Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Blockade of MUC1 expression by glycerol guaiacolate inhibits proliferation of human breast cancer cells.

We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer.

Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation.

BACKGROUND: Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. METHODS: TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitro-generated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. RESULTS: Tumor cell-derived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. CONCLUSIONS: MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer.

We investigated the feasibility of using radioiodine therapy in colon carcinoma cells (HCT 116) following tumor-specific expression of the human sodium iodide symporter (hNIS) using the carcinoembryonic antigen (CEA) promoter. HCT 116 cells were stably transfected with an expression vector, in which hNIS cDNA has been coupled to a CEA promoter fragment. This promoter is responsible for tissue-specific expression of CEA in gastrointestinal tract epithelium, and has been shown to target therapeutic genes to colorectal cancer cells. Functional NIS expression was confirmed by iodide uptake assay, Western blot analysis, immunostaining and in vitro clonogenic assay. The stably transfected HCT 116 cells concentrated (125)I about 10-fold in vitro without evidence of iodide organification. In contrast, transfection of control cancer cells without CEA expression did not result in iodide accumulation. Western blot analysis using a hNIS-specific antibody revealed a band of approximately 90 kDa. In addition, immunostaining of stably transfected HCT 116 cells revealed hNIS-specific membrane-associated immunoreactivity. In an in vitro clonogenic assay approximately 95% of stably transfected HCT 116 cells were killed by exposure to (131)I, while only about 5% of NIS-negative control cells were killed. Further, using an adenovirus carrying the NIS gene linked to the CEA promoter, high levels of tumor-specific radioiodide accumulation were induced in HCT 116 cells. In conclusion, a therapeutic effect of (131)I has been demonstrated in colon carcinoma cells following induction of tumor-specific iodide uptake activity by CEA promoter-directed NIS expression in vitro. This study demonstrates the potential of NIS as a therapeutic gene allowing radioiodine therapy of colon cancer following tumor-specific NIS gene transfer.

The sodium-iodide symporter.

The sodium-iodide symporter (NIS) is an intrinsic plasma membrane protein that mediates active transport of iodide in the thyroid gland and several other extra-thyroidal tissues. This activity has been utilized for many years for imaging the thyroid gland and for treatment of thyroid disease both benign and malignant. Cloning and characterization of NIS has more recently allowed research into its use in non-thyroidal cancers through gene transfer for both diagnosis and treatment.

Amino acid substitution at position 99 affects the rate of CRP subunit exchange.

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Inhibition of growth and metastasis of human pancreatic cancer growing in nude mice by PTK 787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.

Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blockage of VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK 787) decreases the growth and metastasis of human pancreatic carcinoma growing orthotopically in nude mice. Human pancreatic L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice were given daily oral administrations of PTK 787 alone, twice weekly i.p. injections of gemcitabine, or combination therapy. The mice were necropsied when control mice became moribund (day 35). Therapy with PTK 787 alone, gemcitabine alone, or the combination of both agents produced respectively 60%, 70%, and 81% inhibition in the volume of pancreatic cancers. The combination therapy significantly decreased the incidence of lymph node and liver metastasis, leading to a significant increase in survival. Microvessel density (MVD) was significantly decreased in tumors treated with either PTK 787 alone or PTK 787 plus gemcitabine. MVD directly correlated with tumor cell proliferation and inversely correlated with apoptosis of tumor cells and associated endothelial cells. Collectively, our results demonstrate that blockade of VEGF-R signaling may provide an additional approach to the therapy of pancreatic cancer.

Position 127 amino acid substitutions affect the formation of CRP:cAMP:lacP complexes but not CRP:cAMP:RNA polymerase complexes at lacP.

The lacP DNA binding and activation characteristics of CRP having amino acid substitutions at position 127 were investigated. Wild-type (WT) and T127C CRP footprinted lacP DNA in the presence of DNase I in a cAMP-dependent manner. The T127G, T127I, and T127S forms of CRP failed to footprint lacP both in the absence and in the presence of cAMP. Consistent with these data, WT and T127C CRP:cAMP complexes exhibited high affinity for the lacP CRP site whereas T127G, T127I, or T127S CRP:cAMP complexes exhibited low affinity for the lacP CRP site. CRP:cAMP:RNA polymerase (RNAP) complexes formed at lacP in reactions that contained WT, T127C, T127G, T127I, or T127S CRP. These results demonstrate that allosteric changes important for cAMP-mediated CRP activation are differentially affected by amino acid substitution at position 127. Proper cAMP-mediated reorientation of the DNA binding helices required either threonine or cysteine at position 127. However, cAMP-dependent interaction of CRP with RNAP was accomplished regardless of the amino acid at position 127. RNAP:lacP complexes that supported high-level lac RNA synthesis formed rapidly in reactions that contained WT or T127C CRP whereas RNAP:lacP complexes that supported only low-level lac RNA synthesis formed at slower rates in reactions that contained T127I or T127S CRP. The T127G CRP:cAMP:RNAP:lacP complex failed to activate lacP. The results of this study lead us to conclude that threonine 127 plays an important role in transduction of the signal from the CRP cyclic nucleotide binding pocket that promotes proper orientation of the DNA binding helices and only a minor, if any, role in the functional exposure of the CRP RNAP interaction domain.