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Bone marrow transplantation. Nucleated bone marrow cells (5 Â 10 6 ) from Fko or Wt mice were transplanted into 9.5 Gy–irradiated Wt mice.
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The fragile FHIT gene is among the first targets of DNA damage in preneoplastic lesions, and recent studies have shown that Fhit protein is involved in surveillance of genome integrity and checkpoint response after genotoxin exposure. We now find that Fhit-deficient hematopoietic cells, exposed to the genotoxin hydroquinone, are resistant to the su...
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... modulation of expression of checkpoint proteins Hus1 and Chk1, at mid-S checkpoint, modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cells (27). However, the biological significance of Fhit deficiency in environmental carcinogen-related hematopoietic disorders had not been investigated. Here, we report that absence of Fhit in hydroquinone-exposed mouse hematopoietic cells led to resistance to induction of cell death in vitro and escape from bone marrow suppression in transplanted mice. Immunohistochemical analyses of transplanted hydroquinone-exposed, Fhit knockout (Fko) and Wt bone marrow revealed relative absence of apoptosis and senescence markers in Fko bone marrow, in parallel with reduced detection of the oxidized base, 8-hydroxyguanosine (8-OHdG); treatment with the antioxidant N -acetyl- L -cysteine (NAC) alleviated the hydroquinone- induced suppression of colony formation by Wt hematopoietic cells. Accordingly, the long-term survival of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow–derived cells, suggesting that Fhit deficiency leads to unscheduled survival of genotoxin-exposed bone marrow cells, allowing increase of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations. ny formation. The effects of hydroquinone exposure on Fko and Wt bone marrow cells were assessed by in vitro colony formation assays (Fig. 1). To assess the effect of hydroquinone exposure on hematopoietic stem cells (HSC), the KSL fractions from Fko and Wt hematopoietic bone marrow cells were isolated (Fig. 1 A ) and in vitro colony formation potential was assessed (Fig. 1 B ). Unexposed Fko and Wt bone marrow mononuclear cells showed similar BFU-E, CFU-M, CFU-G, and CFU-GM colony-forming efficiency (Fig. 1 C, bottom ). In addition, the appearance and expression of differentiation-specific markers by Fko and Wt committed hematopoietic colonies (Fig. 1 B and C, top ) did not differ significantly in the absence of hydroquinone. The number of CFU-M and CFU-G colonies formed by Wt KSL cells was reduced relative to Fko KSL cells (<0.05) but BFU-E colony numbers did not differ significantly (Fig. 1 D, top ). We next used Lin À bone marrow cells for in vitro colony formation assays (Fig. 1 D, bottom ). The results were similar to those of KSL fractions; that is, CFU-M and CFU-G colony numbers from Wt Lin À cells were reduced relative to Fko Lin À bone marrow cells, a difference not observed without hydroquinone. The results suggest that Fhit plays a role in response to hydroquinone- induced inhibition of hematopoietic colony formation potential, with Fhit deficiency allowing resistance to genotoxic stress- induced suppression of hematopoietic colony formation. Fhit-deficient bone marrow is resistant to genotoxin-induced suppression in vivo . Mononuclear cells of Fko and Wt bone marrow, with or without hydroquinone exposure, were transplanted to Wt mice previously exposed to 9.5 Gy ionizing irradiation (Fig. 2 A, top ). Hematopoietic cell counts in peripheral blood showed that neutrophils, lymphocytes, monocytes, basophils, eosinophils, and platelets increased gradually between 4 and 42 days after transplantation of both Fko and Wt cells (Supplementary Fig. S1), indicating that short-term progenitors are involved in increase and maintenance of Fko and Wt hematopoietic cells in the relatively early posttransplantation phase. Mice receiving non–hydroquinone-exposed Fko or Wt bone marrow cells all survived to 120 days after transplantation (data not shown). In sharp contrast, the recipient mice showed f 80% lethality by 120 days after transplantation of hydroquinone- exposed Wt bone marrow cells; hydroquinone-induced lethality was only 25% by day 120 in Fko bone marrow recipients (Fig. 2 A, bottom ), showing that Fhit deficiency abrogates genotoxin-induced suppression of hematopoiesis. Because the hydroquinone-treated bone marrow transplantation experiments resulted in reduced survival of recipient mice, competitive transplantation assays were performed; untreated or hydroquinone-treated Fko or Wt HSCs were mixed with congenic Ly5.1 mouse bone marrow cells, and the mixtures were transplanted into irradiated mice of Ly5.1 background. The Ly5.1 bone marrow cells would support survival of recipient mice so that the contribution of donor hydroquinone-treated HSCs could be evaluated in the transplant recipient chimeric mice (33, 34). The sorted KSL fractions from Fko and Wt mice were exposed to hydroquinone in vitro , mixed with bone marrow cells from Ly5.1 mice, and transplanted to Ly5.1 recipient mice. All the recipient mice, two receiving Wt and three receiving Fko bone marrow, survived. Assessment of hydroquinone-exposed donor cells in the Ly5.1 background showed that Fko bone marrow cells contributed to increased chimerism (Fig. 2 D, right ). Flow cytometer analysis of peripheral mononuclear cells indicated that Kit + and Sca-1 + cells were increased in mice receiving hydroquinone-exposed Fko donor marrow compared with hydroquinone-exposed Wt marrow (4.6–8.3 times increase in Kit + Fko compared with Kit + Wt; f 3.5 times increase in Sca-1 + cells; Fig. 2 C , representing two recipients of Wt and two of Fko bone marrow). Even in the absence of hydroquinone exposure, Kit + Sca-1 + cells were increased in mice receiving Fko donor marrow compared with Wt marrow (3.2 times in Kit + ; f 1.17 times in Sca-1 + cells; Fig. 2 B ). The fraction of Kit + , Sca-1 , and Lin cells play a significant role in self-renewal and multipotent differentiation of HSCs (33). A serum-free single-cell culture followed by transplantation of cultured cells into lethally irradiated mice indicated that Kit + , Sca-1 + , Lin À cells are highly enriched for murine bone marrow HSCs (34). The above results suggest that ( a ) Fhit-deficient mice maintain a larger fraction of Kit , Sca-1 cells than Wt mice, as apparent after transplantation of hydroquinone-exposed marrow, and ( b ) Fhit-deficient cells pre- serve or retain potential for self-renewal and multipotent differentiation after genotoxic stress. in Fhit-deficient marrow. Histologic assessment of bone marrow was performed by sacrificing recipient mice f 120 days after transplantation of hydroquinone-exposed nucleated cells (Fig. 3 A ). H&E staining of Wt bone marrow showed gross reduction of nucleated bone marrow cells, compatible with the preclinical stage of aplastic anemia and likely related to cause of death of recipient mice. Immunohistochemical analysis of bone marrow after transplantation of hydroquinone-exposed nucleated cells showed patchy positive staining of Bax and phospho-p38 throughout Wt bone marrow, indicating induction of apoptosis and senescence, whereas Fko bone marrow showed relatively healthy cellularity with less Bax and phosho-p38 expression (Fig. 3 A ). The assessment of apoptotic index showed efficient induction of apoptosis in transplanted bone marrow of hydroquinone-exposed Wt cells but much less apoptosis in Fko bone marrow (Fig. 3 B and C ). Assessment of DNA damage by detection of 8-OHdG in genotoxin-exposed Fko and Wt hematopoietic cells showed that 8-OHdG was reduced in Fhit-deficient bone marrow cells, indicating that reduced oxidative stress is associated with enhanced survival of hydroquinone-exposed Fko bone marrow (Fig. 4 A ). We then assessed the effect of oxidative stress on survival of HSCs in vitro . As expected, hydroquinone exposure resulted in a relatively greater reduction of Wt colonies (Fig. 4 B ). Treatment with NAC antioxidant resulted in significant recovery of colony-forming ability of Wt HSCs and, to a lesser extent, of Fko stem cells in long-term in vitro culture, suggesting that treatment with NAC or other antioxidant might prolong the survival of mice receiving hydroquinone-treated Wt bone marrow. The recovery of colony formation by NAC treatment indicates that hydroquinone-induced genotoxic stress, followed by apoptosis, is involved in suppression of colony formation by hematopoietic stem or precursor cells, a process subverted by Fhit deficiency. The data show that Wt stem cell colony-forming ability was suppressed by hydroquinone, likely due to an increase in ROS by hydroquinone treatment (35, 36), which causes DNA damage and apoptosis, whereas in the absence of Fhit ROS-induced DNA damage and apoptosis was reduced, leading to colony growth and mouse survival. This experiment accords with the recent demon- stration that Fhit interacts with the ferredoxin reductase (Fdxr) protein (37), a 54-kDa mitochondrial flavoprotein responsible for transferring electrons from NADPH to cytochrome P 450 via ferredoxin. Leakage of electrons from this shuttling system can result in generation of ROS (38). Following application of oxidative stress, the Fhit-Fdxr interaction leads to ROS generation, an early event in Fhit-triggered apoptosis. In similarly treated Fhit-deficient cells, much less ROS is generated, allowing unscheduled survival of damaged cells (37), a mechanism with relevance to early events in carcinogenesis and to refractoriness to chemotherapy of Fhit- deficient cells. To confirm that hydroquinone treatment leads to decreased ROS production in Fko cells relative to Wt cells, both MEFs and bone marrow cells were treated with 50 or 100 A mol/L of hydroquinone; ROS production was assessed by FACS analysis after staining of bone marrow–derived cells with DCFDA and by fluorescence microscopy and positive cell counting after staining of MEFs with DCFDA. The results of the FACS analysis of the sorted bone marrow cells are shown in Supplementary Fig. S2 and illustrate that, after treatment with 100 mol/L hydroquinone, the Wt hematopoietic cells showed >2-fold more ROS production than Fko hematopoietic cells. For the MEF cells, the fraction of ...
