DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers MSH2 and Cdt2 protein-dependent degradation of the cell cycle and mismatch repair (MMR) inhibitor protein p21Waf1/Cip1

p21(Waf1/Cip1) protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.     

Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.     

MutS inhibits RecA-mediated strand transfer with methylated DNA substrates

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O⁶-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O⁶-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O⁶-methylguanine residues in methylated DNA by HPLC-MS/MS and 5–10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

ATM is activated in response to N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA alkylation

p53 plays an important role in response to ionizing radiation by regulating cell cycle progression and triggering apoptosis. These activities are controlled, in part, by the phosphorylation of p53 by the protein kinase ATM. Recent evidence indicates that the monofunctional DNA alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) also triggers up-regulation and phosphorylation of p53; however, the mechanism(s) responsible for this are unknown. We observed that in MNNG-treated normal human fibroblasts, up-regulation and phosphorylation of p53 was sensitive to the ATM kinase inhibitor wortmannin. ATM-deficient fibroblasts exhibited a delay in p53 up-regulation indicating a role for ATM in triggering the MNNG-induced response. Likewise, a mismatch repair (MMR)-deficient colorectal tumor line failed to show rapid up-regulation of p53. However, unlike ATM-deficient cells, these MMR-deficient cells displayed rapid phosphorylation of the p53 residue serine 15 after MNNG. In vitro kinase assays indicate that ATM is rapidly activated in both normal and MMR-deficient cells in response to MNNG. Using a number of morphological and biochemical approaches, we failed to observe MNNG-induced apoptosis in normal human fibroblasts, suggesting that apoptosis-induced DNA strand breaks are not required for the activation of ATM in response to MNNG. Comet assays indicated that strand breaks accumulated, and p53 up-regulation/phosphorylation occurred quite rapidly (within 30 min) after MNNG treatment, suggesting that DNA strand breaks that arise during the repair process activate ATM. These findings indicate that ATM activation is not limited to the ionizing radiation-induced response and potentially plays an important role in response to DNA alkylation.     

DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage.Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.     

Mismatch Repair Protein Deficiency Compromises Cisplatin-induced Apoptotic Signaling

