normal and FA cells. After 0.1 microgram/ml HN2 (surviving fraction, s.f. = 0.8) normal cells exhibited an S phase accumulation within 6 h, followed by a transient G2 delay. At higher doses of HN2, the S phase delay became more pronounced and there was considerably greater accumulation of cells in G2. HN2 at 0.01 microgram/ml (s.f. = 0.8) induced no detectable S or G2 delay in FA cells. A higher dose, 0.1 microgram/ml (s.f. = 0.13 and 0.29), again induced no S phase delay, but a gradual accumulation of cells in G2 was observed up to 78 h after treatment. The presence of an S phase delay in normal cells after HN2 treatment may be important in allowing time for DNA repair before completion of DNA synthesis. The absence of such a delay in FA cells suggests that an inability to delay S phase traverse in response to DNA damage from bifunctional alkylating agents may contribute to the sensitivity of FA cells to such drugs.

First Announcement of the Fanconi Anemia International Registry

We, Arleen D. Auerbach, USA, and Traute M. Schroeder, West Germany, call upon our colleagues to cooperate with us in order to establish and maintain an International Registry of all known Fanconi anemia (FA) patients. Clinical diagnosis of FA is often difficult to establish due to phenotypic diversity of patients and the occurrence of other disorders that manifest some of the features of FA. Diagnosis may be confirmed by the use of a cytogenetic method based on the sensitivity of FA lymphocytes, fibroblasts, and amniotic fluid cells to chemically induced chromosome breakage (Auerbach, Adler, and Chaganti: Pediatrics 67:128, 1981).