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... investigated. Here, we report that absence of Fhit in hydroquinone-exposed mouse hematopoietic cells led to resistance to induction of cell death in vitro and escape from bone marrow suppression in transplanted mice. Immunohistochemical analyses of transplanted hydroquinone-exposed, Fhit knockout (Fko) and Wt bone marrow revealed relative absence of apoptosis and senescence markers in Fko bone marrow, in parallel with reduced detection of the oxidized base, 8-hydroxyguanosine (8-OHdG); treatment with the antioxidant N -acetyl- L -cysteine (NAC) alleviated the hydroquinone- induced suppression of colony formation by Wt hematopoietic cells. Accordingly, the long-term survival of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow–derived cells, suggesting that Fhit deficiency leads to unscheduled survival of genotoxin-exposed bone marrow cells, allowing increase of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations. ny formation. The effects of hydroquinone exposure on Fko and Wt bone marrow cells were assessed by in vitro colony formation assays (Fig. 1). To assess the effect of hydroquinone exposure on hematopoietic stem cells (HSC), the KSL fractions from Fko and Wt hematopoietic bone marrow cells were isolated (Fig. 1 A ) and in vitro colony formation potential was assessed (Fig. 1 B ). Unexposed Fko and Wt bone marrow mononuclear cells showed similar BFU-E, CFU-M, CFU-G, and CFU-GM colony-forming efficiency (Fig. 1 C, bottom ). In addition, the appearance and expression of differentiation-specific markers by Fko and Wt committed hematopoietic colonies (Fig. 1 B and C, top ) did not differ significantly in the absence of hydroquinone. The number of CFU-M and CFU-G colonies formed by Wt KSL cells was reduced relative to Fko KSL cells (<0.05) but BFU-E colony numbers did not differ significantly (Fig. 1 D, top ). We next used Lin À bone marrow cells for in vitro colony formation assays (Fig. 1 D, bottom ). The results were similar to those of KSL fractions; that is, CFU-M and CFU-G colony numbers from Wt Lin À cells were reduced relative to Fko Lin À bone marrow cells, a difference not observed without hydroquinone. The results suggest that Fhit plays a role in response to hydroquinone- induced inhibition of hematopoietic colony formation potential, with Fhit deficiency allowing resistance to genotoxic stress- induced suppression of hematopoietic colony formation. Fhit-deficient bone marrow is resistant to genotoxin-induced suppression in vivo . Mononuclear cells of Fko and Wt bone marrow, with or without hydroquinone exposure, were transplanted to Wt mice previously exposed to 9.5 Gy ionizing irradiation (Fig. 2 A, top ). Hematopoietic cell counts in peripheral blood showed that neutrophils, lymphocytes, monocytes, basophils, eosinophils, and platelets increased gradually between 4 and 42 days after transplantation of both Fko and Wt cells (Supplementary Fig. S1), indicating that short-term progenitors are involved in increase and maintenance of Fko and Wt hematopoietic cells in the relatively early posttransplantation phase. Mice receiving non–hydroquinone-exposed Fko or Wt bone marrow cells all survived to 120 days after transplantation (data not shown). In sharp contrast, the recipient mice showed f 80% lethality by 120 days after transplantation of hydroquinone- exposed Wt bone marrow cells; hydroquinone-induced lethality was only 25% by day 120 in Fko bone marrow recipients (Fig. 2 A, bottom ), showing that Fhit deficiency abrogates genotoxin-induced suppression of hematopoiesis. Because the hydroquinone-treated bone marrow transplantation experiments resulted in reduced survival of recipient mice, competitive transplantation assays were performed; untreated or hydroquinone-treated Fko or Wt HSCs were mixed with congenic Ly5.1 mouse bone marrow cells, and the mixtures were transplanted into irradiated mice of Ly5.1 background. The Ly5.1 bone marrow cells would support survival of recipient mice so that the contribution of donor hydroquinone-treated HSCs could be evaluated in the transplant recipient chimeric mice (33, 34). The sorted KSL fractions from Fko and Wt mice were exposed to hydroquinone in vitro , mixed with bone marrow cells from Ly5.1 mice, and transplanted to Ly5.1 recipient mice. All the recipient mice, two receiving Wt and three receiving Fko bone marrow, survived. Assessment of hydroquinone-exposed donor cells in the Ly5.1 background showed that Fko bone marrow cells contributed to increased chimerism (Fig. 2 D, right ). Flow cytometer analysis of peripheral mononuclear cells indicated that Kit + and Sca-1 + cells were increased in mice receiving hydroquinone-exposed Fko donor marrow compared with hydroquinone-exposed Wt marrow (4.6–8.3 times increase in Kit + Fko compared with Kit + Wt; f 3.5 times increase in Sca-1 + cells; Fig. 2 C , representing two recipients of Wt and two of Fko bone marrow). Even in the absence of hydroquinone exposure, Kit + Sca-1 + cells were increased in mice receiving Fko donor marrow compared with Wt marrow (3.2 times in Kit + ; f 1.17 times in Sca-1 + cells; Fig. 2 B ). The fraction of Kit + , Sca-1 , and Lin cells play a significant role in self-renewal and multipotent differentiation of HSCs (33). A serum-free single-cell culture followed by transplantation of cultured cells into lethally irradiated mice indicated that Kit + , Sca-1 + , Lin À cells are highly enriched for murine bone marrow HSCs (34). The above results suggest that ( a ) Fhit-deficient mice maintain a larger fraction of Kit , Sca-1 cells than Wt mice, as apparent after transplantation of hydroquinone-exposed marrow, and ( b ) Fhit-deficient cells pre- serve or retain potential for self-renewal and multipotent differentiation after genotoxic stress. in Fhit-deficient marrow. Histologic assessment of bone marrow was performed by sacrificing recipient mice f 120 days after transplantation of hydroquinone-exposed nucleated cells (Fig. 3 A ). H&E staining of Wt bone marrow showed gross reduction of nucleated bone marrow cells, compatible with the preclinical stage of aplastic anemia and likely related to cause of death of recipient mice. Immunohistochemical analysis of bone marrow after transplantation of hydroquinone-exposed nucleated cells showed patchy positive staining of Bax and phospho-p38 throughout Wt bone marrow, indicating induction of apoptosis and senescence, whereas Fko bone marrow showed relatively healthy cellularity with less Bax and phosho-p38 expression (Fig. 