Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53.
OBJECTIVE
Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266).


METHODS
The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73.


RESULTS
p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel.


CONCLUSIONS
Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.
School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia.
Juhura G
JG
Almazi
Swetlana
S
Mactier
O Giles
OG
Best
Ben
B
Crossett
Stephen P
SP
Mulligan
Richard I
RI
Christopherson
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; C081876:CKAP4 protein, human; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D014740:Vidarabine; C024352:fludarabine