Terpyridine platinum compounds induce telomere dysfunction and chromosome instability in cancer cells.

Telomerase/telomere-targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. We recently developed a dual-HAC (Human Artificial Chromosome) assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic HT1080 cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Results: In this study, we used the dual-HAC assay for the analysis of the platinum-derived G4 ligand Pt-tpy and five of its derivatives: Pt-cpym, Pt-vpym, Pt-ttpy, Pt(PA)-tpy, and Pt-BisQ. Our analysis revealed four compounds, Pt-tpy, Pt-ttpy, Pt-vpym and Pt-cpym, that induce a specific loss of a linear but not a circular HAC. Increased CIN after treatment by these compounds correlates with the induction of double-stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere-associated DNA damage. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges (CBs) in late mitosis and cytokinesis. These terpyridine platinum-derived G4 ligands are promising compounds for cancer treatment.

Systematic Analysis of Compounds Specifically Targeting Telomeres and Telomerase for Clinical Implications in Cancer Therapy.

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene; compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds, Cu-ttpy and Pt-ttpy, induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (ultrafine bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.Significance: An assay provides a unique opportunity to screen thousands of chemical compounds for their ability to inactivate replication of telomeric ends in cancer cells and holds potential to lay the foundation for the discovery of new treatments for cancer. Cancer Res; 78(21); 6282-96. ©2018 AACR.

Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System.

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoidtetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoidtetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells.

Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this "loss of signal" assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this "gain of signal" assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The "gain of signal" assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level.

Effects of Anticancer Drugs on Chromosome Instability and New Clinical Implications for Tumor-Suppressing Therapies.

Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a nonessential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Here, we used this assay to rank 62 different anticancer drugs with respect to their effects on chromosome transmission fidelity. Drugs with various mechanisms of action, such as antimicrotubule activity, histone deacetylase inhibition, mitotic checkpoint inhibition, and targeting of DNA replication and damage responses, were included in the analysis. Ranking of the drugs based on their ability to induce HAC loss revealed that paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin were the top 10 drugs demonstrating HAC loss at a high frequency. Therefore, identification of currently used compounds that greatly increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target and leverage the CIN phenotype in cancer cells.

Protecting a transgene expression from the HAC-based vector by different chromatin insulators.

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.

A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells.

BACKGROUND: Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory. METHODS: We have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry. RESULTS: Using the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A. CONCLUSION: Thus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.

Olfactomedin 1 interacts with the Nogo A receptor complex to regulate axon growth.

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.

Exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1-linked families.

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. Our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.

Organization of synthetic alphoid DNA array in human artificial chromosome (HAC) with a conditional centromere.

Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy, and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of the de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoid(tetO)-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoid(tetO)-HAC that has been formed from a ~50 kb synthetic alphoid(tetO)-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoid(tetO)-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells.

Myocilin is a modulator of Wnt signaling.

It is well documented that mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. However, the functions of wild-type myocilin are still not well understood. To study the functions of human myocilin and its two proteolytic fragments, these proteins were expressed in HEK293 cells. Conditioned medium from myocilin-expressing cells, as well as purified myocilin, induced the formation of stress fibers in primary cultures of human trabecular meshwork or NIH 3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin, as well as secreted inhibitors of Wnt signaling, secreted Frizzled-related protein 1 (sFRP1) or sFRP3, and beta-catenin small interfering RNA. Interaction of myocilin with sFRP1, sFRP3, and several Frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different Frizzled and sFRPs. Treatment of NIH 3T3 cells with myocilin and its fragments induced intracellular redistribution of beta-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of beta-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of beta-catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, since myocilin modulates Wnt signaling by interacting with components of this signaling pathway.

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells.

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.