Expression of proliferation‐associated nuclear autoantigens, p330d/CENP‐F and PCNA, in differentiation and in drug‐induced growth inhibition using two‐parameter flow cytometry

Abstract

p330d/CENP-F is a recently described nuclear autoantigen that was detected in PHA-stimulated but not in resting peripheral lymphocytes. This protein accumulates in the nucleus during S-phase and reaches maximum levels during the G2 and M phases of the cell cycles. We compared the expression of p330d/CENP-F and proliferating cell nuclear antigen (PCNA) during the induction of terminal myeloid differentiation of HL-60 tumour cells. HL-60 cells were induced to differentiate with retinoic 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 p330d/CENP-F and PCNA. The percentage of p330d/CENP-F and PCNA positive cells was found to be proportional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330d/CENP-F and PCNA positive cells was reduced 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 G1 and displayed the mature phenotype. The expression of p330d/CENP-F and PCNA was also assessed in the growth inhibition of HT-29 cells induced by various concentrations of camptothecin (CPT), etoposide (VP-16), and aphidicolin (APH). There was a dose-dependent displacement of cells to late S-phase by CPT while VP-16 induced cells to accumulate in G2+M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G1 by APH led to a significant decrease in their expression. The dramatic reduction in p330d/CENP-F levels during differentiation, and the correlation of its expression with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.