3 A ). The assessment of apoptotic index showed efficient induction of apoptosis in transplanted bone marrow of hydroquinone-exposed Wt cells but much less apoptosis in Fko bone marrow (Fig. 3 B and C ). Assessment of DNA damage by detection of 8-OHdG in genotoxin-exposed Fko and Wt hematopoietic cells showed that 8-OHdG was reduced in Fhit-deficient bone marrow cells, indicating that reduced oxidative stress is associated with enhanced survival of hydroquinone-exposed Fko bone marrow (Fig. 4 A ). We then assessed the effect of oxidative stress on survival of HSCs in vitro . As expected, hydroquinone exposure resulted in a relatively greater reduction of Wt colonies (Fig. 4 B ). Treatment with NAC antioxidant resulted in significant recovery of colony-forming ability of Wt HSCs and, to a lesser extent, of Fko stem cells in long-term in vitro culture, suggesting that treatment with NAC or other antioxidant might prolong the survival of mice receiving hydroquinone-treated Wt bone marrow. The recovery of colony formation by NAC treatment indicates that hydroquinone-induced genotoxic stress, followed by apoptosis, is involved in suppression of colony formation by hematopoietic stem or precursor cells, a process subverted by Fhit deficiency. The data show that Wt stem cell colony-forming ability was suppressed by hydroquinone, likely due to an increase in ROS by hydroquinone treatment (35, 36), which causes DNA damage and apoptosis, whereas in the absence of Fhit ROS-induced DNA damage and apoptosis was reduced, leading to colony growth and mouse survival. This experiment accords with the recent demon- stration that Fhit interacts with the ferredoxin reductase (Fdxr) protein (37), a 54-kDa mitochondrial flavoprotein responsible for transferring electrons from NADPH to cytochrome P 450 via ferredoxin. Leakage of electrons from this shuttling system can result in generation of ROS (38). Following application of oxidative stress, the Fhit-Fdxr interaction leads to ROS generation, an early event in Fhit-triggered apoptosis. In similarly treated Fhit-deficient cells, much less ROS is generated, allowing unscheduled survival of damaged cells (37), a mechanism with relevance to early events in carcinogenesis and to refractoriness to chemotherapy of Fhit- deficient cells. To confirm that hydroquinone treatment leads to decreased ROS production in Fko cells relative to Wt cells, both MEFs and bone marrow cells were treated with 50 or 100 A mol/L of hydroquinone; ROS production was assessed by FACS analysis after staining of bone marrow–derived cells with DCFDA and by fluorescence microscopy and positive cell counting after staining of MEFs with DCFDA. The results of the FACS analysis of the sorted bone marrow cells are shown in Supplementary Fig. S2 and illustrate that, after treatment with 100 mol/L hydroquinone, the Wt hematopoietic cells showed >2-fold more ROS production than Fko hematopoietic cells. For the MEF cells, the fraction of Hoechst-positive cells that showed ROS fluorescence (examples shown in Supplementary Fig. S3 A ) were counted under confocal microscope and the quantitative estimates of ROS-positive fractions of Wt and Fko MEFs are shown in Supplementary Fig. S3 B . The results indicate that 2-fold more of the Wt MEFs than Fko MEFs produced measurable ...
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... such as p21 and p53 (8, 9), which inhibit DNA replication and apoptosis (10, 11). Recent studies indicate that p21 cooperates with Chk1 to prevent apoptosis during DNA replication fork stress, which is important for maintaining chromosome stability (11). How chromosomal aberrations, which characterize tumor cells, are induced after exposure to benzene metabolites remains to be elucidated. Common chromosome fragile sites are large regions that preferentially exhibit gaps or breaks, visible in metaphase chromosomes, when cells are exposed to replicative stress conditions and DNA synthesis is perturbed (12). FRA3B and FRA16D, at chromosome regions 3p14.2 and 16q23.3, are the two most active or most fragile of the common human fragile sites and are frequently deleted or altered in environmental carcinogen- associated cancers and in hematopoietic disorders (12, 13). Genomic sequences at fragile sites (14, 15) and protein signal pathways that monitor their stability, including Atr (16), Brca1 (17), Smc1 (18), Fanconi anemia pathway proteins (19), Chk1 (20), Hus1 (21), and Rad51 (22), have been characterized. Homology- dependent recombination (HRR) and nonhomologous end-joining (NHEJ) repair pathways regulate FRA3B and FRA16D fragile site stability, indicating that double-strand breaks are formed at common fragile sites as a result of replication perturbation (22). It is proposed that the fragile sites are so-called replication difficult zones, susceptible to delay of DNA replication, which would be monitored by DNA damage response checkpoints (23). Paradoxically, the FHIT gene product, Fhit, is involved in maintenance of genome integrity at the mid-S checkpoint through the Atr/Chk1 pathway, and Fhit deficiency alters the response to DNA-damaging agents (24–26). The introduction of exogenous Fhit into cells in vitro led to modulation of expression of checkpoint proteins Hus1 and Chk1, at mid-S checkpoint, modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cells (27). However, the biological significance of Fhit deficiency in environmental carcinogen-related hematopoietic disorders had not been investigated. Here, we report that absence of Fhit in hydroquinone-exposed mouse hematopoietic cells led to resistance to induction of cell death in vitro and escape from bone marrow suppression in transplanted mice. Immunohistochemical analyses of transplanted hydroquinone-exposed, Fhit knockout (Fko) and Wt bone marrow revealed relative absence of apoptosis and senescence markers in Fko bone marrow, in parallel with reduced detection of the oxidized base, 8-hydroxyguanosine (8-OHdG); treatment with the antioxidant N -acetyl- L -cysteine (NAC) alleviated the hydroquinone- induced suppression of colony formation by Wt hematopoietic cells. Accordingly, the long-term survival of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow–derived cells, suggesting that Fhit deficiency leads to unscheduled survival of genotoxin-exposed bone marrow cells, allowing increase of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations. ny formation. The effects of hydroquinone exposure on Fko and Wt bone marrow cells were assessed by in vitro colony formation assays (Fig. 