Induction of fusion-competent myoblast-specific gene expression during myogenic differentiation of Drosophila Schneider cells by DNA double-strand breaks or replication inhibition

Differentiation of Drosophila Schneider cells caused by DNA double-strand break (DSB)-inducing topoisomerase II (topo II) inhibitors were attenuated by ICRF-193, a non-DNA-damaging topo II inhibitor. ICRF-193 did not inhibit differentiation induced by neocarzinostatin (NCS), a drug that causes DNA DSBs independent of topo II. Schneider cells differentiated upon treatment with gamma-ray. These results suggest that DNA DSBs induce myogenic differentiation of Schneider cells. We also found DNA replication inhibitors, hydroxyurea (HU), aphidicolin, and ethylmethanesulfonate (EMS) induced myogenic differentiation of Schneider cells. HU-induced differentiation was inhibited upon pretreatment of cells with chemical inhibitors of PP 1/2A, p38 MAPK, JNK, and proteasome. RT-PCR analysis revealed that the expressions of fusion-competent myoblast-specific genes lmd, sns, and del were induced in Schneider cells upon treatment with NCS or HU, whereas expressions of three founder cell-specific genes, duf, ants, and rols, were undetectable. These results indicate that the expression of fusion competent-myoblast-specific genes is induced during myogenic differentiation of Drosophila Schneider cells by DNA DSBs or replication inhibition.     

Nuclear Localization of p38 MAPK in Response to DNA Damage

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.     

DNA Double Strand Breaks but Not Interstrand Crosslinks Prevent Progress through Meiosis in Fully Grown Mouse Oocytes

There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs) induced by ionizing radiation and agents such as neocarzinostatin (NCS), and interstrand crosslinks (ICLs) induced by alkylating agents such as mitomycin C (MMC), are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show γ-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality.     

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.     

A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channels

The complete sequencing of the genome of the fruit fly Drosophila melanogaster offers the prospect of detailed functional analysis of the extensive gene families in this genetic model organism. Comprehensive functional analysis of family members is facilitated by access to a robust, stable and inducible expression system in a fly cell line. Here we show how the Schneider S2 cell line, derived from the Drosophila embryo, provides such an expression system, with the bonus that radioligand binding studies, second messenger assays, ion imaging, patch-clamp electrophysiology and gene silencing can readily be applied. Drosophila is also ideal for the study of new control strategies for insect pests since the receptors and ion channels that many new animal health drugs and crop protection chemicals target can be expressed in this cell line. In addition, many useful orthologues of human disease genes are emerging from the Drosophila genome and the study of their functions and interactions is another area for postgenome applications of S2 cell lines.     

Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint

Delay of cell cycle progression in response to double-strand DNA breaks (DSBs) is critical to allow time for DNA repair and prevent cellular transformation. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is activated in immature thymocytes along with TcRbeta gene V(D)J recombination. Active p38 MAP kinase promotes a G2/M cell cycle checkpoint through the phosphorylation and activation of p53 in these cells in vivo. Inactivation of p38 MAP kinase and p53 is required for DN3 thymocytes to exit the G2/M checkpoint, progress through mitosis and further differentiate. We propose that p38 MAP kinase is activated by V(D)J-mediated DSBs and induces a p53-mediated G2/M checkpoint to allow DNA repair and prevent cellular transformation.     

Site-specific biotinylation of human myeloid differentiation protein 88 in Drosophila melanogaster Schneider 2 cell cytoplasm

In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase alpha subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression level was 1.3 mug 107 cells-1. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a 1-L culture. Exogenous biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88 in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction assays where high sensitivity is required.     

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2-MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.     

Polymorphism and stability of histone gene clusters in Drosophila melanogaster cultured cells

In a previous communication (Saigo, K., Millstein, L. and Thomas, C.A., Jr. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 815–827), the overall structure of histone genes of Schneider line 2 cells was shown to extensively differ from that of Oregon-R embryo from which the cell line was established, and it was speculated that the histone genes might be reshuffled extensively during either the periods of the establishment, or maintenance of cell lines, or both. To establish the validity of this notion the structure of histone genes was examined in Drosophila melanogaster cultured cells. The overall organization of histone gene clusters was found to be stably maintained in both the periods for the establishment and maintenance of cultured cells, indicating that the previous assumption is inadequate. Instead of an extensive rearrangement, minor structural changes were found to occasionally occur probably by simple base substitutions and/or, deletion or insertion of very short DNA pieces. It was also shown that the extensive variation in structures of histone genes in cultured cells such as Schneider line 2 are attributable to polymorphism on the level of individual flies.     

A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster

Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.     

Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.     

Polymorphism and stability of histone gene clusters in Drosophila melanogaster cultured cells

In a previous communication (Saigo, K., Millstein, L. and Thomas, C.A., Jr. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 815-827), the overall structure of histone genes of Schneider line 2 cells was shown to extensively differ from that of Oregon-R embryo from which the cell line was established, and it was speculated that the histone genes might be reshuffled extensively during either the periods of the establishment, or maintenance of cell lines, or both. To establish the validity of this notion the structure of histone genes was examined in Drosophila melanogaster cultured cells. The overall organization of histone gene clusters was found to be stably maintained in both the periods for the establishment and maintenance of cultured cells, indicating that the previous assumption is inadequate. Instead of an extensive rearrangement, minor structural changes were found to occasionally occur probably by simple base substitutions and/or, deletion or insertion of very short DNA pieces. It was also shown that the extensive variation in structures of histone genes in cultured cells such as Schneider line 2 are attributable to polymorphism on the level of individual flies.     

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor,Src, JNK,and p38 MAPK

We report that Aplidin, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. Aplidin-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by Aplidin. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected Aplidin action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, Aplidin also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of Aplidin.     

The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes.

R8 photoreceptor cells play a primary role in the differentiation of Drosophila eyes. In scabrous (sca) mutants, the pattern of R8 photoreceptor differentiation is altered. The sca gene is predicted to encode a secreted protein related in part to fibrinogen and tenascins. Using expression in Drosophila Schneider cells, we showed that sca encoded a dimeric glycoprotein which was secreted and found in soluble form in the tissue culture medium. The sca protein contained both N- and O-linked carbohydrates and interacted with heparin. This Schneider cell protein was similar to protein detected in embryos. We showed that sca mutations, along with conditional alleles of Notch (N) and Delta (Dl), each affected the pattern of cells expressing atonal (ato), the proneural gene required for R8 differentiation. In normal development, about 1 cell in 20 differentiates into an R8 cell; in the others, ato is repressed. N and Dl were required to repress ato in the vicinity of R8 cells, whereas sca had effects over several cell diameters. Certain antibodies detected uptake of sca protein several cells away from its source. The overall growth factor-like structure of sca protein, its solubility, and its range of effects in vivo are consistent with a diffusible role that complements mechanisms involving direct cell contact. We propose that as the morphogenic furrow advances, cell secreting sca protein control the pattern of the next ommatidial column.