Effects of mitomycin C on the rate of DNA synthesis in normal and Faconi anaemia cells

THe effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patient with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.     

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

Summary 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 hetcrozygous cell lines were indistinguishable from normals: the transient inhibition of semi-conservative DNA synthesis was followed by the recovery of a nomral 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.     

Fanconi anemia cells have a normal gene structure for topoisomerase I

Cells from Fanconi anemia (FA) patients have defective DNA repair and are hypersensitive to DNA crosslinking agents such as mitomycin C (MMC). We examined the possibility that topoisomerase I is involved in the DNA crosslink repair system and is deficient in FA group A cells. FA cells and control cells were exposed to MMC with or without camptothecin (CPT), a topoisomerase I inhibitor. The cells did not show any increased sensitivity to killing by MMC with CPT, suggesting that the topoisomerase I is not involved in MMC-damaged DNA repair. However, FA cells showed increased sensitivity to CPT in comparison to control cells, raising the possibility of altered topoisomerase I in FA cells. Therefore, a mutation analysis was performed on topoisomerase I cDNA from FA cells by using chemical cleavage mismatch scanning and nucleotide sequencing. No mutation was detected from GM1309, a group A FA cell line. A base transition (C to T) at position 241, causing an amino acid change (His to Tyr), was found in GM2061, a FA cell line of unknown complementation group. However, allele-specific oligonucleotide hybridization analysis showed that this is a gene polymorphism. We conclude that FA cells have normal gene structure for topoisomerase I.     

Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patients

The repair of mitomycin C (MMC)-induced DNA crosslinking was analyzed by denaturation-renaturation gel electrophoresis in ribosomal RNA genes in lymphoblastoid cell lines from 4 patients with Fanconi's anemia (FA). In comparison to normal lymphoblastoid cell lines, 2 lines of FA cells belonging to complementation group A clearly exhibited higher sensitivity to MMC and an almost identical deficiency in the removal of DNA crosslinking. A complementation group B cell line, HSC 62, exhibited a lower sensitivity than group A cells and a lesser deficiency in crosslink repair. Another 'non-A' group cell line, HSC 230, reproducibly exhibited even higher sensitivity to MMC than group A cells. The results on MMC crosslinkage removal at the molecular level correlated well with cell survival. The observed subtle differences of repair among the 4 FA cell lines might represent possible genetic differences in the respective FA complementation groups.     

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-kappaB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1-10 microM) and inhibited it at high doses (50-200 microM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 microM) was observed. The activity of NF-kappaB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.     

Effect of mitomycin C and bromodeoxyuridine on Fanconi anemia lymphocytes.

The authors studied the effect of mitomycin C (MMC) and bromodeoxyuridine (BrdU) on the induction of chromosome aberrations on lymphocytes of four patients with Fanconi anemia (FA) and of one normal subject. A control culture and six experiments were designed to test the possible synergic effect of MMC and BrdU. Their results revealed no evidence of MMC-BrdU synergism on the induction of chromosome aberrations in FA lymphocytes. However, chromosomes showed more damage when FA cells were harvested 24 h after MMC stress than when cells were harvested shortly after treatment. This can be explained by a DNA repair defect or by a toxic effect of oxygenation of cells during the procedure.     

The Chinese hamster cell mutant V-H4 is homologous to Fanconi anemia (complementation group A)

V-H4, a mitomycin C (MMC)-sensitive Chinese hamster cell mutant, is phenotypically very similar to Fanconi anemia (FA) cells. Genetic complementation analysis shows that V-H4 belongs to the same complementation group as FA group A cells. Proliferating hybrid cell lines obtained after fusion of V-H4 with normal or FA group B cells show an increased resistance to MMC. Absence of complementation was noted in V-H4 x FA group A hybrid cell lines. This was shown not to be due to the absence of a specific human chromosome. The V-H4 mutant represents the first rodent mutant that is genotypically similar to FA complementation group A cells.     

Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.     

Comparison of the sensitivity of Fanconi's anemia and normal fibroblasts to the induction of sister-chromatid exchanges by photoaddition of mono- and bi-functional psoralens

The induction of sister-chromatid exchanges (SCE) by photoaddition of a monofunctional furocoumarin (pyrido[3,4-c]psoralen, PyPs) and a bifunctional furocoumarin (8-methoxypsoralen, 8-MOP) in a normal and three Fanconi anemia (FA) fibroblastic cell lines was investigated. When compared to normal cells, the three FA cell lines demonstrated: a higher sensitivity to 8-MOP photoaddition; an equal or reduced sensitivity to PyPs photoaddition in the low dose range. Normal cells demonstrated a higher sensitivity to photoaddition of PyPs than to 8-MOP in the range of doses used; this is likely to be related to the higher amount of lesions induced by PyPs in DNA. Since FA cells were almost equally sensitive to 8-MOP and PyPs photoaddition and demonstrated a higher sensitivity to SCE induction by 8-MOP than normal cells, it can be concluded that this latter difference is mainly due to cross-links.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.     

