query: B3-Galectin increases cell resistance to tyrosine kinase inhibitors (TKIs) by engaging the alternate KRAS-RalB signaling complex downstream of EGFR.

title: An integrin β3–KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition

text: Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

title: Prognostic and Predictive Value of KRAS Mutations in Advanced Non-Small Cell Lung Cancer

text: Clinical implications of KRAS mutations in advanced non-small cell lung cancer remain unclear. We retrospectively evaluated the prognostic and predictive value of KRAS mutations in patients with advanced NSCLC. Among 484 patients with available results for both KRAS and EGFR mutations, 39 (8%) had KRAS and 182 (38%) EGFR mutations, with two cases having both mutations. The median overall survivals for patients with KRAS mutations, EGFR mutations, or both wild types were 7.7, 38.0, and 15.0 months, respectively (P<0.001). The KRAS mutation was an independent poor prognostic factor in the multivariate analysis (hazard ratio = 2.6, 95% CI: 1.8-3.7). Response rates and progression-free survival (PFS) for the pemetrexed-based regimen in the KRAS mutation group were 14% and 2.1 months, inferior to those (28% and 3.9 months) in the KRAS wild type group. KRAS mutation tended to be associated with inferior treatment outcomes after gemcitabine-based chemotherapy, while there was no difference regarding taxane-based regimen. Although the clinical outcomes to EGFR tyrosine kinase inhibitors (TKIs) seemed to be better in patients with KRAS wild type than those with KRAS mutations, there was no statistical difference in response rates and PFS according to KRAS mutation status when EGFR mutation status was considered. Two patients with both KRAS and EGFR mutations showed partial response to EGFR TKIs. Although G12D mutation appeared more frequently in never smokers, there was no difference in clinical outcomes according to KRAS genotypes. These results suggested KRAS mutations have an independent prognostic value but a limited predictive role for EGFR TKIs or cytotoxic chemotherapy in advanced NSCLC.

title: Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer.

text: Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MAP-ERK kinase (MEK) inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR-mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.

title: Subsequent chemotherapy reverses acquired tyrosine kinase inhibitor resistance and restores response to tyrosine kinase inhibitor in advanced non-small-cell lung cancer

text:
BACKGROUND Patients with advanced or metastatic non-small cell lung cancer (NSCLC) can develop acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. Here, we report the successful treatment with alternating chemotherapy and TKIs of two cases of advanced NSCLC who developed resistance to TKI.    CASE PRESENTATION Two patients with advanced or metastatic NSCLC were treated with palliative chemotherapy followed by erlotinib/gefitinib. When TKI therapy failed, two cycles of chemotherapy were provided, which were followed by re-challenge with erlotinib or gefitinib.
 CONCLUSION NSCLC patients with acquired TKI resistance should be managed aggressively whenever possible. Subsequent chemotherapy and target treatment is one of the reasonable choices for those with an initial dramatic clinical response with erlotinib/gefitinib treatment. Further studies are warranted to substantiate the association of erlotinib /gefitinib treatment with the efficacy of NSCLC patients with acquired TKI failure.

title: NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations.

text: Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.


query: BCL-2 activation antagonizes the apoptotic effects of c-Myc.

title: Antiapoptotic BCL-2 is required for maintenance of a model leukemia.

text: Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).

title: Examining BCL-2 family function with large unilamellar vesicles.

text: The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis(1). Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)(1). After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues(2). In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)(3-6). Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation(7,8). In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members(7,9). As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization(10). LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)(10). This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)(11). As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

title: The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells.

text: Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0-80 microg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC(50) of 40+/-0.08 microg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and (3)H-thymidine uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G(0)/G(1) phase from 74.6 to 84 while had a significant reduction in G(2)/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit proliferation but also induce apoptosis of Eca-109 cells.

title: Wedelolactone exhibits anti-fibrotic effects on human hepatic stellate cell line LX-2.

