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 immunoglobulin 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 β- 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.

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.

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′(b,g-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.

To cite this version:Johanna Räikkönen, Hannu Mönkkönen, Seppo Auriola, Jukka Mönkkönen. Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells. Biochemical Pharmacology, Elsevier, 2009, 79 (5) 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 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. Page 1 of 35A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 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

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