Mismatch repair (MMR) proteins participate in cytotoxicity induced by certain DNA damage-inducing agents, including cisplatin (cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic drug utilized clinically to treat a variety of malignancies. MMR proteins have been demonstrated to bind to CDDP-DNA adducts and initiate MMR protein-dependent cell death in cells treated with CDDP; however, the molecular events underlying this death remain unclear. As MMR proteins have been suggested to be important in clinical responses to CDDP, a clear understanding of MMR protein-dependent, CDDP-induced cell death is critical. In this report, we demonstrate MMR protein-dependent relocalization of cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent cytotoxicity, suggesting that a caspase-dependent signaling mechanism is required for the execution of this cell death. p53 protein levels were up-regulated independently of MMR protein status, suggesting that p53 is not a mediator of MMR-dependent, CDDP-induced death. This work is the first indication of a required signaling mechanism in CDDP-induced, MMR protein-dependent cytotoxicity, which can be uncoupled from other CDDP response pathways, and defines a critical contribution of MMR proteins to the control of cell death.The MMR² system of proteins plays roles in diverse cellular processes, perhaps most notably in preserving genomic integrity by recognizing and facilitating the repair of post-DNA replication base pairing errors. Recognition of these errors and recruitment of repair machinery is performed by the MutSα complex (consisting of the MMR proteins MSH2 and MSH6) or MutSβ complex (consisting of MSH2 and MSH3). Defects in MMR proteins render cells hypermutable and promote microsatellite instability, a hallmark of MMR defects. MMR protein defects are found in a wide variety of sporadic cancers, as well as in hereditary non-polyposis colorectal cancer (1).In addition to their role in DNA repair, MMR proteins also play a role in cytotoxicity induced by specific types of DNA-damaging chemotherapeutic drugs, such as CDDP, which is utilized clinically to treat a number of different cancer types. MutSα recognizes multiple types of DNA damage, including 1,2-intrastrand CDDP adducts and O⁶-methylguanine lesions (2). Treatment of cells with compounds that induce these types of lesions, including CDDP and methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), results in MMR protein-dependent cell cycle arrest and cell death (3–7). This suggests that MMR proteins, in addition to their role in DNA repair, are also capable of initiating cell death in response to certain types of DNA damage.Cells treated with DNA-damaging agents frequently activate an apoptotic cell death pathway mediated by the mitochondria. This intrinsic death signaling pathway predominantly involves the coordinated activity of two groups of proteins: pro-death members of the Bcl-2 family that control the integrity of mitochondrial membranes, and members of the caspase family of cysteinyl proteases that proteolytically cleave intracellular substrates, giving rise to apoptotic morphology and destruction of the cell (8, 9). Pro-death Bcl-2 family members, such as Bax and Bak, target the outer mitochondrial membrane and cause the cytosolic release of pro-death factors residing within the mitochondria of unstressed cells (8). Predominant among these factors is cytochrome c, whose cytoplasmic localization results in the formation of a caspase-activating platform known as the apoptosome (10). This complex includes the adaptor protein Apaf-1, and when formed the apoptosome promotes the cleavage and activation of caspase-9 (11, 12). Once activated, this apical caspase proceeds to cleave and activate caspase-3, the predominant effector protease of apoptosis.A significant amount of evidence has been gathered illustrating MMR protein-dependent pro-death signaling in response to methylating agents (13–16, 3). In contrast, the MMR protein-dependent cytotoxic response to CDDP is largely unknown, with only the p53-related transactivator protein p73 and the c-Abl kinase clearly implicated as potential mediators of CDDP/MMR protein-dependent cell death in human cells (17, 18). Interestingly, ATM, Chk1, Chk2, and p53, which are activated in an MMR protein-dependent manner after treatment of cells with MNNG (3, 13), are not involved in the MMR-dependent response to CDDP (7, 17). In addition, the magnitude of MMR protein-dependent cell death induced by methylating agents and CDDP differs (4). These findings suggest that unique signaling pathways may be engaged by MMR proteins depending upon the type of recognized lesion. As such, there is a requirement for further study of the molecular events underlying MMR protein-dependent cell death and cell cycle arrest for each type of recognized DNA lesion. This is particularly relevant in the case of CDDP, as evidence from a limited number of retrospective clinical studies suggests that MMR proteins play an important role in patient response to CDDP. Several studies examining immunohistochemical staining against MSH2 or MLH1 have demonstrated that levels of these proteins are reduced in ovarian and esophageal tumor samples following CDDP-based chemotherapy (19, 20). Low levels of MMR protein post-chemotherapy seem to be predictive of lower overall survival in a certain subset of tumors (esophageal cancer), but not others (ovarian and non-small cell lung cancer) (19–21). Two recent studies examining MMR protein levels and microsatellite instability in germ cell tumors from patients receiving platinum-based chemotherapy have suggested a prognostic value for pre-chemotherapy MMR protein status in these tumors (22, 23). This potential clinical relevance underscores the need for a greater understanding of MMR protein-dependent mechanisms of CDDP-induced cell death.In this study, we report that CDDP induces an MMR protein-dependent decrease in cell viability and MMR protein-dependent signaling in the form of cytochrome c release to the cytoplasm and cleavage of caspase-9, caspase-3, and PARP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent loss of cell viability, indicating a requirement for caspase activation in this process and uncoupling MMR protein-dependent cytotoxic signaling from other CDDP response pathways. Additionally, the CDDP-induced, MMR protein-dependent cytotoxic response is independent of p53 signaling. Our results demonstrate for the first time an MMR protein-dependent pro-death signaling pathway in cells treated with CDDP.     

ING1b negatively regulates HIF1α protein levels in adipose-derived stromal cells by a SUMOylation-dependent mechanism

Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs.Human mesenchymal stem/stromal cells (MSCs) are able to self-renew and differentiate into various cell types. Recently, MSCs have been developed as tools for tissue engineering and cell-based therapies¹ in particular owing to their trophic and immunosuppressive activities.² Conventionally, the bone marrow MSCs (BM-MSCs) and the adipose-derived stem/stromal cells (ADSCs) have constituted the main sources of MSCs for clinical use. These cells are expanded in vitro prior to their application; however, this long-term culture may allow the emergence of senescence and phenotypic alterations, rendering MSCs unsuitable for clinical purposes.³To overcome these issues, MSC culture in conditions mimicking hypoxic niches has been tested.⁴ Low O₂ tensions promote MSC growth, survival and maintain their self-renewing multipotent state.⁵ However, how hypoxia (1% O₂) affects MSC behavior is unclear. Responses to hypoxia are mainly mediated by hypoxia inducible factors (HIFs). HIF1, 2 and 3α subunits, are constitutively degraded in normoxia and stabilized in hypoxia. Consequently, when stabilized they can dimerize with HIF1β, and then translocate into the nucleus to modulate the expression of selected genes. HIF1α is highly expressed in MSCs, controls their metabolic fate and maintains them in an undifferentiated state.⁶ HIF1α has also been shown to delay the occurrence of senescence in MSCs, by repressing E2A and p21 expression.⁷The inhibitors of growth (ING) family genes act as readers of the epigenetic histone code. Among them, ING1 has been described as a type II tumor suppressor, regulating cell growth, DNA repair, apoptosis, chromatin remodeling and senescence.⁸ To some extent, ING1 and HIF might have opposite effects, (e.g. on tumor progression). Indeed, HIF1α, unlike ING1 that inhibits angiogenesis, promotes angiogenesis.⁹ Furthermore, p53, a well-known ING1b interactor, and HIF1α have been shown in several studies to have antagonistic effects. Following DNA damage, p53 induces apoptosis and inhibits survival of cells by reducing activity and levels of HIF1α.^{10, 11}So far, ING4 has been shown as the only ING protein to regulate the hypoxic response. Indeed, by interacting with HIF prolyl hydroxylase 2 (HPH-2), ING4 has been described to repress some HIF1α activities under hypoxic conditions.¹² Here, we show that ING1b accumulates in ADSCs following DNA damage in hypoxia. According to the opposing roles of ING1b and HIF1α, we hypothesized that ING1b could interfere with HIF1α and participate in the conservation of the genomic integrity of MSCs. Mechanistically, we found that ING1b interacted with HIF1α and promoted its proteasomal degradation in hypoxia. SUMOylation of ING1b played a role since the unSUMOylated form of ING1b was unable to trigger HIF1α degradation. The E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4) participated in HIF1α degradation and ING1b accumulation following a genotoxic stress in 1% O₂. ING1b, subsequently, took part in decreasing PIAS4 levels after DNA damage. Finally, we report that ING1b by decreasing HIF1α level modulated ADSC differentiation potential. These data indicate that ING1b, according to its SUMOylation status, regulates the hypoxic response by contributing to the HIF1α degradation, and therefore may impede HIF1α-related effects on the maintenance of ADSCs stem cell character.     

Expressing MLH1 in HCT116 cells increases cellular resistance to radiation by activating the PRKAC

Mut L homolog-1 (MLH1) is a key DNA mismatch repair protein which participates in the sensitivity to DNA damaging agents. However, its role in the radiosensitivity of tumor cells is less well characterized. In this study, we investigated the role of MLH1 in cellular responses to ionizing radiation (IR) and explored the signaling molecules involved. The isogenic pair of MLH1 proficient (MLH1⁺) and deficient (MLH1^–) human colorectal cancer HCT116 cells was exposed to IR for 24 h at the dose of 3 cGy. The clonogenic survival was examined by the colony formation assay. Cell cycle distribution was analyzed with flow cytometry. Changes in the protein level of MLH1, DNA damage marker γH2AX, and protein kinase A catalytic subunit (PRKAC), a common target for anti-tumor drugs, were examined with Western blotting. The results showed that the HCT116 (MLH1⁺) cells demonstrated increased radio-resistance with increased S population, decreased G2 population, a low level of γH2AX, a reduced ratio of phosphorylated PRKACαβ to total PRKAC, and an elevated level of total PRKAC and phosphorylated PRKACβII following IR compared with the HCT116 (MLH1^–) cells. Importantly, silencing PRKAC in HCT116 (MLH1⁺) cells increased the cellular radiosensitivity. In conclusion, MLH1 may increase cellular resistance to IR by activating PRKAC. Our finding is the first to demonstrate the important role of PRKAC in MLH1-mediated radiosensitivity, suggesting that PRKAC has potential as a biomarker and a therapeutic target for increasing radio-sensitization.     

c-Abl Activates Janus Kinase 2 in Normal Hematopoietic Cells

Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μm imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells.     

