Altered expression and activity of topoisomerases during all-trans retinoic acid-induced differentiation of HL-60 cells.
Regulation of topoisomerase II (TOPO II) isozymes alpha and beta is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIalpha and TOPO IIbeta proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (alpha and beta) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIbeta protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIbeta protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 micromol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 micromol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 micromol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIbeta protein levels are posttranscriptionally regulated and that degradation of TOPO IIbeta is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (alpha + beta) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIbeta may have a specific role in transcription of genes involved in differentiation with RA treatment.
Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH, USA.
M
M
Aoyama
D R
DR
Grabowski
R J
RJ
Isaacs
K A
KA
Krivacic
L A
LA
Rybicki
R M
RM
Bukowski
M K
MK
Ganapathi
I D
ID
Hickson
R
R
Ganapathi
D002454:Cell Differentiation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology*
D007527:Isoenzymes; D009363:Neoplasm Proteins; D014212:Tretinoin; D005047:Etoposide; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D002945:Cisplatin