Nucleotide Excision Repair Is Not Required for the Antiapoptotic Function of Insulin-like Growth Factor 1

The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1's ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repair-deficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1's antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response.     

Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.     

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4 in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.     

STAT3 but Not STAT1 Is Required for Astrocyte Differentiation

The JAK-STAT signaling pathway has been implicated in astrocyte differentiation. Both STAT1 and STAT3 are expressed in the central nervous system and are thought to be important for glial differentiation, as mainly demonstrated in vitro; however direct in vivo evidence is missing. We investigated whether STAT1 and STAT3 are essential for astrocyte development by testing the STAT responsiveness of astrocyte progenitors. STAT3 was absent in the ventricular zone where glial progenitors are born but begins to appear at the marginal zone at E16.5. At E18.5, both phospho-STAT1 and phospho-STAT3 were present in glial fibrillary acidic protein (GFAP)-expressing white matter astrocytes. Overexpression of STAT3 by electroporation of chicks in ovo induced increased numbers of astrocyte progenitors in the spinal cord. Likewise, elimination of STAT3 in Stat3 conditional knockout (cKO) mice resulted in depletion of white matter astrocytes. Interestingly, elimination of STAT1 in Stat1 null mice did not inhibit astrocyte differentiation and deletion of Stat1 failed to aggravate the glial defects in Stat3 cKO mice. Measuring the activity of STAT binding elements and the gfap promoter in the presence of various STAT mutants revealed that transactivation depended on the activity of STAT3 not STAT1. No synergistic interaction between STAT1 and STAT3 was observed. Cortical progenitors of Stat1 null; Stat3 cKO mice generated astrocytes when STAT3 or the splice variant Stat3β was supplied, but not when STAT1 was introduced. Together, our results suggest that STAT3 is necessary and sufficient for astrocyte differentiation whereas STAT1 is dispensable.     

Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.     

c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

The c-Jun NH₂-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1^−/− Jnk2^−/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.The c-Jun NH₂-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. JNK is encoded by two ubiquitously expressed genes (Jnk1 and Jnk2) and by a third gene (Jnk3) that is selectively expressed in neurons (14). Gene disruption studies demonstrate that mice without Jnk1 or Jnk2 are viable, but compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality (14). Murine embryonic fibroblasts (MEFs) isolated from Jnk1^−/− Jnk2^−/− mice exhibit a severe growth retardation phenotype (54). The markedly reduced growth of Jnk1^−/− Jnk2^−/− MEFs is consistent with the finding that JNK is critically required for the regulation of AP1-dependent gene expression (56) that is implicated in cellular proliferation (26). Thus, Jnk1^−/− Jnk2^−/− MEFs express low levels of AP1 proteins (e.g., c-Jun and JunD) and exhibit marked defects in AP1 target gene expression (34, 56). This loss of AP1 function is mediated, in part, by reduced phosphorylation of the activation domain of Jun family proteins and ATF2 (56).More recent studies using a conditional gene ablation strategy have demonstrated that compound JNK deficiency causes rapid senescence (12). This conclusion was confirmed by using chemical genetic analysis with MEFs isolated from mice with a germ line mutation that sensitizes JNK to inhibition by a predesigned small-molecule drug (12, 25). This form of senescence was found to be p53 dependent (12) and resembles the p53-dependent senescence of c-Jun^−/− MEFs (49). These data indicate that JNK plays a critical role in cellular proliferation. Indeed, it is possible that the p53-dependent senescence observed in JNK-deficient cells may contribute to aging. This is because altered p53 function is established to be an important determinant of early aging (36, 55). Importantly, this role of p53 in aging appears to be distinct from p53-mediated tumor suppression and DNA damage responses (21, 39, 43).One aspect of the aging process is a reduction in the regenerative capacity of stem cells (50). Indeed, it has been established that altered p53 activity associated with aging causes decreased stem cell function (8, 18, 42) and that disruption of the p53 pathway can increase stem cell function (1). Since JNK can influence p53-dependent senescence (12), these data indicate that JNK may be important for stem cell proliferation and self-renewal potential.Embryonic stem (ES) cells proliferate and are capable of both self-renewal and differentiation to multiple cell types. Indeed, murine ES cells can differentiate to create all tissues within a mouse. The profound growth retardation and rapid p53-dependent senescence of Jnk1^−/− Jnk2^−/− MEFs (12) suggests that JNK may play a critical role in the normal function of ES cells, including self-renewal and differentiation potential. The purpose of the present study was to test this hypothesis. Our approach was to isolate ES cells from wild-type and JNK-deficient mice. We demonstrate that JNK is not required for self-renewal or the proliferation of ES cells. However, JNK is required for ES cell differentiation.     

