The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells.
We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.
Negri
C
C
Istituto di Genetica Biochemica Evoluzionistica CNR, Pavia, Italy.
Bernardi
R
R
Braghetti
A
A
Ricotti
G C
GC
Scovassi
A I
AI
D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005047:Etoposide / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*
D004273:DNA, Neoplasm; D011064:Poly Adenosine Diphosphate Ribose; D005047:Etoposide; D004247:DNA