Mice with a targeted disruption of the Fanconi anemia homolog Fanca.

Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer predisposition. Bone marrow failure resulting in pancytopenia is the main cause of death of FA patients. Diagnosis of FA is based on their cellular hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic complementation experiments suggest the involvement of at least eight genes in FA. The gene for complementation group A (FANCA) is defective in the majority of FA patients. We show here that mice deficient of FANCA: are viable and have no detectable developmental abnormalities. The hematological parameters showed a slightly decreased platelet count and a slightly increased erythrocyte mean cell volume in mice at young age, but this did not progress to anemia. Consistent with the clinical phenotype of FA patients, both male and female mice showed hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the knock-out mice exhibited spontaneous chromosomal instability and were hyper-responsive to the clastogenic effect of the crosslinker mitomycin C.

Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.

Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance.

Approximately 25% of patients with Fanconi anemia (FA) have evidence of spontaneously occurring mosaicism as manifest by the presence of two subpopulations of lymphocytes, one of which is hypersensitive to cross-linking agents (e.g. mitomycin C) while the other behaves normally in response to these agents. The molecular basis of this phenotypic reversion has not yet been determined. We have investigated 8 FA patients with evidence of mosaicism. Epstein-Barr virus-immortalized lymphoblastoid cell lines established from these patients exhibited an IC50 for mitomycin C of 25 to > 100 nM compared to a mean of 2 +/- 2 nM for 20 nonmosaic FA patients and 49 +/- 11 nM for 8 healthy controls. In 3 patients who were compound heterozygotes for pathogenic FAC gene mutations the molecular mechanism of the mosaicism was investigated by haplotype analysis. The results indicated that an intragenic mitotic recombination must have occurred leading to a segregation of a wild-type allele in the reverted cells and suggested two patterns of recombination. In 1 patient a single intragenic crossover between the maternally and paternally inherited mutations occurred associated with markers located distally to the FAC gene; in the other 2 patients (sibs) the mechanism appears to have been gene conversion resulting in segregants which have lost one pathogenic mutation. In 6 of the 8 patients the hematological symptoms were relatively mild despite an age range of 9-30 years.

Classification of Fanconi anemia patients by complementation analysis: evidence for a fifth genetic subtype.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms, life-threatening progressive panmyelopathy, and cellular hypersensitivity to cross-linking agents. Currently, 4 genetic subtypes or complementation groups (FA-A through FA-D) have been distinguished among 7 unrelated FA patients. We report the use of genetically marked FA lymphoblastoid cell lines representing each of the 4 presently known complementation groups to classify 13 unrelated FA patients through cell fusion and complementation analysis. Twelve cell lines failed to complement cross-linker sensitivity in fusion hybrids with only 1 of the 4 reference cell lines and could thus be unambiguously classified as FA-A (7 patients), FA-C (4 patients), or FA-D (1 patient). One cell line complemented all 4 reference cell lines and therefore represents a new complementation group, designated FA-E. These results imply that at least 5 genes appear to be involved in a pathway that, when defective, causes bone marrow failure in FA patients.

Cytogenetic toxicity of paraquat and streptonigrin in Fanconi's anemia.

We reported earlier that the frequency of chromosomal aberrations observed in Fanconi's anemia lymphocyte cultures depends on the oxygen tension during growth of the cultures, suggesting that "activated oxygen species" (superoxide, O2-; hydrogen peroxide, H2O2; hydoxyl radical, OH; and singlet oxygen, 1O2) or other reactive products generated during oxygen metabolism may be involved in the production of chromosomal damage in this syndrome. Paraquat and streptonigrin, agents that have been proposed as model compounds exerting cellular toxicity through overproduction of superoxide, were tested for their clastogenic potency in lymphocyte cultures from healthy controls and patients with Fanconi's anemia. Paraquat, at concentrations that severely affected mitotic activity (100-200 micrograms/ml), appeared to be a weak clastogen in human lymphocytes, whereas a clastogenic effect of streptonigrin was demonstrable already at a concentration as low as 0.005 microgram/ml. The results indicate that Fanconi's anemia lymphocytes fail to exhibit an increased sensitivity to the antimitotic and clastogenic effects of paraquat and streptonigrin. This suggests that intracellular superoxide is not critically involved in the generation of spontaneous chromosomal aberrations in Fanconi's anemia. We infer from these and previous data that singlet oxygen (1O2) may be a critical contributor to the chromosomal breakage in this disorder.

Oxygen-induced cytogenetic instability in normal human lymphocytes.

Chromosomal aberrations were scored in normal human lymphocytes cultured for 3 to 7 days at 20, 40, 50, and 60% oxygen. Compared to normoxic cultures (20% O2), in hyperoxic cultures (40% to 60% O2) the frequency of aberrant cells was significantly increased, especially at the longer incubation times. The increase was mainly due to chromatid-type damage: chromatid gaps, breaks, and interchanges. In addition, long-term hyperoxic cultures had a remarkably high incidence of endoreduplications, up to 7.4%. It appears that prolonged hyperoxic incubation induces a pattern of cytogenetic abnormalities in normal human lymphocytes similar to that present "spontaneously" in 3-day normoxic cultures from patients with Fanconi anemia.