text: Wedelolactone is a major coumarin of Eclipta prostrata, which is used for preventing liver damage. However the effects of wedelolactone on hepatic fibrosis remained unexplored. The purpose of this study was to demonstrate the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 and the possible underlying mechanisms by means of MTT assay, Hoechst staining, as well as real-time quantitative PCR and western blot. The results showed that wedelolactone reduced the cellular viability of LX-2 in a time and dose-dependent manner. After treatment of wedelolactone, the expressions of collagen I and α-smooth muscle actin, two biomarkers of LX-2 activation, were remarkably decreased. The apoptosis of LX-2 cells was induced by wedelolactone accompanied with the decreasing expression of anti-apoptotic Bcl-2 and increasing expression of pro-apoptotic Bax. In addition, phosphorylated status of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was up-regulated, but not in p38. Moreover, wedelolactone significantly repressed the level of phosphorylated inhibitor of nuclear factor κB (IκB) and p65 in nucleus in spite of tumor necrosis factor-α stimulation. In conclusion, wedelolactone could significantly inhibit the activation of LX-2 cells, the underlying mechanisms of which included inducing Bcl-2 family involved apoptosis, up-regulating phosphorylated status of ERK and JNK expressions, and inhibiting nuclear factor-κB (NF-κB) mediated activity. Wedelolactone might present as a useful tool for the prevention and treatment of hepatic fibrosis.

title: Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2.

text: Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro. Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents. Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

title: Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.

text: Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

title: Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils.

text: The recently identified Fas antigen (Ag) is a cell surface molecule that can mediate apoptosis. The cytoplasmic product of proto-oncogene bcl-2 has been shown to prolong the cellular survival by inhibiting apoptosis. To elucidate the physiologic significance of expression of both molecules, we examined the expression of Fas Ag and bcl-2 on blood leukocyte populations and evaluated their sensitivity to the cytolytic action of anti-Fas antibody. Although Fas Ag was expressed on a fraction of lymphocytes, both neutrophils and monocytes expressed Fas Ag constitutively. In contrast, there was marked difference among these leukocytes regarding bcl-2 expression. Lymphocytes expressed bcl-2 intensely, but monocytes showed weaker bcl-2 expression, and neutrophils were essentially absent for bcl-2 expression. Seemingly reflecting this lack of bcl-2-expression, neutrophils more easily underwent apoptotic cell death in vitro as compared with monocytes and lymphocytes. We showed that anti-Fas antibody affectively accelerated apoptotic cell death in neutrophils. However, the apoptosis-inducing effect of anti-Fas antibody was minimal on monocytes, and lymphocytes were resistant to this antibody. These results suggest that anti-Fas-mediated cell death may, in part, be determined by bcl-2 expression status in Fas+ lymphoid and hematopoietic cells.

title: BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control.

text: BCL-6, a transcriptional repressor frequently translocated in lymphomas, regulates germinal center B cell differentiation and inflammation. DNA microarray screening identified genes repressed by BCL-6, including many lymphocyte activation genes, suggesting that BCL-6 modulates B cell receptor signals. BCL-6 repression of two chemokine genes, MIP-1alpha and IP-10, may also attenuate inflammatory responses. Blimp-1, another BCL-6 target, is important for plasmacytic differentiation. Since BCL-6 expression is silenced in plasma cells, repression of blimp-1 by BCL-6 may control plasmacytic differentiation. Indeed, inhibition of BCL-6 function initiated changes indicative of plasmacytic differentiation, including decreased expression of c-Myc and increased expression of the cell cycle inhibitor p27kip1. These data suggest that malignant transformation by BCL-6 involves inhibition of differentiation and enhanced proliferation.

title: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax

text: BCL-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma1–3. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths4–13. An emerging family of Bcl-2 -related proteins share two highly conserved regions14–20 referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-214. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitu-tion of Gly 145 in BHl domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in inter-leukin-3 deprivation, γ-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homo-dimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.