1). To assess the effect of hydroquinone exposure on hematopoietic stem cells (HSC), the KSL fractions from Fko and Wt hematopoietic bone marrow cells were isolated (Fig. 1 A ) and in vitro colony formation potential was assessed (Fig. 1 B ). Unexposed Fko and Wt bone marrow mononuclear cells showed similar BFU-E, CFU-M, CFU-G, and CFU-GM colony-forming efficiency (Fig. 1 C, bottom ). In addition, the appearance and expression of differentiation-specific markers by Fko and Wt committed hematopoietic colonies (Fig. 1 B and C, top ) did not differ significantly in the absence of hydroquinone. The number of CFU-M and CFU-G colonies formed by Wt KSL cells was reduced relative to Fko KSL cells (<0.05) but BFU-E colony numbers did not differ significantly (Fig. 1 D, top ). We next used Lin À bone marrow cells for in vitro colony formation assays (Fig. 1 D, bottom ). The results were similar to those of KSL fractions; that is, CFU-M and CFU-G colony numbers from Wt Lin À cells were reduced relative to Fko Lin À bone marrow cells, a difference not observed without hydroquinone. The results suggest that Fhit plays a role in response to hydroquinone- induced inhibition of hematopoietic colony formation potential, with Fhit deficiency allowing resistance to genotoxic stress- induced suppression of hematopoietic colony formation. Fhit-deficient bone marrow is resistant to genotoxin-induced suppression in vivo . Mononuclear cells of Fko and Wt bone marrow, with or without hydroquinone exposure, were transplanted to Wt mice previously exposed to 9.5 Gy ionizing irradiation (Fig. 2 A, top ). Hematopoietic cell counts in peripheral blood showed that neutrophils, lymphocytes, monocytes, basophils, eosinophils, and platelets increased gradually between 4 and 42 days after transplantation of both Fko and Wt cells (Supplementary Fig. S1), indicating that short-term progenitors are involved in increase and maintenance of Fko and Wt hematopoietic cells in the relatively early posttransplantation phase. Mice receiving non–hydroquinone-exposed Fko or Wt bone marrow cells all survived to 120 days after transplantation (data not shown). In sharp contrast, the recipient mice showed f 80% lethality by 120 days after transplantation of hydroquinone- exposed Wt bone marrow cells; hydroquinone-induced lethality was only 25% by day 120 in Fko bone marrow recipients (Fig. 2 A, bottom ), showing that Fhit deficiency abrogates genotoxin-induced suppression of hematopoiesis. Because the hydroquinone-treated bone marrow transplantation experiments resulted in reduced survival of recipient mice, competitive transplantation assays were performed; untreated or hydroquinone-treated Fko or Wt HSCs were mixed with congenic Ly5.1 mouse bone marrow cells, and the mixtures were transplanted into irradiated mice of Ly5.1 background. The Ly5.1 bone marrow cells would support survival of recipient mice so that the contribution of donor hydroquinone-treated HSCs could be evaluated in the transplant recipient chimeric mice (33, 34). The sorted KSL fractions from Fko and Wt mice were exposed to hydroquinone in vitro , mixed with bone marrow cells from Ly5.1 mice, and transplanted to Ly5.1 recipient mice. All the recipient mice, two receiving Wt and three receiving Fko bone marrow, survived. Assessment of hydroquinone-exposed donor cells in the Ly5.1 background showed that Fko bone marrow cells contributed to increased chimerism (Fig. 2 D, right ). Flow cytometer analysis of peripheral mononuclear cells indicated that Kit + and Sca-1 + cells were increased in mice receiving hydroquinone-exposed Fko donor marrow compared with hydroquinone-exposed Wt marrow (4.6–8.3 times increase in Kit + Fko compared with Kit + Wt; f 3.5 times increase in Sca-1 + cells; Fig. 2 C , representing two recipients of Wt and two of Fko bone marrow). Even in the absence of hydroquinone exposure, Kit + Sca-1 + cells were increased in mice receiving Fko donor marrow compared with Wt marrow (3.2 times in Kit + ; f 1.17 times in Sca-1 + cells; Fig. 2 B ). The fraction of Kit + , Sca-1 , and Lin cells play a significant role in self-renewal and multipotent differentiation of HSCs (33). A serum-free single-cell culture followed by transplantation of cultured cells into lethally irradiated mice indicated that Kit + , Sca-1 + , Lin À cells are highly enriched for murine bone marrow HSCs (34). The above results suggest that ( a ) Fhit-deficient mice maintain a larger fraction of Kit , Sca-1 cells than Wt mice, as apparent after transplantation of hydroquinone-exposed marrow, and ( b ) Fhit-deficient cells pre- serve or retain potential for self-renewal and multipotent differentiation after genotoxic stress. in Fhit-deficient marrow. Histologic assessment of bone marrow was performed by sacrificing recipient mice f 120 days after transplantation of hydroquinone-exposed nucleated cells (Fig. 3 A ). H&E staining of Wt bone marrow showed gross reduction of nucleated bone marrow cells, compatible with the preclinical stage of aplastic anemia and likely related to cause of death of recipient mice. Immunohistochemical analysis of bone marrow after transplantation of hydroquinone-exposed nucleated cells showed patchy positive staining of Bax and phospho-p38 throughout Wt bone marrow, indicating induction of apoptosis and senescence, whereas Fko bone marrow showed relatively healthy cellularity with less Bax and phosho-p38 expression (Fig. 3 A ). The assessment of apoptotic index showed efficient induction of apoptosis in transplanted bone marrow of hydroquinone-exposed Wt cells but much less apoptosis in Fko bone marrow (Fig. 3 B and C ). Assessment of DNA damage by detection of 8-OHdG in genotoxin-exposed Fko and Wt hematopoietic cells showed that 8-OHdG was reduced in Fhit-deficient bone marrow cells, indicating that reduced oxidative stress is associated with enhanced survival of hydroquinone-exposed Fko bone marrow (Fig. 4 A ). We then assessed the effect of oxidative stress on survival of HSCs in vitro . As expected, hydroquinone exposure resulted in a relatively greater reduction of Wt colonies (Fig. 4 B ). Treatment with NAC antioxidant resulted in significant recovery of colony-forming ability of Wt HSCs and, to a lesser extent, of Fko stem cells in long-term in vitro culture, suggesting that treatment with NAC or other antioxidant might prolong the survival of mice receiving hydroquinone-treated Wt bone marrow. The recovery of colony formation by NAC treatment indicates that hydroquinone-induced genotoxic stress, followed by apoptosis, is ...