Assembly of the CD8α/p56 protein complex in stably expressing rat epithelial cells

We have previously characterized the biogenesis of the human CD8α protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8α/p56^lck protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56^lck, or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8α, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8α established hetero-oligomeric complexes with p56^lck, as revealed by co-immunoprecipitation assays performed with anti-CD8α antibody. Moreover, p56^lck bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56^lck binds rapidly to CD8α and most of the p56^lck is bound to CD8α at steady state; (2) CD8α/p56^lck protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56^lck reaches the cell surface.     

The BH3-only protein Bik/Blk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNgamma-induced cell death

IFNγ induces cell death in epithelial cells, but the mediator for this death pathway has not been identified. In this study, we find that expression of Bik/Blk/Nbk is increased in human airway epithelial cells (AECs [HAECs]) in response to IFNγ. Expression of Bik but not mutant BikL61G induces and loss of Bik suppresses IFNγ-induced cell death in HAECs. IFNγ treatment and Bik expression increase cathepsin B and D messenger RNA levels and reduce levels of phospho–extracellular regulated kinase 1/2 (ERK1/2) in the nuclei of bik^+/+ compared with bik^−/− murine AECs. Bik but not BikL61G interacts with and suppresses nuclear translocation of phospho-ERK1/2, and suppression of ERK1/2 activation inhibits IFNγ- and Bik-induced cell death. Furthermore, after prolonged exposure to allergen, hyperplastic epithelial cells persist longer, and nuclear phospho-ERK is more prevalent in airways of IFNγ^−/− or bik^−/− compared with wild-type mice. These results demonstrate that IFNγ requires Bik to suppress nuclear localization of phospho-ERK1/2 to channel cell death in AECs.     

Induction of kinetochore positive and negative micronuclei in V79 cells by N-methyl-N′-nitro-N-nitrosoguanidine: the protective effect of the pyridoindole antioxidant stobadine

The induction of micronuclei by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and their reduction by the cardioprotective synthetic antioxidant, stobadine were studied in hamster V79 cells cultured in vitro. The micronuclei derived from acentric fragments or from whole chromosomes were evaluated with the help of an immunofluorescent staining using antikinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Our results showed that MNNG (0.5 μg/ml) induced mainly kinetochore-negative micronuclei. At 6, 24 and 48 h after MNNG treatment, we measured a 2.7-, 4.3- and 7.0-fold increase, respectively, of kinetochore-negative micronuclei over the controls. The increase of kinetochore-positive micronuclei was rather low and represented at 6, 24 and 48 h, respectively 0.9-, 1.8- and 2.6-fold increases over the controls. Stobadine decreased the level of kinetochore-negative micronuclei at 6, 24 and 48 h to approximately one-half; the frequency of kinetochore-positive micronuclei was reduced only at 6 h. We suppose that the antioxidant stobadine reduces the induction of micronuclei by MNNG by scavenging of MNNG-induced highly reactive OH radicals which cause chromosomal damage.     

Handcuffing intrinsically disordered regions in Mlh1–Pms1 disrupts mismatch repair

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker''s yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.     

Chemical Carcinogen,N-methyl-N’-nitro-N-nitrosoguanidine,is Specific Activator of Oncogenic Ras

N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is a well-known chemical carcinogen that is widely used for animal carcinogenesis model. Treatment of MNNG, through drinking-water, can evoke multiple tumors in gastro-intestinal tract. In addition, MNNG shows the synergic effect with infection such as H. pylori on gastric cancer formation. Although tumorigenic ability of MNNG is known to be related with DNA alkylation, however, recent reports suggested that MNNG-induced tumors do not show the difference in DNA methylation, and genetic mutation profile is quite different from similar DNA alkylating agent, MNU-inducing cancer. Otherwise, genetic mutation of Ras is frequently detected in MNNG-induced tumors. Considering them, tumorigenic property of MNNG would be related with Ras. So we checked the effect of MNNG on Ras pathway. In this study, we demonstrated that MNNG could activate Ras-MAPK pathway as oncogenic Ras dependent manner. Activation of Erk by MNNG could not suppressed by cycloheximide and ALLN. In addition, Inhibition of PI3K, p38/HOG1, Raf, and CDK could not block the MNNG-induced p-Erk activation, whereas U0126 and PD98059 abolished it. Moreover, MNNG could reduce the expression of E-cadherin and promote dissociation of β-catenin from E-cadherin through oncogenic-Ras-MAPK pathway. These results strongly suggested that oncogenic Ras would be direct target of MNNG and provided new insight that carcinogen also possesses it specific target.