Vinblastine, a chemotherapeutic drug, inhibits palmitoylation of tubulin in human leukemic lymphocytes.

BACKGROUND: We have previously shown that tubulin, the major protein of microtubules, is posttranslationally modified by palmitoylation. In addition, we demonstrated that palmitoylation of tubulin is inhibited in vitro by stoichiometric levels of the chemotherapeutic drug, vinblastine. Here, we sought to determine whether a clinically relevant dose of vinblastine inhibits palmitoylation of tubulin in vivo. METHODS: Human CEM leukemic lymphocytes were incubated with [3H]palmitate in the presence and absence of a low, clinically relevant dose of vinblastine. [3H]palmitoylated tubulin was identified by two-dimensional PAGE and autoradiography. RESULTS: We found, first, that tubulin was palmitoylated in CEM cells. Second, the clinically relevant dose of vinblastine inhibited palmitoylation of tubulin in vivo in CEM cells. In addition, microtubules were disassembled and cells became apoptotic. CONCLUSION: This study identifies a previously unknown mechanism of action of vinblastine, the depalmitoylation of tubulin, and suggests that depalmitoylation of tubulin may be a target for new chemotherapeutic drugs.

Systematic identification and classification of adverse events in human research.

Widely accepted standards and safeguards for research participants now include systematic surveillance and recording of adverse events. In the absence of a uniform regulation or structure for such reporting, each institution must now establish suitable yet efficient procedures to accomplish this task. We report herein our single center experience with a customized data collection, storage and review system specifically designed to identify and react appropriately to adverse events. Adverse events are classified by each investigator using three criteria in specific order: seriousness, expectedness and relatedness to the investigational intervention. Once classified, events are entered into an online database that includes collation, retrieval and search capabilities. Events meeting specified criteria are reviewed and adjudicated on a weekly basis by The University of Connecticut Research Adverse Events Committee, which makes advisory recommendations to the hospital's two Institutional Research Boards ranging from modification of informed consent to study suspension. Three hundred and seventy-one serious adverse events from > 900 studies were reviewed in the previous academic year. Our system, which combines timely on-line reporting with regular surveillance, provides a potential model that meets the need for comprehensive yet practical adverse events assessment and reporting.

Characterization and partial purification of protein fatty acyltransferase activity from rat liver.

The acylation of proteins through the addition of palmitate to cysteine residues is a common posttranslational modification for a variety of proteins, but the enzymology of this reversible modification has resisted elucidation. We developed a strategy to purify protein fatty acyltransferase (PAT) activity from rat livers that took advantage of recent knowledge on the cellular location and inhibition of PAT activity. We determined that three different thiolases have PAT activity in the presence of imidazole and therefore started the purification with a plasma membrane fraction to minimize the contamination with these enzymes. After detergent extraction of the plasma membrane fraction, the PAT activity was enriched about 90-fold by sequential chromatography including affinity chromatography to a cerulenin-based inhibitor of palmitoylation. The partially purified PAT activity (1) was lost with treatments to degrade or denature proteins, (2) could acylate tubulin, Galpha(i) and RGS16 and (3) showed a preference for palmitate and to a lesser degree other long-chain fatty acids. This purification procedure is a significant advance over previous efforts at PAT purification and a starting point for a proteomic approach for identification of mammalian PAT.