Ubenimex (Bestatin) is a potent inhibitor of aminopeptidases (APase) including APase N (EC 3.4.11.2), a widely distributed membrane-bound metalloprotease. Binding of Ubenimex (UBX) to cells has been implicated in a variety of its biological activities, while little evidence has yet been provided as to any subsequent mechanisms of intracellular signal transduction. We now examined the possible involvement of protein kinase C (PKC), a key regulator in transmembrane signaling. Human leukemia K562 cells were cultured in the presence or absence of UBX (1 to 50 micrograms.ml-1, 1 to 72 h), and the subcellular distribution as well as phorbol-12, 13-dibutyrate (PDBu)-induced redistribution of PKC activities were assessed. The membrane-bound enzymatic activity tended to increase in the presence of UBX, while a significant loss of the activity was demonstrable upon subsequent exposure to PDBu (100 nM, 10 min) in both the cytosolic and membrane fractions. Specific binding of [3H]PDBu to intact K562 cells was also down-modulated with UBX concentration- and time-dependently, suggesting loss of PKC enzyme protein on the cell surface. Western blot analysis of the total cell extracts disclosed no appreciable alteration in the amount of PKC protein. APase inhibition with UBX was observable independently of PKC modulation. The present findings were discussed with reference to the possible differential mechanisms of PKC-mediated regulation of cellular responses depending on cell types.

Aminopeptidases↗

PubMedDate not supplied

Reduced topoisomerase II-mediated DNA cleavage in VP-16 resistant human leukemic cell line.

Drug resistance in the VP-16 resistant human leukemic cell line (THP-1/E) was studied the possible relevance of topoisomerase II activity. Strand-passing activity in crude nuclear extract from sensitive and resistant cells was comparable and equally sensitive to inhibition by VP-16. However, it was demonstrated that VP-16-mediated pBR322 DNA cleavage in the presence of nuclear extract from resistant cells was reduced to one-tenth of that from sensitive cells. These results suggested that the resistance of THP-1/E cells to VP-16 was due to reduced DNA cleavage activity.

Cell Line↗