Cellular uptake of cobalamin in clinically-derived blood samples, normal and cancerous brain cells

The cellular uptake and sequestration of fluorescently labeled cobalamin by cancer cells and normal cells was studied by fluorescence flow cytometry and fluorescence microscopy. A fluorescent analog of cobalamin was synthesized by attaching the Oregon Green 488RTM fluorophore to the cobalt atom of cobalamin.;The sequestration of cobalamin by 35 blood samples from patients with myeloid leukemia was quantified by flow cytometry. Blood samples from patients with the M1-subtype of acute myelogenous leukemia exhibited the most aggressive uptake and sequestration of cobalamin. Less uptake of fluorescent cobalamin was exhibited by the M2, M3 and M4/M5 subtypes of leukemia cells that have increasing levels of cellular maturation. This is consistent with the hypothesis that cancer cells that are closer to progenitor cells will have the greatest demand for cobalamin.;The uptake of fluorescent cobalamin by human astrocytes and T98G human glioblastoma cells was quantified by confocal fluorescence microscopy and flow cytometry. Neoplastic glioblastoma cells sequester more fluorescent cobalamin than normal astrocytes. Cobalamin-targeted delivery of cytotoxic drugs may be a useful method to treat brain tumors.;The cellular pharmacokinetic behavior of four naturally-occurring forms of cobalamin was studied to determine the form of cobalamin with the longest mean-residence-time in cells. Methylcobalamin exhibited a shorter mean residence time than adenosylcobalamin or cyanocobalamin in all cell types.;The reaction of amyl nitrite with naturally-occurring forms of cobalamin was studied. The co-morbidity of amyl nitrite abuse with the development of AIDS and Kaposi's sarcoma has yet to be explained. Amyl nitrite reacts rapidly with hydroxocobalamin and methylcobalamin, but no reaction with adenosylcobalamin and cyanocobalamin could be detected. The depletion of free methylcobalamin and hydroxocobalamin in blood is consistent with the suggestion that methylcobalamin may play a role in regulation of the innate immune system.;The ability of cobalamin to deliver a pentapeptide from the N-terminus of the apoptosis-activating protein, Smac, was investigated. Cobalamin was not an effective delivery vehicle for the AIPVA pentapeptide, as premature cleavage of the peptide occurs in lysosomes.