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... in environmental carcinogen- associated cancers and in hematopoietic disorders (12, 13). Genomic sequences at fragile sites (14, 15) and protein signal pathways that monitor their stability, including Atr (16), Brca1 (17), Smc1 (18), Fanconi anemia pathway proteins (19), Chk1 (20), Hus1 (21), and Rad51 (22), have been characterized. Homology- dependent recombination (HRR) and nonhomologous end-joining (NHEJ) repair pathways regulate FRA3B and FRA16D fragile site stability, indicating that double-strand breaks are formed at common fragile sites as a result of replication perturbation (22). It is proposed that the fragile sites are so-called replication difficult zones, susceptible to delay of DNA replication, which would be monitored by DNA damage response checkpoints (23). Paradoxically, the FHIT gene product, Fhit, is involved in maintenance of genome integrity at the mid-S checkpoint through the Atr/Chk1 pathway, and Fhit deficiency alters the response to DNA-damaging agents (24–26). The introduction of exogenous Fhit into cells in vitro led to modulation of expression of checkpoint proteins Hus1 and Chk1, at mid-S checkpoint, modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cells (27). However, the biological significance of Fhit deficiency in environmental carcinogen-related hematopoietic disorders had not been investigated. Here, we report that absence of Fhit in hydroquinone-exposed mouse hematopoietic cells led to resistance to induction of cell death in vitro and escape from bone marrow suppression in transplanted mice. Immunohistochemical analyses of transplanted hydroquinone-exposed, Fhit knockout (Fko) and Wt bone marrow revealed relative absence of apoptosis and senescence markers in Fko bone marrow, in parallel with reduced detection of the oxidized base, 8-hydroxyguanosine (8-OHdG); treatment with the antioxidant N -acetyl- L -cysteine (NAC) alleviated the hydroquinone- induced suppression of colony formation by Wt hematopoietic cells. Accordingly, the long-term survival of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow–derived cells, suggesting that Fhit deficiency leads to unscheduled survival of genotoxin-exposed bone marrow cells, allowing increase of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations. ny formation. The effects of hydroquinone exposure on Fko and Wt bone marrow cells were assessed by in vitro colony formation assays (Fig. 1). To assess the effect of hydroquinone exposure on hematopoietic stem cells (HSC), the KSL fractions from Fko and Wt hematopoietic bone marrow cells were isolated (Fig. 1 A ) and in vitro colony formation potential was assessed (Fig. 1 B ). Unexposed Fko and Wt bone marrow mononuclear cells showed similar BFU-E, CFU-M, CFU-G, and CFU-GM colony-forming efficiency (Fig. 1 C, bottom ). In addition, the appearance and expression of differentiation-specific markers by Fko and Wt committed hematopoietic colonies (Fig. 1 B and C, top ) did not differ significantly in the absence of hydroquinone. The number of CFU-M and CFU-G colonies formed by Wt KSL cells was reduced relative to Fko KSL cells (<0.05) but BFU-E colony numbers did not differ significantly (Fig. 1 D, top ). We next used Lin À bone marrow cells for in vitro colony formation assays (Fig. 1 D, bottom ). The results were similar to those of KSL fractions; that is, CFU-M and CFU-G colony numbers from Wt Lin À cells were reduced relative to Fko Lin À bone marrow cells, a difference not observed without hydroquinone. The results suggest that Fhit plays a role in response to hydroquinone- induced inhibition of hematopoietic colony formation potential, with Fhit deficiency allowing resistance to genotoxic stress- induced suppression of hematopoietic colony formation. Fhit-deficient bone marrow is resistant to genotoxin-induced suppression in vivo . Mononuclear cells of Fko and Wt bone marrow, with or without hydroquinone exposure, were transplanted to Wt mice previously exposed to 9.5 Gy ionizing irradiation (Fig. 2 A, top ). Hematopoietic cell counts in peripheral blood showed that neutrophils, lymphocytes, monocytes, basophils, eosinophils, and platelets increased gradually between 4 and 42 days after transplantation of both Fko and Wt cells (Supplementary Fig. S1), indicating that short-term progenitors are involved in increase and maintenance of Fko and Wt hematopoietic cells in the relatively early posttransplantation phase. Mice receiving non–hydroquinone-exposed Fko or Wt bone marrow cells all survived to 120 days after transplantation (data not shown). In sharp contrast, the recipient mice showed f 80% lethality by 120 days after transplantation of hydroquinone- exposed Wt bone marrow cells; hydroquinone-induced lethality was only 25% by day 120 in Fko bone marrow recipients (Fig. 2 A, bottom ), showing that Fhit deficiency abrogates genotoxin-induced suppression of hematopoiesis. Because the hydroquinone-treated bone marrow transplantation experiments resulted in reduced survival of recipient mice, competitive transplantation assays were performed; untreated or hydroquinone-treated Fko or Wt HSCs were mixed with congenic Ly5.1 mouse bone marrow cells, and the mixtures were transplanted into irradiated mice of Ly5.1 background. The Ly5.1 bone marrow cells would support survival of recipient mice so that the contribution of donor hydroquinone-treated HSCs could be evaluated in the transplant recipient chimeric mice (33, 34). The sorted KSL fractions from Fko and Wt mice were exposed to hydroquinone in vitro , mixed with bone marrow cells from Ly5.1 mice, and transplanted to Ly5.1 recipient mice. All the recipient mice, two receiving Wt and three receiving Fko bone marrow, survived. Assessment of hydroquinone-exposed donor cells in the Ly5.1 background showed that Fko bone marrow cells contributed to increased chimerism (Fig. 2 D, right ). Flow cytometer analysis of peripheral mononuclear cells indicated that Kit + and Sca-1 + cells were increased in mice receiving hydroquinone-exposed Fko donor marrow compared with hydroquinone-exposed Wt marrow (4.6–8.3 times increase in Kit + Fko compared with Kit + Wt; f 3.5 times increase in Sca-1 + cells; Fig. 2 C , representing two recipients of Wt and two of Fko bone marrow). Even in the absence of hydroquinone exposure, Kit + Sca-1 + cells were increased in mice receiving Fko donor marrow compared with Wt marrow (3.2 times in Kit + ; f 1.17 times in Sca-1 + cells; Fig. 2 B ). The fraction of Kit + , Sca-1 , and Lin cells play a significant role in self-renewal and multipotent differentiation of HSCs (33). A serum-free single-cell culture followed by transplantation of cultured cells into lethally irradiated mice indicated that Kit + , Sca-1 + , Lin À cells are highly enriched for murine bone marrow HSCs (34). The above results suggest that ( a ) Fhit-deficient mice maintain a larger fraction of Kit , Sca-1 cells than Wt mice, as apparent after transplantation of hydroquinone-exposed marrow, and ( b ) Fhit-deficient cells pre- serve or retain potential for self-renewal and multipotent differentiation after genotoxic stress. in Fhit-deficient marrow. Histologic assessment of bone marrow was performed by sacrificing recipient mice f 120 days after transplantation of hydroquinone-exposed nucleated cells (Fig. 3 A ). H&E staining of Wt bone marrow showed gross reduction of nucleated bone marrow cells, compatible with the preclinical stage of aplastic anemia and likely related to cause of death of recipient mice. Immunohistochemical analysis of bone marrow after transplantation of hydroquinone-exposed nucleated cells showed patchy positive staining of Bax and phospho-p38 throughout Wt bone marrow, indicating induction of apoptosis and senescence, whereas Fko bone marrow showed relatively healthy cellularity with less Bax and phosho-p38 expression (Fig. 3 A ). The assessment of apoptotic index showed efficient induction of apoptosis in transplanted bone marrow of hydroquinone-exposed Wt cells but much less apoptosis in Fko bone marrow (Fig. 3 B and C ). Assessment of DNA damage by detection of 8-OHdG in genotoxin-exposed Fko and Wt hematopoietic cells showed that 8-OHdG was reduced in Fhit-deficient bone marrow cells, indicating that reduced oxidative stress is associated with enhanced survival of hydroquinone-exposed Fko bone marrow (Fig. 4 A ). We then assessed the effect of oxidative stress on survival of HSCs in vitro . As expected, hydroquinone exposure resulted in a relatively greater reduction of Wt colonies (Fig. 4 B ). Treatment with NAC antioxidant resulted in significant recovery of colony-forming ability of Wt HSCs and, to a lesser extent, of Fko stem cells in long-term in vitro culture, suggesting that treatment with NAC or other antioxidant might prolong the survival of mice receiving hydroquinone-treated Wt bone marrow. The recovery of colony formation by NAC treatment indicates that hydroquinone-induced genotoxic stress, followed by apoptosis, is involved in suppression of colony formation by hematopoietic stem or precursor cells, a process subverted by Fhit deficiency. The data show that Wt stem cell colony-forming ability was suppressed by hydroquinone, likely due to an increase in ROS by hydroquinone treatment (35, 36), which causes DNA damage and apoptosis, whereas in the absence of Fhit ROS-induced DNA damage and apoptosis was reduced, leading to colony growth and mouse survival. This experiment accords with the recent demon- stration that Fhit interacts with the ferredoxin reductase (Fdxr) protein (37), a 54-kDa mitochondrial flavoprotein responsible for transferring electrons from NADPH to cytochrome P 450 via ferredoxin. Leakage of electrons from this shuttling system can result in generation of ROS (38). Following ...
