Table I

Activity of Topoisomerases in Mitotic and Interphase A431 Cells. Mean Results of Three Independent Experiments

Log cultureMetaphase*
Cell cycle distribution
 G1, %‡       400
 S, %‡       430
 G2/M, %‡       17100
 Mitotic index, %‡     < 196
Topoisomerase I
 pg/nucleus‖ 0.10.22
 Molecules/nucleus 6.2 × 10513.8 × 105
 U/nucleus 2.8 × 10−3 1.3 × 10−3
 U/mg**    28 × 106 6.9 × 106
 Band depletion by10085
 30 μM camptothecin, %††
 Extractability, %§§       9896
Topoisomerase IIα
 fr/nucleus‖       1320
 Molecules/ nucleus 4.7 × 104 7.3 × 104
 Band depletion by10050
 200 μM etoposide, %††
 Extractability, %§§       8852
Topoisomerase IIβ
 fg/nucleus‖       1530
 Molecules/nucleus 5.2 × 10410.4 × 104
 Band depletion by10098
 200 μM etoposide, %††
 Extractability, %§§       9897
Topoisomerase IIα + β
 U/nucleus‖‖ 8.3 × 10−5 4.8 × 10−5
 U/mg¶¶2.96 × 106 1.2 × 106
  • Standard errors of the mean were <20%.  

  • *  Cultured with 0.26 μM demecolcine for 16 h.  

  •  Flow cytometry of bisbenzimide stained cells.  

  • §  Scored by fluorescence microscopy of bisbenzimide stained cells.  

  •  Comparative densitometry of immunoblots with purified enzyme as standard.  

  •  pBR 322 relaxation activity in the presence of orthovanadate.  

  • **  Activity related to extractable amount of topoisomerase I antigen.  

  • ††  Comparative densitometry. Density of immunoreactive enzyme band obtained in untreated cells was set to 0% and background value of identical position in an empty lane to 100%.  

  • §§  Density of immunoreactive band obtained with whole nuclei was compared to that obtained with nuclear remnant after extraction with 350 mM NaCl and DNase digestion.  

  •  

  •  P4 DNA unknotting activity.  

  • ¶¶  Activity related to extractable antigen amount of both isoenzymes of topoisomerase II.