EXPRESSION OF PROLIFERATION-ASSOCIATED NUCLEAR AUTOANTIGENS, P330(D) CENP-F AND PCNA, IN DIFFERENTIATION AND IN DRUG-INDUCED GROWTH-INHIBITION USING 2-PARAMETER FLOW-CYTOMETRY/

p330(d)/CENP-F is a recently described nuclear autoantigen that was de tected in PHA-stimulated but not in resting peripheral lymphocytes. Th is protein accumulates in the nucleus during S-phase and reaches maxim um levels during the G(2) and M phases of the cell cycles. We compared the expression of p330(d)/CENP-F and proliferating cell nuclear antig en (PCNA) during the induction of terminal myeloid differentiation of HL-60 tumour cells. HL-60 cells were induced to differentiate with ret inoic acid (RA), dimethyl sulfoxide (DMSO), and 3-nitrobenzothiazolo [ 3,2-]quinolinium (NBQ), and collected at different intervals. Control and treated cells were analyzed by two-parameter flow cytometry using propidium iodide and antibodies to p330(d)/CENP-F and PCNA. The percen tage of p330(d)/CENP-F and PCNA positive cells was found to be proport ional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330(d)/CENP-F and PCNA positive cells was r educed proportionately to the number of cells that had differentiated. Reduction in the expression of both antigens was completed after 120 h when 80% to 85% of the cells were arrested in G(1) and displayed the mature phenotype. The expression of p330(d)/CENP-F and PCNA was also assessed in the growth inhibition of HT-29 cells induced by various co ncentrations of camptothecin (CPT), etoposide (VP-16), and aphidicolin (APH). There was a dose-dependent displacement of cells to late S-pha se by CPT while VP-16 induced cells to accumulate in G(2) + M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G(1) by APH led to a signifi cant decrease in their expression. The dramatic reduction in p330(d)/C ENP-F levels during differentiation, and the correlation of its expres sion with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.