JNK pathway inhibition selectively primes pancreatic cancer stem cells to TRAIL-induced apoptosis without affecting the physiology of normal tissue resident stem cells.

OBJECTIVE: Successful treatment of solid cancers mandates targeting cancer stem cells (CSC) without impact on the physiology of normal tissue resident stem cells. C-Jun N-terminal kinase (JNK) signaling has been shown to be of importance in cancer. We test whether JNK inhibition would sensitize pancreatic CSCs to induction of apoptosis via low-dose TNFα-related apoptosis-inducing ligand (TRAIL). DESIGN: Effects of JNK inhibition (JNKi) were evaluated in vitro in functional assays, through mRNA and protein expression analysis, and in in vivo mouse studies. CSCs were enriched in anoikis-resistant spheroid culture and analyzed accordingly. RESULTS: We confirmed that the JNK pathway is an important regulatory pathway in pancreatic cancer stem cells and further found that JNK inhibition downregulates the decoy receptor DcR1 through IL-8 signaling while upregulating pro-apoptotic death receptors DR4/5, thereby sensitizing cells - even with acquired TRAIL-resistance - to apoptosis induction. Treatment of orthotopic pancreatic cancer xenografts with either gemcitabine, JNKi or TRAIL alone for 4 weeks showed only modest effects compared to control, while the combination of JNKi and TRAIL resulted in significantly lower tumor burden (69%; p < 0.04), reduced numbers of circulating tumor cells, and less distant metastatic events, without affecting the general health of the animals. CONCLUSIONS: The combination of JNKi and TRAIL significantly impacts on CSCs, but leaves regular tissue-resident stem cells unaffected - even under hypoxic stress conditions. This concept of selective treatment of pancreatic CSCs warrants further evaluation.

Targeting piggyBac transposon integrations in the human genome.

DNA based transposon systems offer a technology for active and efficient delivery of genes into human cells. An emerging field is directed at manipulating such systems to achieve site-directed integration as compared to un-targeted integration which occurs with native or unmodified transposon systems. The naturally active piggyBac transposon system is derived from insects but has been shown to be very efficient in gene-modifying human cells. Recent efforts have utilized the fusion of DNA binding domains to the piggyBac transposase enzyme with the goal of targeting integration to specific locations in the human genome. In this chapter, we describe methodology for engineering and characterizing chimeric piggyBac transposase enzymes, including experimental approaches for evaluating activity and targeting specificity in the human genome.

Manipulating piggyBac transposon chromosomal integration site selection in human cells.

The ability to direct gene delivery to a user-defined chromosomal location would greatly improve gene transfer applications. The piggyBac transposon system is a nonviral gene transfer system proven effective in a variety of cells and tissues, including human cells. We fused a highly site-specific synthetic zinc-finger DNA-binding domain (ZFP) to the N-terminus of the piggyBac transposase and evaluated site-directed genomic integration in human cells. Chimeric ZFP-piggyBac transposase exhibited robust gene transfer activity, targeted binding to a cognate endogenous chromosomal ZFP site in the human genome, and site-directed transposon integration into an episomal plasmid target containing a single ZFP site in human cells. We evaluated the ability of ZFP-piggyBac to direct gene integration into an engineered chromosomal ZFP target site in the human genome and consistently observed a higher degree of ZFP-piggyBac site-directed genomic integration when compared to native piggyBac. Chromatin immunoprecipitation (ChIP) experiments revealed binding of native piggyBac to our engineered TTAA-containing chromosomal target which supported integration, but not a TTAA-deficient chromosomal target which lacked integration. Our results offer insight into the requirements for using a chimeric zinc finger-piggyBac transposase to direct integration into a user-defined chromosomal location.

Genome-wide mapping of PiggyBac transposon integrations in primary human T cells.

The piggyBac transposon system represents a promising nonviral tool for gene delivery and discovery, and may also be of value for clinical gene therapy. PiggyBac is a highly efficient integrating vector that stably transfects (approximately 40%) of primary human T cells for potential adoptive immunotherapy applications. To evaluate the potential genotoxicity of piggyBac, we compared 228 integration sites in primary human T cells to integrations in 2 other human-derived cell lines (HEK293 and HeLa) and randomly simulated integrations into the human genome. Our results revealed distinct differences between cell types. PiggyBac had a nonrandom integration profile and a preference for transcriptional units (approximately 50% into RefSeq genes in all cell types), CpG islands (18% in T cells and 8% in other human cells), and transcriptional start sites (<5 kb, 16% to 20% in all cell types). PiggyBac also preferred TTAA but not AT-rich regions of the human genome. We evaluated the expression of mapped genes into which piggyBac integrated, and found selection of more active genes in primary human T cells compared with other human cell types, possibly due to concomitant T-cell activation during transposition. Importantly, we found that in comparison to what has been reported for gammaretroviral and human lenitviral vectors, piggyBac had decreased integration frequency into or within 50 kb of the transcriptional start sites of known proto-oncogenes. Hence the piggyBac nonviral gene delivery system seems to represent a promising gene transfer system for clinical applications using human T lymphocytes.

Cardiac type 2 inositol 1,4,5-trisphosphate receptor: interaction and modulation by calcium/calmodulin-dependent protein kinase II.

The type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP(3)R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP(3)R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP(3)R2 associates with Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta), the major isoform expressed in cardiac myocytes. Recombinant InsP(3)R2 and CaMKIIdelta(B) also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP(3)R2 were sufficient for interaction with CaMKIIdelta(B) and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP(3)R2, as demonstrated by (32)P labeling. Incorporation of CaMKII-treated InsP(3)R2 into planar lipid bilayers revealed that InsP(3)-mediated channel open probability is significantly reduced ( approximately 11 times) by phosphorylation via CaMKII. We concluded that the InsP(3)R2 and CaMKIIdelta likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP(3)R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP(3)R2 function.

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions.

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (approximately 55%) in mammalian and amphibian channels. Two amplitudes, differing by approximately 35%, were found in amphibian channel studies, probably corresponding to alpha and beta RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (approximately 40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy.

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.