Alterations of growth fraction and DNA content in K562 cells by differentiating agents

Abstract

The growth fraction, estimated by the monoclonal antibody Ki-67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with GI, S, and G, + M contents in Ki-67-positive and Ki-67-negative cells to be evaluated.Butyric acid (BUT) was used as positive control. The fraction of Ki-positive cells decreased with the BUT concentration, while the proportion of cells with G, DNA content increased only in the Ki-negative cells.Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki-positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki-negative cells displayed a G,, but also a G, and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.Key terms: FCM, proliferation-associated antibody Ki-67, glycophorin A, erythroid differentiationIn cancer therapy, it is of interest to evaluate the tumour growth fraction (GF). It reflects the rate of tumour growth which is a factor of prognosis (8,16,29) and the fraction of cells sensitive to anticancer agents. Non-cycling cells have been described with a GI but also with a G2 and even an S DNA content, both in vitro (4,13,15) and in vivo (11); "recruitment" and "synchronizing" protocols will be better scheduled if the DNA content distributions of the proliferating and quiescent cells are known. A recent strategy in cancer chemotherapy has been to induce cell maturation and inhibit cell growth (21,23) without cell killing. In this case, it will be interesting to know precisely the cell cycle parameters, i.e., the GF and the DNA distributions of both cycling and non-cycling cells. Classical methods to determine GF by using radioactive DNA precursors (11,311, the bromodeoxyuridine (BrdU) antibody technique (3,27), or dye staining of both DNA and RNA (2,4,13) are difficult and time consuming. Recently, methods using monoclonal antibodies (MoAb) have been described. They are directed against human alpha DNA polymerase (11, a heterochromatin-associated antigen (MoAb 244-7) ( 5 ) , a proliferating cell nuclear matrix antigen (32), a nuclear antigen (M 150,000) (9), nuclear antigen p105 (161, the transferrin receptor (25), the ribonucleotide reductase M1 subunit (20), and especially a nuclear-matrix-associated proliferation-related antigen (MoAb Ki-67) (18,30). Different attempts were carried out to stain simultaneously cells with Ki-67 MoAb and propidium iodide (3,12,24). We used simultaneously Ki-67 and ethidium bromide (EB) and then separately measured the DNA content of cells according to their reactivity with Ki-67 MoAb.

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

“…Although they could display slightly different nuclear structures, G 0 cells were not analysed separately, as they represent a minor component of such cell populations in growth phase (less than 1% as evaluated by Ki‐67 staining, i.e. below the resolution of the analysis system) ( Gorisse et al . 1990 ).…”