Context 5
... Fhit knockout (Fko) and Wt bone marrow revealed relative absence of apoptosis and senescence markers in Fko bone marrow, in parallel with reduced detection of the oxidized base, 8-hydroxyguanosine (8-OHdG); treatment with the antioxidant N -acetyl- L -cysteine (NAC) alleviated the hydroquinone- induced suppression of colony formation by Wt hematopoietic cells. Accordingly, the long-term survival of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow–derived cells, suggesting that Fhit deficiency leads to unscheduled survival of genotoxin-exposed bone marrow cells, allowing increase of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations. ny formation. The effects of hydroquinone exposure on Fko and Wt bone marrow cells were assessed by in vitro colony formation assays (Fig. 1). To assess the effect of hydroquinone exposure on hematopoietic stem cells (HSC), the KSL fractions from Fko and Wt hematopoietic bone marrow cells were isolated (Fig. 1 A ) and in vitro colony formation potential was assessed (Fig. 1 B ). Unexposed Fko and Wt bone marrow mononuclear cells showed similar BFU-E, CFU-M, CFU-G, and CFU-GM colony-forming efficiency (Fig. 1 C, bottom ). In addition, the appearance and expression of differentiation-specific markers by Fko and Wt committed hematopoietic colonies (Fig. 1 B and C, top ) did not differ significantly in the absence of hydroquinone. The number of CFU-M and CFU-G colonies formed by Wt KSL cells was reduced relative to Fko KSL cells (<0.05) but BFU-E colony numbers did not differ significantly (Fig. 1 D, top ). We next used Lin À bone marrow cells for in vitro colony formation assays (Fig. 1 D, bottom ). The results were similar to those of KSL fractions; that is, CFU-M and CFU-G colony numbers from Wt Lin À cells were reduced relative to Fko Lin À bone marrow cells, a difference not observed without hydroquinone. The results suggest that Fhit plays a role in response to hydroquinone- induced inhibition of hematopoietic colony formation potential, with Fhit deficiency allowing resistance to genotoxic stress- induced suppression of hematopoietic colony formation. Fhit-deficient bone marrow is resistant to genotoxin-induced suppression in vivo . Mononuclear cells of Fko and Wt bone marrow, with or without hydroquinone exposure, were transplanted to Wt mice previously exposed to 9.5 Gy ionizing irradiation (Fig. 2 A, top ). Hematopoietic cell counts in peripheral blood showed that neutrophils, lymphocytes, monocytes, basophils, eosinophils, and platelets increased gradually between 4 and 42 days after transplantation of both Fko and Wt cells (Supplementary Fig. S1), indicating that short-term progenitors are involved in increase and maintenance of Fko and Wt hematopoietic cells in the relatively early posttransplantation phase. Mice receiving non–hydroquinone-exposed Fko or Wt bone marrow cells all survived to 120 days after transplantation (data not shown). In sharp contrast, the recipient mice showed f 80% lethality by 120 days after transplantation of hydroquinone- exposed Wt bone marrow cells; hydroquinone-induced lethality was only 25% by day 120 in Fko bone marrow recipients (Fig. 2 A, bottom ), showing that Fhit deficiency abrogates genotoxin-induced suppression of hematopoiesis. Because the hydroquinone-treated bone marrow transplantation experiments resulted in reduced survival of recipient mice, competitive transplantation assays were performed; untreated or hydroquinone-treated Fko or Wt HSCs were mixed with congenic Ly5.1 mouse bone marrow cells, and the mixtures were transplanted into irradiated mice of Ly5.1 background. The Ly5.1 bone marrow cells would support survival of recipient mice so that the contribution of donor hydroquinone-treated HSCs could be evaluated in the transplant recipient chimeric mice (33, 34). The sorted KSL fractions from Fko and Wt mice were exposed to hydroquinone in vitro , mixed with bone marrow cells from Ly5.1 mice, and transplanted to Ly5.1 recipient mice. All the recipient mice, two receiving Wt and three receiving Fko bone marrow, survived. Assessment of hydroquinone-exposed donor cells in the Ly5.1 background showed that Fko bone marrow cells contributed to increased chimerism (Fig. 2 D, right ). Flow cytometer analysis of peripheral mononuclear cells indicated that Kit + and Sca-1 + cells were increased in mice receiving hydroquinone-exposed Fko donor marrow compared with hydroquinone-exposed Wt marrow (4.6–8.3 times increase in Kit + Fko compared with Kit + Wt; f 3.5 times increase in Sca-1 + cells; Fig. 2 C , representing two recipients of Wt and two of Fko bone marrow). Even in the absence of hydroquinone exposure, Kit + Sca-1 + cells were increased in mice receiving Fko donor marrow compared with Wt marrow (3.2 times in Kit + ; f 1.17 times in Sca-1 + cells; Fig. 2 B ). The fraction of Kit + , Sca-1 , and Lin cells play a significant role in self-renewal and multipotent differentiation of HSCs (33). A serum-free single-cell culture followed by transplantation of cultured cells into lethally irradiated mice indicated that Kit + , Sca-1 + , Lin À cells are highly enriched for murine bone marrow HSCs (34). The above results suggest that ( a ) Fhit-deficient mice maintain a larger fraction of Kit , Sca-1 cells than Wt mice, as apparent after transplantation of hydroquinone-exposed marrow, and ( b ) Fhit-deficient cells pre- serve or retain potential for self-renewal and multipotent differentiation after genotoxic stress. in Fhit-deficient marrow. Histologic assessment of bone marrow was performed by sacrificing recipient mice f 120 days after transplantation of hydroquinone-exposed nucleated cells (Fig. 3 A ). H&E staining of Wt bone marrow showed gross reduction of nucleated bone marrow cells, compatible with the preclinical stage of aplastic anemia and likely related to cause of death of recipient mice. Immunohistochemical analysis of bone marrow after transplantation of hydroquinone-exposed nucleated cells showed patchy positive staining of Bax and phospho-p38 throughout Wt bone marrow, indicating induction of apoptosis and senescence, whereas Fko bone marrow showed relatively healthy cellularity with less Bax and phosho-p38 expression (Fig. 3 A ). The assessment of apoptotic index showed efficient induction of apoptosis in transplanted bone marrow of hydroquinone-exposed Wt cells but much less apoptosis in Fko bone marrow (Fig. 3 B and C ). Assessment of DNA damage by detection of 8-OHdG in genotoxin-exposed Fko and Wt hematopoietic cells showed that 8-OHdG was reduced in Fhit-deficient bone marrow cells, indicating that reduced oxidative stress is associated with enhanced survival of hydroquinone-exposed Fko bone marrow (Fig. 4 A ). We then assessed the effect of oxidative stress on survival of HSCs in vitro . As expected, hydroquinone exposure resulted in a relatively greater reduction of Wt colonies (Fig. 4 B ). Treatment with NAC antioxidant resulted in significant recovery of colony-forming ability of Wt HSCs and, to a lesser extent, of Fko stem cells in long-term in vitro culture, suggesting that treatment with NAC or other antioxidant might prolong the survival of mice receiving hydroquinone-treated Wt bone marrow. The recovery of colony formation by NAC treatment indicates that hydroquinone-induced genotoxic stress, followed by apoptosis, is involved in suppression of colony formation by hematopoietic stem or precursor cells, a process subverted by Fhit deficiency. The data show that Wt stem cell colony-forming ability was suppressed by hydroquinone, likely due to an increase in ROS by hydroquinone treatment (35, 36), which causes DNA damage and apoptosis, whereas in the absence of Fhit ROS-induced DNA damage and apoptosis was reduced, leading to colony growth and mouse survival. This experiment accords with the recent demon- stration that Fhit interacts with the ferredoxin reductase (Fdxr) protein (37), a 54-kDa mitochondrial flavoprotein responsible for transferring electrons from NADPH to cytochrome P 450 via ferredoxin. Leakage of electrons from this shuttling system can result in generation of ROS (38). Following application of oxidative stress, the Fhit-Fdxr interaction leads to ROS generation, an early event in Fhit-triggered apoptosis. In similarly treated Fhit-deficient cells, much less ROS is generated, allowing unscheduled survival of damaged cells (37), a mechanism with relevance to early events in carcinogenesis and to refractoriness to chemotherapy of Fhit- deficient cells. To confirm that hydroquinone treatment leads to decreased ROS production in Fko cells relative to Wt cells, both MEFs and bone marrow cells were treated with 50 or 100 A mol/L of hydroquinone; ROS production was assessed by FACS analysis after staining of bone marrow–derived cells with DCFDA and by fluorescence microscopy and positive cell counting after staining of MEFs with DCFDA. The results of the FACS analysis of the sorted bone marrow cells are shown in Supplementary Fig. S2 and illustrate that, after treatment with 100 mol/L hydroquinone, the Wt hematopoietic cells showed >2-fold more ROS production than Fko hematopoietic cells. For the MEF cells, the fraction of Hoechst-positive cells that showed ROS fluorescence (examples shown in Supplementary Fig. S3 A ) were counted under confocal microscope and the quantitative estimates of ROS-positive fractions of Wt and Fko MEFs are shown in Supplementary Fig. S3 B . The results indicate that 2-fold more of the Wt MEFs than Fko MEFs produced measurable ROS after 50 A mol/L hydroquinone ( P V 0.022) and 100 A mol/L hydroquinone ( P V 0.001) treatment. Repair pathways in Fhit-deficient bone marrow cells. Recent studies have indicated that Fhit is involved in maintenance of genome integrity at the mid-S checkpoint and ...