title: tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1

text: Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of β-oxidation flux by labeled palmitate demonstrates that tBid inhibits β-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on β-oxidation. The unexpected role of tBid in the regulation of lipid β-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

title: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells

text: The aim of this study was to investigate the mechanism through which Sphingosine kinase-1 (SPHK1) exerts its anti-apoptosis activity in glioma cancer cells. We here report that dysregulation of SPHK1 alters the sensitivity of glioma to apoptosis both in vitro and in vivo. Further mechanistic study examined the expression of Bcl-2 family members, including Bcl-2, Mcl-1, Bax and Bim, in SPHK1-overexpressing glioma cells and revealed that only pro-apoptotic Bim was downregulated by SPHK1. Moreover, the transcriptional level of Bim was also altered by SPHK1 in glioma cells. We next confirmed the correlation between SPHK1 and Bim expression in primary glioma specimens. Importantly, increasing SPHK1 expression in glioma cells markedly elevated Akt activity and phosphorylated inactivation of FOXO3a, which led to downregulation of Bim. A pharmacological approach showed that these effects of SPHK1 were dependent on phosphatidylinositol 3-kinase (PI3K). Furthermore, effects of SPHK1 on Akt/FOXO3a/Bim pathway could be reversed by SPHK1 specific RNA interference or SPHK1 inhibitor. Collectively, our results indicate that regulation of the Akt/FOXO3a/Bim pathway may be a novel mechanism by which SPHK1 protects glioma cells from apoptosis, thereby involved in glioma tumorigenesis.

title: p53 mutation in breast cancer. Correlation with cell kinetics and cell of origin.

text:
AIM Several studies have investigated the expression of the cytokeratins (CKs), vimentin, the epithelial growth factor receptor (EGFR), the oestrogen receptor (ER), and the progesterone receptor (PgR), in breast cancer, but no study has directly compared p53 mutations with these phenotypic and differentiation markers in the same case. The present study was designed to provide some of this information.
 METHODS The expression of the p53 and bcl-2 proteins was evaluated by immunohistochemistry in relation to phenotypic characteristics and cellular kinetic parameters (mitotic index and apoptotic index) in 37 cases of ductal carcinoma in situ (DCIS) and 27 cases of infiltrating ductal carcinoma (IDC) of the breast. In addition, p53 gene mutation was examined by polymerase chain reaction single strand conformation polymorphism analysis (SSCP).
 RESULTS Thirteen cases (eight DCIS and five IDC) showed expression of CK8, CK14, CK18, vimentin, and EGFR, consistent with a stem cell phenotype, whereas 44 cases (27 DCIS and 17 IDC) showed expression of CK8 and CK1, weak or negative expression of CK18, but were negative for vimentin and EGFR, consistent with a luminal cell phenotype. DCIS and IDC cases with a stem cell phenotype were ER/PgR negative and intermediately or poorly differentiated. In contrast, the cases with luminal cell phenotype were ER/PgR positive and well or intermediately differentiated. In addition, intermediately or poorly differentiated cases with a stem cell phenotype showed higher proliferative activity (per cent of MIB-l positive cells) than did intermediately or well differentiated cases with a luminal cell phenotype. Both DCIS and IDC cases with a stem cell phenotype were p53 positive and bcl-2 negative by immunohistochemistry. In IDC, p53 expression was associated with a reduction of both mitotic index and apoptotic index compared with DCIS. Most of the tumours showing a more differentiated phenotype (luminal) were p53 negative and bcl-2 positive. In these cases, cell kinetic parameters increased from DCIS to IDC. These data suggest the existence of subsets of DCIS and IDC that, because of their phenotypic characteristics, could be derived from subpopulations of normal breast cells having different control mechanisms of cell proliferation and neoplastic progression.
 CONCLUSIONS These results are compatible with the hypothesis that the phenotype of the cell of origin constrains both tumour phenotype and the choice of genetic events; however, the occurrence of p53 mutants by chance during neoplastic transformation cannot be excluded.

title: At the gates of death.

text: Apoptosis that proceeds via the mitochondrial pathway involves mitochondrial outer membrane permeabilization (MOMP), responsible for the release of cytochrome c and other proteins of the mitochondrial intermembrane space. This essential step is controlled and mediated by proteins of the Bcl-2 family. The proapoptotic proteins Bax and Bak are required for MOMP, while the antiapoptotic Bcl-2 proteins, including Bcl-2, Bcl-xL, Mcl-1, and others, prevent MOMP. Different proapoptotic BH3-only proteins act to interfere with the function of the antiapoptotic Bcl-2 members and/or activate Bax and Bak. Here, we discuss an emerging view, proposed by Certo et al. in this issue of Cancer Cell, on how these interactions result in MOMP and apoptosis.

