Fluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear
DNA topoisomerase I in individual CEM human
leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21-amino-acid
peptide representing
amino acids 219-239 of human
topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst
dye 33342, allowing
DNA content to be determined in each cell. Cell cycle analysis showed that nuclear
topoisomerase I content doubled (2.2-fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size,
topoisomerase I content per nuclear area remained almost constant (1.3-fold increase). For comparison, we measured the amount of
proliferating cell nuclear antigen (
PCNA), a
protein whose expression fluctuates during the cell cycle. Nuclear
PCNA content increased 2.7-fold from G1 to S phase, then declined in G2/M- phases, whereas
PCNA content per nuclear area increased 1.7-fold from G1 to S phase. We also measured
topoisomerase I content in
leucine-deprived cells to determine if altered growth conditions affect
topoisomerase I protein expression. Compared to CEM cells in logarithmic growth,
leucine-deprived CEM cells had 1.8-fold less
topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed
topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast,
leucine-deprived cells exhibited a perinuclear distribution of
topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of
topoisomerase I as well as other
nuclear proteins in individual cells.