Citations
... For example, FHIT-deficient bone marrow cells (BMC) have lower levels of ROS after hydroquinone challenge. A reduction in intracellular levels of ROS are detected in FHIT negative BMCs relative to wild-type, and this reduction in ROS accumulation is linked to failure to induce apoptosis as the phenotype is reversed with application of the antioxidant N-acetyl-L-cysteine. 1,5,[7][8][9]69 Several studies that connect Fhit expression to increased sensitivity to anti-cancer drugs may also serve to substantiate the link between Fhit function and oxidative stress. Re-expression of Fhit in Fhit-negative cancerderived cell lines increases sensitivity to mitomycin C (MMC), camptothecin (CPT), and cisplatin. ...
Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.
... Examination of cells that have lost FHIT revealed that the protein has some functional roles in response to DNA damage (Saldivar et al., 2010). In particular, kidney epithelial cells established from Fhit-/-mice exhibited >2-fold increased chromosome breaks at fragile sites vs. corresponding Fhit+/+ cells (Turner et al., 2002), and the frequency of mutations following replicative and oxidative stress in Fhit-deficient cells was 2 to 5-fold greater than in Fhit-expressing cells (Ishii et al., 2008;Ottey et al., 2004). Despite these findings and strong evidence that Fhit acts as a tumor suppressor (Joannes et al., 2010;Pekarsky et al., 1998;Siprashvili et al., 1997) it has been proposed that deletions within the FHIT locus are secondary alterations rather than cancer-driving mutations (Bignell et al., 2010). ...
Keywords: CFSs, common fragile sites, aphidicolin, genomic instability, FRA3B, FRA16D, CFS tumor suppressor genes
... In this context, HQ has been studied in vitro in hematopoietic cells and lymphocytes . HQ was found to induce genotoxicity such as DNA damages mediated by oxidative stress in these cell models (Andreoli et al. 1999; Ishii et al. 2008; Peng et al. 2012; Wan and Winn 2007). Similar findings were also found in other cell models such as V79 cells (Silva et al. 2003) and Chinese hamster ovary (CHO) cells (Winn 2003). ...
Hydroquinone (HQ) is found in natural and anthropogenic sources including food, cosmetics, cigarette smoke, and industrial products. In addition to ingestion and dermal absorption, human exposure to HQ may also occur by inhaling cigarette smoke or polluted air. The adverse effects of HQ on respiratory systems have been studied, but genotoxicity HQ on human lung cells is unclear. The aim of this study was to investigate the cytotoxicity and genotoxicity of HQ in human lung alveolar epithelial cells (A549). We found that HQ induced a dose response in cell growth inhibition and DNA damage which was associated with an increase in oxidative stress. Cytotoxicity results demonstrated that HQ was most toxic after 24 h (LC50 = 33 μM) and less toxic after 1 h exposure (LC50 = 59 μM). Genotoxicity of HQ was measured using the Comet assay, H2AX phosphorylation, and chromosome aberration formation. Results from the comet assay revealed that DNA damage was highest during the earlier hours of exposure (1 and 6 h) and thereafter was reduced. A similar pattern was observed for H2AX phosphorylation suggesting that damage DNA may be repaired in later exposure hours. An increase in chromosomal aberration corresponded with maximal DNA damage which further confirmed the genotoxic effects of HQ. To investigate whether oxidative stress was involved in the cytotoxic and genotoxic effects of HQ, cellular glutathione and 8-Oxo-deoguanisone (8-Oxo-dG) formation were measured. A decrease in the reduced glutathione (GSH) and an increase oxidized glutathione (GSSG) was observed during the early hours of exposure which corresponded with elevated 8-Oxo-dG adducts. Together these results demonstrate that HQ exerts its cytotoxic and genotoxic effects in A549 lung cells, probably through DNA damage via oxidative stress.
... Paradoxically, examination of cells that have lost the FHIT gene product has revealed that Fhit protein has functional roles in response to DNA damage [13]: 1) kidney epithelial cells established from Fhit 2/2 mice exhibited .2fold increased chromosome breaks at fragile sites vs corresponding Fhit +/+ kidney cells [14]; 2) the frequency of mutations following replicative or oxidative stress in Fhit-deficient transformed and cancer cells was 2 to 5-fold greater than in Fhit-expressing cells [15,16]. Despite these findings and strong evidence that Fhit exerts tumor suppressor activity [17,18], it has been argued that deletions within the FHIT locus in transformed cells are passenger alterations rather than cancer-driving mutations [19]. ...
Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.
... Interestingly, the DNA damage-susceptible FRA3B/ FHIT chromosome fragile site encodes a protein required for protecting cells from accumulated DNA damage through its ability to modulate the checkpoint proteins Atr and Chk1, whereas inactivation of Fhit contributes to the accumulation of abnormal checkpoint phenotypes during cancer development (61)(62)(63). The absence of Fhit protein in stem cells, a reduction in oxidative stress, and efficient but not error-free DNA damage repair, facilitate the unscheduled long-term survival of genotoxin-exposed Fhit-deficient hematopoietic stem cells that carry deleterious mutations (64). Thus, the small noncoding RNAs are nucleotides that can affect cancer risk. ...
Epigenetic modifications play crucial roles in cancer initiation and development. Complete reprogramming can be achieved through the introduction of defined biological factors such as Oct4, Sox2, Klf4, and cMyc into mouse and human fibroblasts. Introduction of these transcription factors resulted in the modification of malignant phenotype behavior. Recent studies have shown that human and mouse somatic cells can be reprogrammed to become induced pluripotent stem cells using forced expression of microRNAs, which completely eliminates the need for ectopic protein expression. Considering the usefulness of RNA molecules, microRNA-based reprogramming technology may have future applications in regenerative and cancer medicine.
