Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).     

Dose-dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.     

Autophagy facilitates the progression of ERα-positive breast cancer cells to antiestrogen resistance

A major impediment to the successful treatment of estrogen receptor α (ERα)-positive breast cancer is the development of antiestrogen resistance. Tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to ERα and blocking the growth- promoting action of estrogen-bound ERα in breast cancer cells. Tamoxifen treatment primarily induces cytostasis (growth arrest) and the surviving breast cancer cells commonly acquire tamoxifen resistance. Numerous clinically-relevant mechanisms of acquired antiestrogen resistance have been identified by in vitro studies. Our recent studies (Mol Cancer Ther 2008: 7:2977-87) now demonstrate that autophagy (also referred to as macroautophagy) is critical to the development of antiestrogen resistance. Under conditions of compromised autophagy, including treatments with pharmacological inhibitors and RNAi targeting of the beclin 1 gene, the cytotoxicity (death-inducing effects) of the antiestrogen 4-hydroxytamoxifen (4-OHT) was significantly increased. 4-OHT is an active metabolite of tamoxifen commonly used for in vitro studies. A step-wise drug selection protocol, using 4-OHT as the selecting drug, established antiestrogen-resistant breast cancer cell lines. Analysis of a representative resistant cell line showed an increased ability of the cells to sustain high levels of antiestrogen-induced autophagy without progression to death. Importantly, blockade of autophagosome function in the 4-OHT-treated, antiestrogen-resistant cells induced a robust death response. These data provide strong evidence that autophagy is a key mechanism of cell survival during antiestrogen challenge and progression to antiestrogen resistance. We discuss the potential benefit of blocking autophagosome function to significantly reduce the emergence of antiestrogen-resistant breast cancer cells.     

The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.     

NF-κB suppression provokes the sensitization of hormone-resistant breast cancer cells to estrogen apoptosis

The progression of breast cancer cells to estrogen-independent growth may be accompanied with the paradoxical cell sensitization to estrogen apoptotic action; however, the mechanism of this phenomenon is still unclear. In the present study, we have shown that the sensitization of hormone-resistant breast cancer cells to estrogen apoptotic action is accompanied with the gradual NF-κB suppression. Using the chemical inhibitors of NF-κB as well as the dominant-negative NF-κB constructs, we have proved the sufficiency of NF-κB inhibition for the sensitization of the resistant cells to estrogen apoptosis. Estradiol treatment results in the additional suppression of NF-κB, demonstrating the possible NF-κB involvement in the regulation of cell response to estrogens. Totally, the results presented suggest that the constitutive NF-κB suppression in the estrogen-independent cells may be considered as one of the factors resulting in a imbalance between pro- and anti-apoptotic pathways and enhancement in estrogen apoptotic action in the cells.     

Comparative studies of 5α-reductase inhibitors within MCF-7 human breast cancer cells

We have compared the inhibitory effects of six synthetic steroid analogs (17β-carboxy-4-androsten-3-one benzylanilide (VP-1), 17α -acetoxy-6-methylene-4-pregnene-3, 20-dione (VP-2), 6-methylene-4-pregnene-3, 20-dione (VP-3), 17β-acetoxy-6-methylene-4-androsten-3-one (VP-4), 17β -acetoxy-16, 16-dimethyl-6-methylene-4-androsten-3-one (VP-5), and 3β-hydroxy-16-methylene-5-androsten-17-one (VP-6)) upon 5α-reductase activity within MCF-7 human breast cancer cells and rat prostate. Enzyme assays were performed by quantifying the amounts of [³H]5α-androstan-3α-17β-diol and/or [³H]dihydrotestosterone formed from 40 nM [³H]testosterone within each system. Five μM concentrations of VP-2 and VP-3 inhibited prostatic 5α-reductase by 55 and 65%, respectively, whereas the other analogs showed little activity. In contrast, each of the six analogs was active against MCF-7 homogenate 5α reductase activity. VP-2 and VP-4 demonstrated approx 65 and 70% inhibitions, respectively, whereas the other four compounds inhibited enzyme activity by 40–55% in this system. These results suggest that rat prostate and MCP-7 cells contain different 5α-reductase isozymes. When these agents were examined for 5α-reductase inhibitory activity following 1 h preincubations with intact MCF-7 cultures, VP-1 and 3 demonstrated potencies similar to those in MCF-7 homogenate. The other compounds, however, were far less active under these conditions. Longer culture preincubations (16 h) were associated with substantially increased VP-6 potency, moderate increases for VP-4 and 5, but no change in VP-2 activity. Additional studies examining the abilities of these agents to bind to MCF-7 androgen receptor (AR) and progesterone receptor (PR) revealed moderate AR binding activities of VP-2, 3, and 4, and substantial PR binding for VP-2 and 3. Finally, VP-4 failed to inhibit estrogen-dependent MCF-7 PR synthesis, suggesting that it has no androgenic activity despite its ability to interact with MCF-7 AR.     

