Modulation of peritumoral fibroblasts with a membrane-tethered tissue inhibitor of metalloproteinase (TIMP) for the elimination of cancer cells.

BACKGROUND: Peritumoral fibroblasts are key components of the tumor microenvironment. Through remodeling of the extracellular matrix (ECM) and secretion of pro-tumorigenic cytokines, peritumoral fibroblasts foster an immunosuppressive milieu conducive to tumor cell proliferation. In this study, we investigated if peritumoral fibroblasts could be therapeutically engineered to elicit an anti-cancer response by abolishing the proteolytic activities of membrane-bound metalloproteinases involved in ECM modulation. METHODS: A high affinity, glycosylphosphatidylinositol (GPI)-anchored Tissue Inhibitor of Metalloproteinase (TIMP) named "T1PrαTACE" was created for dual inhibition of MT1-MMP and TACE. T1PrαTACE was expressed in fibroblasts and its effects on cancer cell proliferation investigated in 3D co-culture models. RESULTS: T1PrαTACE abrogated the activities of MT1-MMP and TACE in host fibroblasts. As a GPI protein, T1PrαTACE could spontaneously detach from the plasma membrane of the fibroblast to co-localize with MT1-MMP and TACE on neighboring cancer cells. In a 3D co-culture model, T1PrαTACE promoted adherence between the cancer cells and surrounding fibroblasts, which led to an attenuation in tumor development. CONCLUSION: Peritumoral fibroblasts can be modulated with the TIMP for the elimination of cancer cells. As a novel anti-tumor strategy, our approach could potentially be used in combination with conventional chemo- and immunotherapies for a more effective cancer therapy.

Transcription Factor AP4 Mediates Cell Fate Decisions: To Divide, Age, or Die.

Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) is a basic-helix-loop-helix-leucine-zipper transcription factor that was first identified as a protein bound to SV40 promoters more than 30 years ago. Almost 15 years later, AP4 was characterized as a target of the c-Myc transcription factor, which is the product of a prototypic oncogene that is activated in the majority of tumors. Interestingly, AP4 seems to represent a central hub downstream of c-Myc and N-Myc that mediates some of their functions, such as proliferation and epithelial-mesenchymal transition (EMT). Elevated AP4 expression is associated with progression of cancer and poor patient prognosis in multiple tumor types. Deletion of AP4 in mice points to roles of AP4 in the control of stemness, tumor initiation and adaptive immunity. Interestingly, ex vivo AP4 inactivation results in increased DNA damage, senescence, and apoptosis, which may be caused by defective cell cycle progression. Here, we will summarize the roles of AP4 as a transcriptional repressor and activator of target genes and the contribution of protein and non-coding RNAs encoded by these genes, in regulating the above mentioned processes. In addition, proteins interacting with or regulating AP4 and the cellular signaling pathways altered after AP4 dysregulation in tumor cells will be discussed.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

A Simple Algorithm for Population Classification.

A single-nucleotide polymorphism (SNP) is a variation in the DNA sequence that occurs when a single nucleotide in the genome differs across members of the same species. Variations in the DNA sequences of humans are associated with human diseases. This makes SNPs as a key to open up the door of personalized medicine. SNP(s) can also be used for human identification and forensic applications. Compared to short tandem repeat (STR) loci, SNPs have much lower statistical testing power for individual recognition due to the fact that there are only 3 possible genotypes for each SNP marker, but it may provide sufficient information to identify the population to which a certain samples may belong. In this report, using eight SNP markers for 641 samples, we performed a standard statistical classification procedure and found that 86% of the samples could be classified accurately under a two-population model. This study suggests the potential use of SNP(s) in population classification with a small number (n ≤ 8) of genetic markers for forensic screening, biodiversity and disaster victim controlling.

Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated ß-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells.

Effects of three-layered nanodisk size on cell detection sensitivity of plasmon resonance biosensors.

The double resonance plasmonic biosensors based on Au nanodisks (NDs) with a thin SiO2 spacer between the top and bottom Au layers were employed to detect MCF-7 breast cancer cells. The hybridized modes between the localized surface plasmon resonance of Au NDs and the gap coupling resonance of NDs with the Au film underneath have been observed. These multiple metallic layer NDs exhibit higher sensitivity than the common single metallic layer NDs. The extinction spectra showed double resonance bands that could be tailored by varying the ND size. Three sizes of multiple layer NDs ranging from 60 to 200 nm diameter (dia.) were generated and their refractive sensitivity to the surrounding media were analyzed for cell detection. Nanodisks with 120 dia. showed the highest refractive sensitivity up to 230 nm/refractive index unit. These sensors could be used to detect a broad range of MCF-7 cells from a low cell concentration down of 1.0×10(3)cells/ml up to a high cell concentration of 1.7×10(7) cells/ml.

AP4 activates cell migration and EMT mediated by p53 in MDA-MB-231 breast carcinoma cells.

