Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells.

Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 mAb stimulation because IL-2 is released by ß(-) Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophosphates but does not bind the intermediates with high affinity.

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells.

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K(isom)) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K(isom) values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway. As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K(isom) calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.

siRNA transfection with calcium phosphate nanoparticles stabilized with PEGylated chelators.

Despite the enormous therapeutic potential of siRNAs, their delivery is still problematic due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. However, rapid particle growth has largely prevented the translation of this method for in vivo purposes. It has recently been shown that bisphosphonate derivatives can physically stabilize calcium phosphate nanoparticles while still allowing for efficient cell transfection with plasmid DNA. Herein, two novel PEGylated chelating agents (PEG-alendronate and PEG-inositolpentakisphosphate) with enhanced stabilizing properties are introduced, and it is demonstrated that the bisphosphonate-stabilized nanoparticles can efficiently deliver siRNA in vitro. The nanoparticles are mainly taken up by clathrin-dependent endocytosis, and acidification of the endosomal compartment is required to release the entrapped siRNA into the cytosol. Furthermore, particle uptake enhances the inhibition of the mevalonate pathway by the bisphosphonate in macrophages.

Viability of freeze dried microencapsulated human retinal pigment epithelial cells.

Encapsulated human retinal pigment epithelial cell line ARPE-19 has been successfully used in experimental cell therapy of retinal degenerations and Parkinson's disease, but the long-term storage of encapsulated cells is still an unresolved question. Reconstitution of viable encapsulated cells from dry form would benefit the development of cell therapy products. We freeze dried and reconstituted microencapsulated ARPE19 and ARPE19-SEAP cells. Cross-linked alginate matrix with polycation (poly-l-lysine, cationic starch) coating was used for microencapsulation. Cell viability was assessed with fluorescence microscopy and oxygen consumption of the cells. Freeze dried and reconstituted cell microcapsules were imaged using scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM). We show partial viability of microencapsulated cells after freeze-drying. Unlike poly-l-lysine (PLL) coating, cationic starch supported microcapsule shape and cell viability during freeze-drying. Trehalose pre-treatment augmented cell viability. Likewise, some lyoprotectants (trehalose, glycerol) enabled preservation of cell viability. Upon reconstitution the freeze dried cell microcapsules rapidly regained their original spherical shape. This proof-of-concept study demonstrates that microencapsulated cells can retain their viability during freeze-drying. Therefore, this approach can be further optimized for the benefit of cell therapy product development.

Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells.

Increasing evidence is accumulating that zoledronic acid (ZOL), a nitrogen-containing bisphosphonate (N-BP), is able to affect tumor cells by inhibiting the enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway (MVP). The consequent accumulation of unprenylated proteins is believed to largely account for the cytotoxic effects of ZOL. FPPS inhibition leads also to the accumulation of isopentenyl pyrophosphate (IPP) and the apoptotic ATP analog, ApppI, but the role of this mechanism in the cytotoxic action of bisphosphonates is less clear. Since treatment with MVP intermediates has been shown to overcome N-BP-induced apoptosis via rescuing protein prenylation, our aim here was to determine their mechanism of action on ZOL-induced IPP/ApppI accumulation. Interestingly, the results revealed that ZOL-induced IPP/ApppI accumulation in MCF-7 cells were decreased by farnesol, and almost completely blocked by geranylgeraniol and geranylpyrophosphate. The functionality of the regulatory enzymes of IPP and ApppI, IPP isomerase and aminoacyl-tRNA-synthase, respectively, or protein levels of FPPS were not affected by the treatments. However, the protein levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and unprenylated Rap1A were observed to be strongly downregulated by geranylgeraniol and geranylpyrophosphate. This study represents a novel insight into the mechanism of action of MVP intermediates on the regulation of MVP after FPPS inhibition. The data implies that in addition to the previously reported effects on rescuing protein prenylation, MVP intermediates can preserve cell activity by inhibiting the accumulation of IPP/ApppI via HMGR downregulation. This supports the hypothesis that IPP/ApppI formation is a significant mechanism in the anticancer action of ZOL.

Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry.

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC-MS(2) for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030microM (1.35fmol on-column) and for ApppI and ApppD 0.020microM (0.9fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS(2) were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS(3) spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25muM zoledronic acid (an N-BP) for 24h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4nmol/mg of protein and amount of ApppI and ApppD was 1.1nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.

Zoledronic acid induces formation of a pro-apoptotic ATP analogue and isopentenyl pyrophosphate in osteoclasts in vivo and in MCF-7 cells in vitro.

BACKGROUND AND PURPOSE: Bisphosphonates (BPs) are highly effective inhibitors of bone resorption. Nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid, induce the formation of a novel ATP analogue (1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester triphosphoric acid; ApppI), as a consequence of the inhibition of farnesyl pyrophosphate synthase and the accumulation of isopentenyl pyrophosphate (IPP). ApppI induces apoptosis, as do comparable metabolites of non-nitrogen-containing bisphosphonates (non-N-BPs). In order to further evaluate a pharmacological role for ApppI, we obtained more detailed data on IPP/ApppI formation in vivo and in vitro. Additionally, zoledronic acid-induced ApppI formation from IPP was compared with the metabolism of clodronate (a non-N-BP) to adenosine 5'(beta,gamma-dichloromethylene) triphosphate (AppCCl2p). EXPERIMENTAL APPROACH: After giving zoledronic acid in vivo to rabbits, IPP/ApppI formation and accumulation was assessed in isolated osteoclasts. The formation of ApppI from IPP was compared with the metabolism of clodronate in MCF-7 cells in vitro. IPP/ApppI and AppCCl2p levels in cell extracts were analysed by mass spectrometry. KEY RESULTS: Isopentenyl pyrophosphate/ApppI were formed in osteoclasts in vivo, after a single, clinically relevant dose of zoledronic acid. Furthermore, exposure of MCF-7 cells in vitro to zoledronic acid at varying times and concentrations induced time- and dose-dependent accumulation of IPP/ApppI. One hour pulse treatment was sufficient to cause IPP accumulation and subsequent ApppI formation, or the metabolism of clodronate into AppCCl2p. CONCLUSIONS AND IMPLICATIONS: This study provided the first conclusive evidence that pro-apoptotic ApppI is a biologically significant molecule, and demonstrated that IPP/ApppI analysis is a sensitive tool for investigating pathways involved in BP action.