title: LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.

text: Murine ES cells can be maintained as a pluripotent, self-renewing population by LIF/STAT3-dependent signaling. The downstream effectors of this pathway have not been previously defined. In this report, we identify a key target of the LIF self-renewal pathway by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3beta dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, our results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency.

title: Molecular mechanisms of (-)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line.

text: Dietary polyphenols have been associated with reduced risk of chronic diseases, but the precise molecular mechanisms of protection remain unclear. This work was aimed at studying the effect of (-)-epicatechin (EC) and chlorogenic acid (CGA) on the regulation of apoptotic and survival/proliferation pathways in a human hepatoma cell line (HepG2). EC or CGA treatment for 18 h had a slight effect on cell viability and decreased reactive oxygen species formation, and EC alone promoted cell proliferation, whereas CGA increased glutathione levels. Phenols neither induced the caspase cascade for apoptosis nor affected expression levels of Bcl-xL or Bax. A sustained activation of the major survival signals AKT/PI-3-kinase and ERK was shown in EC-treated cells, rather than in CGA-exposed cells. These data suggest that EC and CGA have no effect on apoptosis and enhance the intrinsic cellular tolerance against oxidative insults either by activating survival/proliferation pathways or by increasing antioxidant potential in HepG2.

title: Long non-coding RNA HOTTIP promotes BCL-2 expression and induces chemoresistance in small cell lung cancer by sponging miR-216a

text: Despite progress in treatment of small cell lung cancer (SCLC), its multidrug chemoresistance and poor prognosis still remain. Recently, we globally assessed long non-coding RNAs (lncRNAs) for contributions to SCLC chemoresistance using microarray data, in vitro and in vivo assays. Here we reported that HOTTIP, encoding a lncRNA that is frequently amplified in SCLC, was associated with SCLC cell chemosensitivity, proliferation, and poor prognosis of SCLC patients. Moreover, mechanistic investigations showed that HOTTIP functioned as an oncogene in SCLC progression by binding miR-216a and abrogating its tumor-suppressive function in this setting. On the other hand, HOTTIP increased the expression of anti-apoptotic factor BCL-2, another important target gene of miR-216a, and jointly enhanced chemoresistance of SCLC by regulating BCL-2. Taken together, our study established a role for HOTTIP in SCLC progression and chemoresistance suggest its candidacy as a new diagnostic and prognostic biomarker for clinical management of SCLC.

title: Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion.

text: Sustained signaling from the T cell receptor (TCR) and costimulatory molecules is thought necessary for generating high numbers of effector T cells. Here, we show that Survivin is controlled in peripheral T cells by OX40 cosignaling via sustained PI3k and PKB activation. Survivin is induced by OX40 independent of mitotic progression in late G1, and blocking Survivin suppresses S-phase transition and division of T cells and leads to apoptosis. Moreover, Survivin expression alone is sufficient to restore proliferation and to antagonize apoptosis in costimulation-deficient T cells and can rescue T cell expansion in vivo. Survivin allows effector T cells to accumulate in large numbers, but Bcl-2 family proteins are required for T cell survival after the phase of active division. Thus, sustained Survivin expression from costimulatory signaling maintains T cell division over time and regulates the extent of clonal expansion.

title: Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis.

text: Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

title: CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters.

text: CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

title: B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver.

text: Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.


query: BCL-2 promotes the apoptotic effects of c-Myc.

title: Antiapoptotic BCL-2 is required for maintenance of a model leukemia.

text: Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).

title: Examining BCL-2 family function with large unilamellar vesicles.

text: The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis(1). Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)(1). After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues(2). In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)(3-6). Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation(7,8). In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members(7,9). As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization(10). LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)(10). This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)(11). As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

title: The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells.

text: Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0-80 microg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC(50) of 40+/-0.08 microg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and (3)H-thymidine uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G(0)/G(1) phase from 74.6 to 84 while had a significant reduction in G(2)/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit proliferation but also induce apoptosis of Eca-109 cells.