Clastogenicity of cyclophosphamide in Fanconi's anemia lymphocytes without exogenous metabolic activation.

The indirect mutagen cyclophosphamide (CP) is known to induce chromosomal aberrations in human lymphocyte cultures only after exogenous metabolic activation. We document here that lymphocytes from five patients with Fanconi's anemia (FA) exhibited extensive chromosomal breakage when cultured in the continuous presence of 1 mM CP without deliberate activation of the drug, whereas, cultures from healthy subjects were not significantly susceptible to breakage under these conditions. CP exposure also enhanced the frequency of sister chromatid exchanges (SCE), but to similar extents in control and FA subjects, suggesting that there is a similar low level of CP metabolism in FA and normal lymphocytes. The direct clastogenic effect of nonactivated CP as demonstrated here in FA lymphocyte cultures may be of value in screening for possible hypersensitivity in patients considered for CP therapy.

Some characteristics of hyperoxia-adapted HeLa cells. A tissue culture model for cellular oxygen tolerance.

By culturing HeLa cells at stepwise increased oxygen tensions over a prolonged period of time (approximately 21 months) we selected a substrain capable of growing under 80% O2/19% N2/1% CO2, an oxygen level that is lethal to normal HeLa cells, adapted to 20% O2/79% N2/1% CO2. The 80% O2-adapted cells exhibited the following characteristics. At the ultrastructural level an abnormal mitochondrial morphology was observed: compared to normal cells, mitochondria of the hyperoxia-adapted cells exhibited a 3-fold larger mean profile area in sections and were slightly decreased in number; the relative mitochondrial volume was increased 2-fold, whereas the size of both cell types was the same. Mitochondrial matrix appeared less dense in the hyperoxia-adapted cells; no structural damage was detected. Compared to the 20% O2-adapted cells O2 consumption per cell was approximately 40% decreased in the 80% O2-adapted cells. Under hyperoxic conditions 20% O2-adapted and 80% O2-adapted cells exhibited very similar cyanide-resistant respiration rates (0.16 +/- 0.04 and 0.15 +/- 0.02 fmoles/cell/minute, respectively), suggesting that the increased O2 tolerance of the 80% O2-adapted cells was not due to a decreased cellular production of activated oxygen species at hyperoxia. Cellular levels of the enzymes directly involved in protection against activated oxygen species, i.e., superoxide dismutases, catalase, and glutathione peroxidase, were normal or slightly below normal in the 80% O2-adapted cells, implying that these enzymes were of no significance for the increased O2 tolerance. In addition, the specific activity of glucose-6-phosphate dehydrogenase, a key enzyme for cellular production of NADPH, was not related to the degree of O2 tolerance. Our results suggest that the increased O2 tolerance of the 80% O2-adapted cells is neither based on cellular properties controlling the formation or removal of intracellular activated oxygen species nor on the cellular capacity to repair or replace damaged cellular components. We speculate that the increased O2 tolerance is largely due to a genetically determined increased resistance of oxygen-sensitive cellular targets.

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary. Antioxygenic enzyme levels and ultrastructural morphometry of peroxisomes and mitochondria.

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.

Cytogenetic toxicity of D2O in human lymphocyte cultures. Increased sensitivity in Fanconi's anemia.

Chromosomal aberrations were scored in lymphocyte cultures from healthy individuals, patients with Bloom syndrome, and patients with Fanconi's anemia, after 4-5 h exposure to culture medium containing 90% heavy water (D2O). D2O treatment resulted in occasional pulverization of metaphases, and increased frequencies of chromosomal breakage. Patients with Fanconi's anemia were particularly sensitive to the chromosome breaking effect of D2O.

Effect of oxygen tension on chromosomal aberrations in Fanconi anaemia.

Blood samples from four healthy individuals and from seven Fanconi anaemia (FA) patients were cultured at oxygen tensions ranging from 3% to 45% O2. Cultures were harvested at 72 h and scored for chromosomal aberrations. In the majority of FA patients the aberration frequency showed a tendency to increase as a function of oxygen tension over the culture, whereas the aberration frequency in healthy individuals was not affected. However, the response in FA cultures was variable among patients and in individual cases when assayed on different occasions. A much stronger effect of oxygen tension was observed when the FA blood samples had been treated with mitomycin C (0.25 microgram/ml, 30 min) before culture initiation.

Oxygen-dependence of chromosomal aberrations in Fanconi's anaemia.

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by a high frequency of 'spontaneous' chromosomal aberrations and an increased risk of cancer. If, as seems plausible, the microscopically visible chromosomal aberrations in this disorder result from DNA or chromatin damage that would normally be repairable, the questions arise as to which step (or steps) in the repair process is deficient and whether the deficiency is intrinsic or the result of secondary factors. We report here that the frequency of chromosomal aberrations in FA lymphocyte cultures is positively related to oxygen tension and suggest that the site primarily affected by the FA mutation is in the complex system of defence (that is, protection and repair) against the genetic toxicity of oxygen.