CELL-CYCLE ANALYSIS OF AMOUNT AND DISTRIBUTION OF NUCLEAR-DNA TOPOISOMERASE-I AS DETERMINED BY FLUORESCENCE DIGITAL IMAGING MICROSCOPY

Fluorescence digital imaging microscopy (FDIM) has been used to perfor m a cell cycle analysis of both the amount and the distribution of nuc lear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclona l antiserum generated with a 21-amino-acid peptide representing amino acids 219-239 of human topoisomerase I. Immunohistochemical staining w as followed by staining with Hoechst dye 33342, allowing DNA content t o be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2-fold increase) as the cells progr essed from G(1) to G(2)/M phases of the cell cycle. However, when norm alized for nuclear size, topoisomerase I content per nuclear area rema ined almost constant (1.3-fold increase). For comparison, we measured the amount of proliferating cell, nuclear antigen (PCNA), a protein wh ose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7-fold from G(1) to S phase, then de-clined in G(2)/M- pha ses, whereas PCNA content per nuclear area increased 1.7-fold from G(1 ) to S phase. We also measured topoisomerase I content in leucine-depr ived cells to determine if altered growth conditions affect topoisomer ase 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 nuc leoli throughout the cell cycle. In contrast, leucine-deprived cells e xhibited a perinuclear distribution of topoisomerase I. Our results sh ow that FDIM is a useful technique in determining the cell cycle posit ion and both the content and the distribution of topoisomerase I as we ll as other nuclear proteins in individual cells. (C) 1995 Wiley-Liss, Inc.