A four-gene signature predicts the efficacy of paclitaxel-based neoadjuvant therapy in human epidermal growth factor receptor 2–negative breast cancer

Neoadjuvant chemotherapy (NAC) is the major preoperative treatment of breast cancer (BC) with negative human epidermal growth factor receptor 2 (HER2), and the efficacy of NAC and the optimization of regimen are under intensive research. The current study aimed to define the predictive biomarkers for paclitaxel (PTX) response in NAC of HER2-negative BC. Data from GSE25065, GSE26065, GSE41998, as well as drug sensitivity data of breast and ovarian cancer cell line from NCI60, were used. Through logistic regression, COX regression, and correlation analysis with bootstrapping, we found that four genes (CDK8, FAM64A, MARC2, and OCEL1) were associated with drug sensitivity of PTX. The four gene “≥3” model had the best classification accuracy. Subgroup analysis found that the model performed well in the hormone receptor positive, HER2-negative subgroup and did not perform well in the triple-negative subgroup. Decision curve analysis showed that the model could enhance the predictive effect of clinical features. Subsequent gene set enrichment analysis, network analysis showed that these genes may be related to the cell cycle, mitosis and other pathways. The current study demonstrated the promising potential of the novel four-gene signature as a predictive biomarker for pathological complete response of HER2-negative BC patients and indicated the drug sensitivity of PTX.     

Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity

ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80% of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K_M) rather than the catalytic step (k_cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K_M and k_cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.     

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.     

Curcumin suppresses the TPA-induced invasion through inhibition of PKCα-dependent MMP-expression in MCF-7 human breast cancer cells

Curcumin (diferuloylmethane) is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. Although anti-carcinogenic, anti-oxidant, anti-inflammation, and anti-angiogenic properties have been reported, the effect of curcumin on breast cancer metastasis is unknown. Matrix metalloproteinase-9 (MMP-9) is a major component in cancer cell invasion. In this study, we investigated the inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion and the molecular mechanisms involved in MCF-7 cells. Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Also, curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that curcumin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells. Curcumin may have potential value in restricting breast cancer metastasis.     

Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.     

Using the “reverse Warburg effect” to identify high-risk breast cancer patients

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H2O2), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “Reverse Warburg Effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immuno-staining of human breast cancer tissue microarrays (TMAs; > 180 triple-negative patients) to directly assess the prognostic value of the “Reverse Warburg Effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis, and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (< 18% survival at 10 y post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-y survival rates of ~97% (p-value < 10^?32). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10^?14), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “Reverse Warburg Effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable-target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977, and AZD-3965).     

Radiation-induced modification of human somatic cells’ chromosome sensitivity to the testing mutagenic exposure of bleomycin in vitro in lung cancer patients in delayed terms following chernobyl accident

The above-background level of cytogenetic effect has been studied using the modified G₂-bleomycin sensitivity assay, the value of which was a marker of hidden chromosome instability (HCI). The following three groups were examined: (1) Chernobyl disaster liquidators (CDLs) (occupational group I), (2) patients with lung cancer (LC) who denied voluntary contact with ionizing radiation (comparative group); and (3) patients with LC participating in the liquidation of the Chernobyl disaster (occupational group 2). Significant interindividual variations in the cytogenetic effects induced by bleomycin and the absence of a positive correlation with the background and above-background frequency of chromosome aberrations have been revealed for all the three groups. It was established that occupational group 2 experienced the highest level of above-background cytogenetic effect and therefore had the highest number of patients with HCI. The data allowed us to hypothesize the existence of an association between a radiation-induced increase in individual susceptibility to testing mutagenic exposure and the development of cancer pathologies in patients exposed to ionizing radiation. The results showed the feasibility of using the G₂-bleomycin sensitivity assay for the examination of irradiated contingents for the detection of HCI as an informative marker of predisposition to cancer.     

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.