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  • Title: Uptake of methotrexate linked to polyclonal and monoclonal antimelanoma antibodies by a human melanoma cell line.
    Author: Uadia P, Blair AH, Ghose T, Ferrone S.
    Journal: J Natl Cancer Inst; 1985 Jan; 74(1):29-35. PubMed ID: 3855485.
    Abstract:
    [3H] Methotrexate [( 3H]MTX) was covalently linked to monoclonal antibody (MoAb) 225.28S against human melanoma, to a rabbit anti-human melanoma IgG absorbed either with human red blood cells (AHMGR) or with red blood cells and a variety of normal human tissues (AHMGR + T), or to normal rabbit IgG (NRG). Human melanoma M21 cells were incubated at 0 degrees C or 37 degrees C with 10 microM free MTX or 10 microM MTX linked to one of the above carriers. The order of net uptake of MTX during 6 hours was MTX-MoAb 225.28S greater than MTX-AHMGR greater than MTX-AHMGR + T greater than MTX-NRG greater than or equal to MTX. This order of uptake by the three antibody conjugates corresponded to the amount of conjugate bound at equilibrium at 0 degrees C and to the immunofluorescence titers. Binding sites for MoAb 225.28S were more efficient for internalization of MTX than were those for the two polyclonal antibody preparations. When M21 cells preloaded with MTX by incubation at a drug concentration of 1.0 or 10 microM were incubated in drug-free medium, the amount of cell-associated MTX rapidly declined to 1.8 pmol/mg protein, i.e., the level of intracellular dihydrofolate reductase (DHFR). However, when cells preloaded to a drug content of 112 pmol/mg protein by incubation with 10 microM MTX linked to AHMGR were transferred to conjugate-free medium, 65 pmol MTX/mg remained cell associated after 12 hours. The efflux was inhibited by chloroquine. Both the efflux medium and M21 cells after a 9.5-hour incubation period had MTX-containing catabolic fragments that inhibited DHFR.
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