A non-radioactive, sensitive method for the detection of DNA fragmentation in apoptotic cells (rat pheochromocytoma PC12 and rat cortical cells).
A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.
Lauc
G
G
Department of Medical Biochemistry, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia.
Perovic
S
S
Dapper
J
J
Flögel
M
M
Iskric
S
S
Müller
W E
WE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001710:Biotin / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry*; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004256:DNA Polymerase I / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D015699:HIV Envelope Protein gp120 / Q000494:pharmacology; D016716:PC12 Cells / Q000737:chemistry*; D051381:Rats; D013194:Staining and Labeling; D014220:Trialkyltin Compounds / Q000494:pharmacology
D003856:Deoxyuracil Nucleotides; D015699:HIV Envelope Protein gp120; D014220:Trialkyltin Compounds; C027078:deoxyuridine triphosphate; C011559:tributyltin; D001710:Biotin; D004247:DNA; D004256:DNA Polymerase I; D004996:Ethidium