... FRA3B, the most active or most fragile of the human common fragile sites, is frequently altered in environmental carcinogen-associated cancers and in hematopoietic disorders [23,44]. We reported that absence of FHIT in mouse hematopoietic cells exposed to hydroquinone, a genotoxic benzene metabolite, led to resistance to induction of cell death in vitro and escape from bone marrow suppression in transplanted mice [45]. Immunohistochemical analyses of transplanted hydroquinone-exposed, FHIT −/− , and FHIT +/+ bone marrow revealed absence of apoptosis and senescence markers in KO bone marrow. ...
... Immunohistochemical analyses of transplanted hydroquinone-exposed, FHIT −/− , and FHIT +/+ bone marrow revealed absence of apoptosis and senescence markers in KO bone marrow. Accordingly, the long-term survival of hydroquinone-exposed FHIT-deficient bone marrow-transplanted mice allowed accumulation of inaccurately repaired DNA lesions and premalignant alterations in bone marrow-derived cells, suggesting that FHIT deficiency leads to unscheduled survival of genotoxinexposed bone marrow cells, and increases the population of stem or precursor cells with damaged genomes and resultant accumulation of genomic alterations [45]. ...
Chromosomal common fragile sites (CFSs) are specific mammalian genomic regions that show an increased frequency of gaps and breaks when cells are exposed to replication stress in vitro. CFSs are also consistently involved in chromosomal abnormalities in vivo related to cancer. Interestingly, several CFSs contain one or more tumor suppressor genes whose structure and function are often affected by chromosomal fragility. The two most active fragile sites in the human genome are FRA3B and FRA16D where the tumor suppressor genes FHIT and WWOX are located, respectively. The best approach to study tumorigenic effects of altered tumor suppressors located at CFSs in vivo is to generate mouse models in which these genes are inactivated. This paper summarizes our present knowledge on mouse models of cancer generated by knocking out tumor suppressors of CFS.
... TN cancers do show evidence of more genomic alterations than other breast cancer subtypes [38]. It has even been suggested that reduced expression of BRCA1 and Fhit may be hallmarks of stem cells [37,39]. The persistent expression of DDR-associated proteins observed in this study in TN cancers probably has an important role in development of these cancers. ...
Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread.
... In this regard, we recently disclosed that FHIT protein is involved in surveillance of genome integrity and checkpoint response after genotoxin exposure. 12 Tissues of genotoxinexposed FHIT-deficient bone marrow-transplanted mice exhibited preneoplastic alterations, including accumulation of DNA damage. Those carcinogenic stimuli allowed longterm survival of genotoxin-exposed FHIT-deficient hematopoietic stem cells with deleterious mutations. ...
We focused on the mechanism by which FHIT suppresses neoplastic transformation in normal but damaged esophageal epithelial cells exposed to inflammatory stimuli in vivo and to chemo-radiotherapy in clinical samples. For in vitro analysis, Adenoviral-FHIT (Ad-FHIT) in TE4 and TE2 were used for microarray analysis. For in vivo analysis, wild-type (WT) FHIT and FHIT-deficient (KO) C57BL/6 mice were exposed to N-nitrosomethylbenzylamine (NMBA) and to a cyclooxygenase-2 inhibitor (COXI). Considering DNA damage on clinical samples, expressions of FHIT, BAX and PCNA were evaluated by comparing between 3 cases of esophageal cancer cases of the chemo-radiotherapy responder and 7 cases of the non-responder. In in vitro analysis, we listed the down-regulated genes in Ad-FHIT that significantly control Lac-Z infected cells, such as prostaglandin E receptor 4, cyclooxygenase-1 and cyclooxygenase-2. In in vivo analysis, FHIT-KO mice were much more susceptible to tumorigenesis than were FHIT-WT mice. A significant difference in PGE2 activation was observed between FHIT-WT mice (5.2 ng/mL) and FHIT-KO mice (28.4 ng/mL) after exposure to NMBA in the absence of COXI as determined by ELISA assay (P less than 0.01). BAX expression was significantly higher in FHIT-WT (1.0±0.43) than in FHIT-KO (0.17±0.17) (P less than 0.05). The IHC score for FHIT and BAX expression was significantly higher in responders than the others (P less than 0.05). FHIT possesses tumor suppressor activity by induction of apoptosis in damaged cells after exposure to inflammatory carcinogens and DNA damaging chemo-radiotherapy.
... In this regard, we recently disclosed that FHIT protein is involved in surveillance of genome integrity and checkpoint response after genotoxin exposure. 12 Tissues of genotoxinexposed FHIT-deficient bone marrow-transplanted mice exhibited preneoplastic alterations, including accumulation of DNA damage. Those carcinogenic stimuli allowed longterm survival of genotoxin-exposed FHIT-deficient hematopoietic stem cells with deleterious mutations. ...
We focused on the mechanism by which FHIT suppresses neoplastic transformation in normal but damaged esophageal epithelial cells exposed to inflammatory stimuli in vivo and to chemo-radiotherapy in clinical samples. For in vitro analysis, Adenoviral-FHIT (Ad-FHIT) in TE4 and TE2 were used for microarray analysis. For in vivo analysis, wild-type (WT) FHIT and FHIT-deficient (KO) C57BL/6 mice were exposed to N-nitrosomethylbenzylamine (NMBA) and to a cyclooxygenase-2 inhibitor (COXI). Considering DNA damage on clinical samples, expressions of FHIT, BAX and PCNA were evaluated by comparing between 3 cases of esophageal cancer cases of the chemo-radiotherapy responder and 7 cases of the non-responder. In in vitro analysis, we listed the down-regulated genes in Ad-FHIT that significantly control Lac-Z infected cells, such as prostaglandin E receptor 4, cyclooxygenase-1 and cyclooxygenase-2. In in vivo analysis, FHIT-KO mice were much more susceptible to tumorigenesis than were FHIT-WT mice. A significant difference in PGE2 activation was observed between FHIT-WT mice (5.2 ng/mL) and FHIT-KO mice (28.4 ng/mL) after exposure to NMBA in the absence of COXI as determined by ELISA assay (P less than 0.01). BAX expression was significantly higher in FHIT-WT (1.0±0.43) than in FHIT-KO (0.17±0.17) (P less than 0.05). The IHC score for FHIT and BAX expression was significantly higher in responders than the others (P less than 0.05). FHIT possesses tumor suppressor activity by induction of apoptosis in damaged cells after exposure to inflammatory carcinogens and DNA damaging chemo-radiotherapy.
... We have reported previously that Fhit is involved in the DNA damage response through modulation of checkpoint proteins ( Ishii et al., 2008;Okumura et al., in press). To determine if Nit1 may also have a role in responses to DNA damage, we examined the expression of γH2AX and pChk2 in wt, Fhit −/− and DKO non-neoplastic tissues of treated and non NMBA-treated mice; pChk2 and γH2AX were expressed in NMBA-treated Fhit −/− , nontreated and treated DKO forestomach tissues, but not in untreated Fhit −/− tissue (Fig. 5A), suggesting that the untreated DKO tissues express an activated checkpoint in the nonneoplastic DKO forestomach tissue, consistent with a preneoplastic condition ( Bartkova et al., 2005;Gorgoulis et al., 2005). ...
The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit(-/-)Nit1(-/-) mice and observed that double knockout mice develop more spontaneous and carcinogen-induced tumors than Fhit(-/-) mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney-derived double-deficient cells appear not to activate the pChk2 pathway and when treated with H(2)O(2), show little evidence of DNA damage, compared with wild type and Fhit(-/-) cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas.
