We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 μm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.
Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)—proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.
We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through a unique mechanism. A biotinylated form of diazonamide A affinity purifies ornithine ␦-amino transferase (OAT), a mitochondrial enzyme, from HeLa cell and Xenopus egg extracts. In the latter system, the interaction between diazonamide A and OAT is regulated by RanGTP. We find that specific OAT knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death, the effects largely phenocopying diazonamide A treatment in these cell lines. Our experiments reveal an unanticipated, paradoxical role for OAT in mitotic cell division and identify the protein as a target for chemotherapeutic drug development.A variety of small molecules block progression through M phase of the cell cycle. The most common are tubulin ligands. Tubulin-binding toxins have helped elucidate the structure and organization of the mitotic spindle, and certain of these toxins are clinically effective as cancer chemotherapy. However, systemic disruption of the tubulin cytoskeleton has drawbacks. Microtubule poisons disturb nonmitotic functions of the cytoskeleton in both replicating cells and differentiated nondividing cells. Wasting, neutropenia, and peripheral neuropathy are severe dose-limiting toxicities common to this family of drugs in vivo (1). As a result, considerable effort has been made to identify alternative antineoplastics that target mitotic regulatory factors or components of the spindle other than tubulin (2). The development of specific inhibitors of the aurora kinases (3) and the kinesin motor protein Eg5 (2) are notable recent examples.
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