Tumor metastasis is the primary cause of mortality in most cancer patients. Before disassociation from the tumors, most of malignant tumor cells undergo the epithelial-mesenchymal transition to break away from the adhesions between the cells and the surrounding extracellular matrix. Recently, activating enhancer-binding protein (AP4) has been shown to be a mediator of EMT in colorectal cancer and high level of AP4 correlates with poor prognosis in cancer patients. It has been found that AP4 upregulates the genes involved in EMT and cell proliferation in colorectal cancer cells and that the aggressive human breast cancer cells MDA-MB-231 are highly metastatic. Therefore, we tested the hypothesis that AP4 may also affect cell migration and EMT in this cell type. Three different assays, including the wound-healing assay, the Boyden chamber assay, and the cell tracking assay, were employed to confirm that AP4 activated both cell migration and invasion. Immunofluorescence staining and Western blot analysis revealed that the cells underwent EMT when AP4 was upregulated. In contrast, overexpression of dominant-negative AP4, lacking the DNA-binding domain, inactivated the DNA-binding ability of endogenous AP4 and led to lower cell motility. Furthermore, we found that AP4 enhanced p53 expression at both transcriptional and translational levels. Knockdown of p53 by siRNA significantly diminished the activation of cell migration by AP4, indicating that AP4 can regulate cell migration via the activity of p53.

Efficient co-delivery of a Pt(IV) prodrug and a p53 activator to enhance the anticancer activity of cisplatin.

A nanoplatform targeting DNA and p53 simultaneously is assembled. Layered double hydroxide nanoparticles are co-loaded with a Pt(IV) prodrug and a p53 activator. Once inside cells, cisplatin is released to attack genomic DNA and kill cancer cells; simultaneously, the p53 activator results in active p53, a key protein involved in the apoptotic pathways initiated by platinum drugs. The anticancer efficacy of cisplatin is significantly improved through this synergistic application.

Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth.

BACKGROUND: Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection. RESULTS: In this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo. CONCLUSIONS: We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

Luminescent ruthenium(II) complex bearing bipyridine and N-heterocyclic carbene-based C∧N∧C pincer ligand for live-cell imaging of endocytosis.

Luminescent ruthenium(II)-cyanide complex with N-heterocyclic carbene pincer ligand C(∧)N(∧)C = 2,6-bis(1-butylimidazol-2-ylidene)pyridine and 2,2'-bipyridine (bpy) shows minimal cytotoxicity to both human breast carcinoma cell (MCF-7) and human retinal pigmented epithelium cell (RPE) in a wide range of concentration (0.1-500 µM), and can be used for the luminescent imaging of endocytosis of the complex in these cells.

Chalcoplatin, a dual-targeting and p53 activator-containing anticancer platinum(IV) prodrug with unique mode of action.

Complexation of cisplatin with a p53 activator as a single anticancer agent resulted in synergistically improved cytotoxicity in p53 wild-type but not p53 null human cancer cells. Mechanistic investigation was carried out on this dual-targeting Pt(IV) prodrug, chalcoplatin. The prodrug effectively entered cancer cells and arrested the cell cycle at the S and G2/M phases, distinctive of that from cisplatin. Chalcoplatin significantly induced p53 activation as well as the subsequent apoptosis pathways. This unique mode of action renders chalcoplatin remarkably cytotoxic and makes this compound among the first examples of a Pt(IV) prodrug that directly interacts with the downstream pathway after the formation of Pt-DNA lesions.

Identification of Vß7.1_H3F7 as a therapeutic gene encoding TCR specific to hepatocellular carcinoma.

The feasibility of T-Cell receptor (TCR) gene therapy using a MART-1-specific TCR has been previously demonstrated in melanoma patients. However, it remains a challenge without a defined tumor-specific antigen in the therapy of hepatocellular carcinoma (HCC). In this study, through the analysis of clonal expansion of TCR Vß subfamily and DNA sequencing, we identified TCR Vß7.1_H3F7 as a potential therapeutic gene specifically for the HCC patients. Peripheral blood monouclear cells (PBMC) transfected with TCRV ß7.1_H3F7 gene were specifically cytotoxic against HCC cells in vitro. Adoptive transfer of this transfected PBMC resulted in a marked suppression of HCC tumor development in the animal model. These results demonstrated the value of TCRV ß7.1_H3F7 as a therapeutic gene specifically for HCC. More importantly, it provides a novel strategy for screening tumor-specific TCR genes, which may pave the way for TCR gene therapy in cancer patients currently without the defined tumor-specific antigens.

Effects of nanoparticle size and cell type on high sensitivity cell detection using a localized surface plasmon resonance biosensor.