text: Wedelolactone is a major coumarin of Eclipta prostrata, which is used for preventing liver damage. However the effects of wedelolactone on hepatic fibrosis remained unexplored. The purpose of this study was to demonstrate the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 and the possible underlying mechanisms by means of MTT assay, Hoechst staining, as well as real-time quantitative PCR and western blot. The results showed that wedelolactone reduced the cellular viability of LX-2 in a time and dose-dependent manner. After treatment of wedelolactone, the expressions of collagen I and α-smooth muscle actin, two biomarkers of LX-2 activation, were remarkably decreased. The apoptosis of LX-2 cells was induced by wedelolactone accompanied with the decreasing expression of anti-apoptotic Bcl-2 and increasing expression of pro-apoptotic Bax. In addition, phosphorylated status of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was up-regulated, but not in p38. Moreover, wedelolactone significantly repressed the level of phosphorylated inhibitor of nuclear factor κB (IκB) and p65 in nucleus in spite of tumor necrosis factor-α stimulation. In conclusion, wedelolactone could significantly inhibit the activation of LX-2 cells, the underlying mechanisms of which included inducing Bcl-2 family involved apoptosis, up-regulating phosphorylated status of ERK and JNK expressions, and inhibiting nuclear factor-κB (NF-κB) mediated activity. Wedelolactone might present as a useful tool for the prevention and treatment of hepatic fibrosis.

title: Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells.

text: Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.

title: At the gates of death.

title: Apoptosis in breast cancer: relationship with other pathological parameters.

text: Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. The number of apoptotic cells as a percentage of cells present, or the number of apoptotic cells per square millimetre of neoplastic tissue, is usually described as the apoptotic index (AI). In breast cancer, the AI is not related to tumour size, axillary lymph node metastasis or distant metastasis at diagnosis. It is greater in invasive ductal carcinomas than in other histological types. High AI is also related to high histological grade, high nuclear grade, comedo-type necrosis, lack of tubule formation, and dense infiltration of the tumour by lymphocytes. Sex steroid receptor-negative tumours have greater AIs than the sex steroid receptor-positive ones. Aneuploid breast cancers with high S-phase fractions (SPFs) also have high AI values compared with diploid tumours with low SPFs. p53-Positive breast cancers have high AIs, whereas tumours that are Bcl-2 positive have low AIs. The AI shows a strong positive correlation to all direct or indirect indicators of cell proliferation, such as mitotic index and Ki67 immunolabelling. Univariate survival analyses show that a high AI is linked with unfavourable disease outcome in axillary lymph node-negative and -positive breast cancer, but multivariate analyses indicate that AI is not an independent prognostic factor. In conclusion, a high AI is related to malignant cellular features and indicators of invasiveness and cell proliferation in breast cancer.

title: Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.

text: We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.


query: BLM gene encodes RecQ-class DNA helicase.

title: The Bloom's syndrome gene product is homologous to RecQ helicases

text: The Bloom's syndrome (BS) gene, BLM, plays an important role in the maintenance of genomic stability in somatic cells. A candidate for BLM was identified by direct selection of a cDNA derived from a 250 kb segment of the genome to which BLM had been assigned by somatic crossover point mapping. In this novel mapping method, cells were used from persons with BS that had undergone intragenic recombination within BLM. cDNA analysis of the candidate gene identified a 4437 bp cDNA that encodes a 1417 amino acid peptide with homology to the RecQ helicases, a subfamily of DExH box-containing DNA and RNA helicases. The presence of chain-terminating mutations in the candidate gene in persons with BS proved that it was BLM.

title: Human RecQ helicases in DNA repair, recombination, and replication.

text: RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability.

title: The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA

text: The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg2+, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.

