The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay has been modified for real-time, quantitative PCR analysis. Here, we describe cost-effective procedures for detection of telomerase activity using a fluorescent-based assay as well as by using real-time PCR. These modified TRAP assays can be accomplished within 4 h (from lysis of samples to analysis of telomerase products).

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3 0 -P5 0 thiophosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first-(GRN163) and secondgeneration (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC 50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipidconjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Purpose: Maintenance of telomeres by telomerase is critical for the continuing proliferation of most advanced cancer cells. Telomerase activity has been detected in the vast majority of cancer cells but not most normal cells, making the enzyme an attractive target for anticancer therapy. The aim of this study was to address the breast cancer translational potential of the novel telomerase inhibitor, GRN163L. Experimental Design: In the present study, we investigated the effects of GRN163L treatment on a panel of breast cancer cells representing different tumor subtypes with varying genetic backgrounds, including ER+, ERÀ, HER2+, BRCA1mutant breast tumor cells as well as doxorubicinresistant cancer cells.To investigate the in vivo effects of GRN163L, we employed a breast cancer xenograft and metastasis model that simulates a clinical situation in which a patient arrives with a primary tumor that may be then treated or surgically removed. Results: GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in all breast cancer cell lines resulting in progressive telomere shortening. A mismatch control oligonucleotide showed no effect on telomerase activity and GRN163L did not significantly affect telomere shortening in normal human mammary epithelial cells or in endothelial cells. Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases (P = 0.017) of MDA-MB-231cells in vivo after 4 weeks of treatment. Conclusions:These results show in vivo effectiveness of GRN163L in breast cancer and support its promising clinical potential for breast cancer treatment.

The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.Entamoeba histolytica is the causative agent of amebiasis and prevails in areas of poor sanitation. The organism is estimated to be responsible for 50 million infections and 100,000 deaths each year. Infection can lead to amebic dysentery, resulting from trophozoites invading the intestinal wall. Amebic liver abscess and other extraintestinal lesions can result through the spread of trophozoites from the intestine into the bloodstream. The host and parasite factors determining infection outcome have yet to be fully elucidated.We hypothesize that alteration in transcription of certain crucial genes may contribute to the expression of the virulence phenotype. Distinct patterns of E. histolytica gene expression have been observed under a variety of experimental conditions (3,6,14,18,19,23,24,27,33,43,44,67). Previously we catalogued the gene expression profile of E. histolytica associated with amebic colitis in the murine model (27). mRNA transcripts more abundant in vivo (mouse colon) than in vitro (laboratory culture) included putative DNA/RNA regulatory factors, representing a pool of potential phenotype-specific transcription factors. The transcript of the locus XM_652200/EHI_093800 was upregulated more than twofold at day 1 of amebic colitis. This gene showed significant sequence similarity to the high-mobility-group box chromosomal protein 1 (HMGB1).HMGB1 is an abundant nonhistone nuclear protein and is a member of the HMG superfamily. The protein is highly conserved in all metazoans, plants, and yeasts and in the parasites Tr...

The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway® cloning system to generate the E. histolytica expression vector pAHDEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was Strep-Myctagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway® system should facilitate investigation into the functions of novel proteins in E. histolytica. Keywords Entamoeba histolytica; Gateway® Vector; EHI_144490Entamoeba histolytica is an enteric aerotolerant parasite that can colonize the large intestine, and invade through the intestinal epithelium to cause amebic colitis and liver abscess [1]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. fairly large data sets [2][3][4][5][6][7]. An efficient and high throughput method is needed for cloning and analyzing the function of the proteins encoded by these transcripts. NIH Public AccessThe Gateway® (Invitrogen) system has been used successfully to stably express open reading frames (ORFs) from P. falciparum by recombinational cloning, which like E. histolytica, contains an AT-rich genome [8][9][10]. The Gateway® system also has the potential to rapidly transfer cloned inserts from one vector to another [11]. To construct the E. histolytica 'Destination' plasmid we used the well-characterized plasmid pGir308 as the backbone of the new construct [12]. This vector uses the E. histolytica ferredoxin promoter, which drives strong expression of cloned genes in vivo and in vitro [2,13]. The vector also carries a hygromycin resistance gene, which permits the use of this vector in co-infection studies with other E. histolytica shuttle vectors that carry G418-selectable markers [14].The vector pGir308 was digested with Xb...