A localized surface plasmon resonance (LSPR) effect was used to distinguish cell concentration on ordered arrays of Au nanoparticles (NPs) on glass substrates. Human-derived retinal pigment epithelial RPE-1 cells with flatter bodies and higher confluency were compared with breast cancer MCF-7 cells. Nanosphere lithography was used to form Au NPs with average diameters of 500 and 60 nm in order to compare cell detection range, resonance peak shift, and cell concentration sensitivity. A larger cell concentration range was detected on the larger 500 nm Au NPs compared to 60 nm Au NPs (8.56 × 10(3)-1.09 × 10(6) vs. 3.43 × 10(4)-2.73 × 10(5)cells/ml). Resonance peak shift could distinguish RPE-1 from MCF-7 cells on both Au NPs. RPE-1 cells consistently displayed larger resonance peak shifts compared to MCF-7 cells until the detection became saturated at higher concentration. For both types of cells, higher concentration sensitivity in the range of ~10(4)-10(6)cells/ml was observed on 500 nm compared to 60 nm Au NPs. Our results show that cells on Au NPs can be detected in a large range and at low concentration. Optimal cell sensing can be achieved by altering the dimensions of Au NPs according to different cell characteristics and concentrations.

Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli.

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

Unique marker finder algorithm generates molecular diagnostic markers.

By taking advantage of the power of comparative genomics, we devised an algorithm, Unique Marker Finder (U-MarFin), to generate a collection of unique DNA sequences from a target organism. The whole target genome is partitioned into a scoring pool of less 4000 base-pair fragments, which are then subjected to elimination of homologous sequences in other bacterial genomes by BLAST alignment, and looked for all open reading frames as they may be applied as unique markers. Through regular, nested, multiplex and real time PCR and microarray technology, we empirically demonstrated that the sequences discovered were highly specific to the species that they are derived from, and they can serve as molecular biomarkers for diagnostic purpose.

Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b) in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.

BACKGROUND: CITED proteins belong to a family of non-DNA-binding transcriptional co-regulators that are characterized by a conserved ED-rich domain at the C-terminus. This family of genes is involved in the regulation of a variety of transcriptional responses through interactions with the CBP/p300 integrators and various transcription factors. In fish, very little is known about the expression and functions of CITEDs. RESULTS: We have characterized two closely related but distinct CITED3 genes, gcCited3a and gcCited3b, from the hypoxia-tolerant grass carp. The deduced gcCITED3a and gcCITED3b proteins share 72% amino acid identity, and are highly similar to the CITED3 proteins of both chicken and Xenopus. Northern blot analysis indicates that the mRNA expression of gcCited3a and gcCited3b is strongly induced by hypoxia in the kidney and liver, respectively. Luciferase reporter assays demonstrated that both gene promoters are activated by gcHIF-1. Further, ChIP assays comparing normal and hypoxic conditions reveal differential in vivo binding of gcHIF-1 to both gene promoters in kidney and liver tissues. HRE-luciferase reporter assays demonstrated that both gcCITED3a and gcCITED3b proteins inhibit gcHIF-1 transcriptional activity, and GST pull-down assays confirmed that both proteins bind specifically to the CH1 domain of the grass carp p300 protein. CONCLUSION: The grass carp gcCITED3a and gcCITED3b genes are differentially expressed and regulated in different fish organs in response to hypoxic stress. This is the first report demonstrating in vivo regulation of two closely-related CITED3 isogenes by HIF-1, as well as CITED3 regulation of HIF-1 transcriptional activity in fish. Overall, our findings suggest that unique molecular mechanisms operate through these two gcCITED3 isoforms that likely play an important regulatory role in the hypoxic response in the grass carp.

Complementary quantitative proteomics reveals that transcription factor AP-4 mediates E-box-dependent complex formation for transcriptional repression of HDM2.

Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI.SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in a transcriptional-regulating complex at the HDM2-P2 promoter in response to DNA damage.

Dextran sulfate provides a quantitative and quick microarray hybridization reaction.

Microarray technology is a powerful tool to speed up genomics study, yet many technical aspects need to be improved. The hybridization reaction of microarray experiments is carried out for 16h or overnight in order to obtain reasonably strong signals for analysis in the presence of high salt buffer, like SSC. However, the quantitative aspect of microarray hybridization has seldom been investigated. In this study, we showed that higher overall signals from hybridization were achieved in a buffer system containing dextran sulfate, which can accelerate the kinetics of reaction by increasing the local concentration of the reactants. The dextran sulfate containing hybridization solution increases the reaction 4-fold (median) for cDNA microarray and 29-fold for oligonucleotide microarray. More importantly, the solution also provides a quantitative hybridization reaction, where the hybridization signals are proportional to the abundance of transcript added. The enhancement in the kinetics of hybridization is due to both dextran sulfate and formamide present in the solution, but the effect is not due to the higher temperature used during the reaction. With a slightly longer reaction time the hybridization reaction with the solution allows the detection of hybridization signals from rare transcripts that is not possible with regular hybridization buffers. With appropriate washing, the enhancement of kinetics by the solution does not increase the background signals at all, allowing higher signal-to-noise ratios to be achieved.

Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.