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.     

Proliferative kinetics and mitomycin C-induced chromosome damage in Fanconi's anemia lymphocytes

Lymphocytes from two sisters with Fanconi's anemia (FA) were studied for cell cycle kinetics, sister chromatid exchanges (SCEs), and chromosomal aberrations when they had undergone one, two, or three or more divisions in mitomycin C (MMC)-treated cultures. Lymphocytes from the parents, another sister of the probands, and a healthy unrelated adult were examined as controls. Analyses of cell cycle kinetics by the sister chromatid differential staining method revealed that the relative frequency of metaphase cells at their third or subsequent divisions was much smaller in untreated FA cultures than in normal cultures fixed at 96 h after phytohemagglutinin stimulation. These data indicate that FA cells proliferate much more slowly than normal cells. MMC treatments of FA and normal cells led to a clearly dose-related delay in cell turnover times, the duration of delay being much longer in FA than in normal cells. FA cells had about 1.4 times higher frequencies of SCEs than normal cells in both MMC-treated and untreated cultures. FA cells also showed several times higher frequencies of chromosomal aberrations than normal cells, and the frequency of chromosomal aberrations decreased through subsequent mitoses by approximately 60% in both FA and normal cells.     

Fanconi Anemia C Protein Acts at a Switch between Apoptosis and Necrosis in Mitomycin C-Induced Cell Death

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Δψ), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Δψ. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Δψ, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.     

Fluocinolone acetonide: A potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis

The relationship between the inhibition of mouse skin tumor promotion and the inhibition of epidermal DNA synthesis by the steroidal anti-inflammatory agent, fluocinolone acetonide (FA), was investigated. Simultaneous doses of either 10, 1, or 0.1 μg of FA and phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in an almost complete inhibition of promotion, whereas 0.01 and 0.001 μg of FA resulted in inhibition rates of 82% and 15%, respectively. Likewise, simultaneous doses of 10 or 1 μg of fluclorolone acetonide (FCA) and TPA caused a nearly complete inhibition of promotion, whereas 0.1 μg of FCA decreased promotion by 62%. In general, as the dose of both steroids was increased, an increase in the tumor latency period was observed. With the exception of the borderline effect of 0.001 μg of FA, the above doses of FA inhibited epidermal DNA synthesis by at least 60% for a 24-h period. Topical treatment with 10 μg of FA resulted in an almost complete inhibition of DNA synthesis for 6 days. The administration of 10 μg of FA 24 h after TPA treatment brought about a maximal inhibition of DNA synthesis of 65%, as compared with a 98% inhibition in control mice whose DNA synthesis had not been prestimulated. That is, FA was not quite as effective on S-phase cells as on G-1 cells. There appears to be a relationship between the inhibition of tumor promotion and epidermal DNA synthesis.     

Radioresistant DNA synthesis: an intrinsic feature of ataxia telangiectasia

DNA synthesis in 6 ataxia langiectasia (AT) cell strains was much more resistant to X-irradiation than was DNA synthesis in normal human diploid cells. 3 of the cell strains tested have been classified as proficient in repair replication. These data, along with those reported elsewhere, strongly suggest that radioresistant DNA synthesis is an intrinsic feature of this disease.The radioresistance of DNA synthesis in AT cells is primarily due to a reduced inhibition of replicon initiation compared to that occuring in normal cells, but DNA chain elongation is also more radioresistant in AT cells. The small inhibition of DNA synthesis that does occur in AT cells at doses up to 2000 rad is almost exclusively due to inhibition of replicon initiation and not to inhibition of chain elongation, as would be expected from results with normal human cells or from previous studies with established cell lines.     

Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.     

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (? = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (? = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.     

The fate of 8-methoxypsoralen-photoinduced DNA interstrand crosslinks in Fanconi's anemia cells of defined genetic complementation groups

The fate of 8-methoxypsoralen (8-MOP)-photoinduced DNA interstrand crosslinks was followed by alkaline elution in Fanconi's anemia (FA) fibroblasts belonging to complementation groups A (FA 150 and FA 402) and B (FA 145) in comparison to a normal (1 BR/3) and a heterozygote (F 311) cell line. Clonogenic cell survival to 8-MOP photoaddition was established in parallel for all cell lines. In comparison to normal cells, group A FA cells demonstrated a higher photosensitivity than group B cells (sensitivity index 2.3 and 1.5, respectively), the heterozygote cell line being only slightly more sensitive. FA cells from both groups A and B demonstrated an incision capacity of crosslinks, the kinetics and extent of which being, however, different from that of normal or heterozygote cells. The incision is slower in FA cells and, at 24 h of post-treatment incubation, the amount of crosslinks incised is clearly lower than that observed in normal cells for group A cells, whereas in group B cells incision approaches the level of normal cells. These results correlate with survival as well as with rates of DNA semi-conservative synthesis after 8-MOP photoaddition.