An ultrastructural and functional study was performed during methotrexate (MTX) induced apoptosis on HL-60 leukemia cells. The fine preservation of plasma membrane architecture and organellar components was present until latest apoptotic stages, despite strong nuclear changes. This observation suggests the presence of a residual cell metabolic activity, which is here investigated. DNA agarose gel electrophoresis demonstrated its fragmentation, which was also confirmed in situ by nick translation confocal analysis. Nuclear purification was subsequently performed to investigate DNA polymerase activities. DNA polymerase alpha activity appeared strongly reduced from the early phases of methotrexate treatment, while the rate of DNA polymerase beta synthesis was found to increase progressively along with the time of drug treatment. Low levels of DNA polymerase gamma activity revealed both in control and in treated cells, suggesting the irrelevant involvement of this enzyme in the DNA metabolism of this model. DNA polymerase beta appears thus to be the enzyme preminently correlated to cell attempts to repair the methotrexate-induced DNA damage.
Morphological patterns and DNA polymerase regulation in apoptotic HL60 cells.
CATALDI, Amelia;DI BALDASSARRE, Angela;ROBUFFO I;MISCIA, Sebastiano
1996-01-01
Abstract
An ultrastructural and functional study was performed during methotrexate (MTX) induced apoptosis on HL-60 leukemia cells. The fine preservation of plasma membrane architecture and organellar components was present until latest apoptotic stages, despite strong nuclear changes. This observation suggests the presence of a residual cell metabolic activity, which is here investigated. DNA agarose gel electrophoresis demonstrated its fragmentation, which was also confirmed in situ by nick translation confocal analysis. Nuclear purification was subsequently performed to investigate DNA polymerase activities. DNA polymerase alpha activity appeared strongly reduced from the early phases of methotrexate treatment, while the rate of DNA polymerase beta synthesis was found to increase progressively along with the time of drug treatment. Low levels of DNA polymerase gamma activity revealed both in control and in treated cells, suggesting the irrelevant involvement of this enzyme in the DNA metabolism of this model. DNA polymerase beta appears thus to be the enzyme preminently correlated to cell attempts to repair the methotrexate-induced DNA damage.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