Nucleotide Excision Repair Is a Predominant Mechanism for Processing Nitrofurazone-Induced DNA Damage in Escherichia coli

Nitrofurazone is reduced by cellular nitroreductases to form N²-deoxyguanine (N²-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N²-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.Replication in the presence of DNA damage is thought to produce most of the mutagenesis, genomic rearrangements, and lethality that occur in all cells. UV-induced photoproducts, X-ray-induced strand breaks, psoralen- or cis-platin-interstrand cross-links, oxidized bases from reactive oxygen species, and base depurination are just a few of the structurally distinct challenges that the replication machinery must overcome. It seems likely that the mechanisms that process these lesions will vary depending on the nature of the impediment.While a number of the lesions described above are known to block replication, the events associated with UV-induced damage have been the most extensively characterized. UV irradiation causes the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts in DNA that block the progression of the replication fork (16, 29, 30, 37). Following the arrest of replication at UV-induced damage, RecA and several RecF pathway proteins are required to process the replication fork such that the blocking lesion is removed or bypassed (2, 5, 6, 8-10). Cells lacking either RecA or any of several RecF pathway proteins are hypersensitive to UV-induced damage and fail to recover replication following disruption by the lesions (2, 6, 10). RecBCD is an exonuclease/helicase complex that is involved in repairing double-strand breaks (38). It also is required for resistance to UV-induced damage, although it is not required to process or restore disrupted replication forks, and the substrates it acts upon after UV irradiation currently remain unclear (3, 10, 19).Survival and the ability to resume DNA synthesis following UV-induced damage depend predominantly on the removal of the lesions by nucleotide excision repair (5, 7, 36). Cells deficient in nucleotide excision repair are unable to remove UV-induced DNA lesions and exhibit elevated levels of mutagenesis, strand exchanges, rearrangements, and cell lethality (16, 33, 34). In cases where replication fork processing or lesion repair is prevented, the recovery of replication and survival become entirely dependent on translesion synthesis by DNA polymerase V (Pol V) (6). However, in repair-proficient cells, the contribution of translesion synthesis to recovery and survival is minor and is detected only following UV doses that exceed the repair capacity of the cell (5, 6).Less is known about how replication recovers from other forms of DNA damage. We chose to characterize nitrofurazone, because a number of studies suggested that N²-deoxyguanine (N²-dG) adducts induced by this and other agents would be processed differently than UV-induced lesions. Nitrofurazone is a topical antibacterial agent that historically has been used for treating burns and skin grafts in patients and animals (14, 15, 32). Nitrofurazone toxicity is known to require activation by cellular nitroreductases (25, 42). However, the mechanism and targets of its antimicrobial properties have yet to be fully elucidated. In addition to its antimicrobial properties, the reduced nitrofurazone metabolites also target DNA and have been shown to induce free radical damage, strand breaks, and N²-dG adducts (26, 40, 42, 45), and they are mutagenic and carcinogenic in rodent models (1, 15, 24, 39).Whereas nucleotide excision repair is the predominant mechanism required for survival after UV-induced damage, a number of studies suggest that translesion synthesis plays a larger role in survival after nitrofurazone-induced DNA damage. dinB mutants lacking Pol IV were shown to be hypersensitive to nitrofurazone compared to cells that constitutively express the polymerase (17). Biochemically, Pol IV and a number of Pol IV homologs from other organisms have been shown to efficiently replicate over a range of N²-dG adducts in vitro (17, 35, 44). In addition, several studies have reported that uvrA mutants, which are defective in nucleotide excision repair, do not exhibit any hypersensitivity to nitrofurazone or other agents that induce similar adducts in vivo (12, 21, 27). Early studies also observed a direct correlation between nitrofurazone-induced mutations and lethality, suggesting that mutagenic lesions persist in the DNA to cause toxicity (21, 23, 27, 43). Consistent with these observations, nitrofuran-induced lesions were found to be poor substrates for nucleotide excision repair in vitro (46).Taken together, these observations suggest to us that the cellular response to nitrofurazone will be distinct from its response to UV irradiation. However, no study has examined the relative contributions that nucleotide excision repair, translesion synthesis, or recombination has in recovering from nitrofurazone-induced damage. In this study, we characterized the mechanism by which nitrofurazone inhibits DNA replication and identified the genes that contribute to the recovery, survival, and mutagenesis of Escherichia coli treated with nitrofurazone. In contrast to previous studies, we found that survival following nitrofurazone-induced damage depends predominantly on nucleotide excision repair. Similarly to UV-induced DNA damage, both the RecF and RecBC pathways contribute to survival following nitrofurazone-induced DNA damage. The contribution of translesion polymerases to survival was minor and was mediated by Pol IV. In addition, we found that nitrofurazone can act to inhibit DNA replication directly when used at higher concentrations. The direct inhibition of replication is reversible and occurs independently of DNA damage, suggesting that DNA is not the primary target of its antimicrobial properties.     

MicroRNA-223 Regulates Granulopoiesis but Is Not Required for HSC Maintenance in Mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.     

Increased Tyrosine Kinase Activity but Not Calcium Mobilization Is Required for Ceramide-Induced Apoptosis

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.     

Dynein-2 Affects the Regulation of Ciliary Length but Is Not Required for Ciliogenesis in Tetrahymena thermophila

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.     

Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1^−/− lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP^Sc detection, we identified PrP^Sc deposits associated with HEVs in TNFR1^−/− lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.