Intranuclear distribution of the human myeloid cell nuclear differentiation antigen in HL-60 cells

✍ Scribed by David M. Duhl; Marek Gaczynski; Ryszard Olinski; Robert C. Briggs

• Publisher: John Wiley and Sons
• Year: 1989
• Tongue: English
• Weight: 688 KB
• Volume: 141
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041410122

✦ Synopsis

Based on solubility properties, the human myeloid cell nuclear differentiation antigen exists as at least two distinct populations. Most is easily extracted from isolated nuclei in 0.35 M NaCI, while 20 percent resists such treatment. Compared to undigested nuclei, both the amount of myeloid cell nuclear differentiation antigen (MNDA) released from nuclei after DNase l treatment and the amount resisting further extraction in 0.35 M NaCl increased after DNA was digested with DNase I. Under these conditions, there was a concomitant decrease in the amount of M N D A that was extractable with 0.35 M NaCl. Mixing nuclear protein extracts that contain MNDA with nuclei from cells that do not express this protein demonstrated that the M N D A redistributes from the freely soluble form to the nuclear residual fraction as a consequence of DNase I digestion. These data are consistent with a model in which the amount of M N D A that is tightly bound to salt-washed nuclei is held constant in the presence of an excess of unassociated M N D A in the nucleus, and that the level of M N D A binding to this nuclear fraction increases in proportion to the extent of DNA damage resulting from DNase I digestion.

MATERIALS AND METHODS Growth and preparation of cells

Cells were maintained and grown in RPMI 1640 medium (Grand Island Biological Company, Grand Island, NY 1 containing 15% defined-supplemented calf serum (Hyclone Laboratories, Logan, UT). HL-60 cells were seeded at a cell density of 2 x lo5 cellsiml and KG-la cells at 1 x lo5 cellsiml. Cells were grown at 37°C in a humidified incubator containin 5% COz until they were harvested or subcultured. reached a density of 6-9 x 10 fi cellsiml, when they