title: The BLM helicase contributes to telomere maintenance through processing of late-replicating intermediate structures

text: Werner's syndrome (WS) and Bloom's syndrome (BS) are cancer predisposition disorders caused by loss of function of the RecQ helicases WRN or BLM, respectively. BS and WS are characterized by replication defects, hyperrecombination events and chromosomal aberrations, which are hallmarks of cancer. Inefficient replication of the G-rich telomeric strand contributes to chromosome aberrations in WS cells, demonstrating a link between WRN, telomeres and genomic stability. Herein, we provide evidence that BLM also contributes to chromosome-end maintenance. Telomere defects (TDs) are observed in BLM-deficient cells at an elevated frequency, which is similar to cells lacking a functional WRN helicase. Loss of both helicases exacerbates TDs and chromosome aberrations, indicating that BLM and WRN function independently in telomere maintenance. BLM localization, particularly its recruitment to telomeres, changes in response to replication dysfunction, such as in WRN-deficient cells or after aphidicolin treatment. Exposure to replication challenge causes an increase in decatenated deoxyribonucleic acid (DNA) structures and late-replicating intermediates (LRIs), which are visible as BLM-covered ultra-fine bridges (UFBs) in anaphase. A subset of UFBs originates from telomeric DNA and their frequency correlates with telomere replication defects. We propose that the BLM complex contributes to telomere maintenance through its activity in resolving LRIs.

title: WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair.

text: Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3' --> 5' exonuclease and 3' --> 5' helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN-/-) complemented with either wtWRN, exonuclease-defective WRN (E-), helicase-defective WRN (H-) or exonuclease/helicase-defective WRN (E-H-). The single E-and H- mutants each partially complemented the NHEJ abnormality of WRN-/- cells. Strikingly, the E-H- double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E- and H- single mutants increased HR to levels higher than those restored by either E-H- or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.

title: The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks.

text: Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.

title: Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

text: RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability.

title: The processing of Holliday junctions by BLM and WRN helicases is regulated by p53.

text: BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser(376) or Ser(378) completely abolishes this inhibition. Following blockage of DNA replication, Ser(15) phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases.

title: BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

text: Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome-associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

title: RecQ helicases: caretakers of the genome

text: RecQ helicases are highly conserved from bacteria to man. Germline mutations in three of the five known family members in humans give rise to debilitating disorders that are characterized by, amongst other things, a predisposition to the development of cancer. One of these disorders — Bloom's syndrome — is uniquely associated with a predisposition to cancers of all types. So how do RecQ helicases protect against cancer? They seem to maintain genomic stability by functioning at the interface between DNA replication and DNA repair.

title: Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks.

text: The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.

title: BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges.

text: Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.

title: PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

text: The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.

title: Homologous recombination resolution defect in werner syndrome.

text: Werner syndrome (WRN) is an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer. We used three different experimental strategies to show that WRN cellular phenotypes of limited cell division potential, DNA damage hypersensitivity, and defective homologous recombination (HR) are interrelated. WRN cell survival and the generation of viable mitotic recombinant progeny could be rescued by expressing wild-type WRN protein or by expressing the bacterial resolvase protein RusA. The dependence of WRN cellular phenotypes on RAD51-dependent HR pathways was demonstrated by using a dominant-negative RAD51 protein to suppress mitotic recombination in WRN and control cells: the suppression of RAD51-dependent recombination led to significantly improved survival of WRN cells following DNA damage. These results define a physiological role for the WRN RecQ helicase protein in RAD51-dependent HR and identify a mechanistic link between defective recombination resolution and limited cell division potential, DNA damage hypersensitivity, and genetic instability in human somatic cells.

title: 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

text: Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.

title: Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans.

text: Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.

title: The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.

text: The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

title: The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage

text:
BACKGROUND G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo.
 RESULTS We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites.
 CONCLUSIONS In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.

title: G-quadruplexes significantly stimulate Pif1 helicase-catalyzed duplex DNA unwinding.

text: The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation.

title: TAL effectors are remote controls for gene activation.

text: TAL (transcription activator-like) effectors constitute a novel class of DNA-binding proteins with predictable specificity. They are employed by Gram-negative plant-pathogenic bacteria of the genus Xanthomonas which translocate a cocktail of different effector proteins via a type III secretion system (T3SS) into plant cells where they serve as virulence determinants. Inside the plant cell, TALs localize to the nucleus, bind to target promoters, and induce expression of plant genes. DNA-binding specificity of TALs is determined by a central domain of tandem repeats. Each repeat confers recognition of one base pair (bp) in the DNA. Rearrangement of repeat modules allows design of proteins with desired DNA-binding specificities. Here, we summarize how TAL specificity is encoded, first structural data and first data on site-specific TAL nucleases.