Immunochemical detection of cell cycle synchronization in a human erythroleukemia cell line, K562.

Cells of the human erythroleukemic line K562 can be induced by manipulation of culture conditions to arrest within the G1 phase of the cell cycle, and subsequently to enter S phase synchronously after release from G1. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescent staining. This protocol offers a method for synchronization of K562 cells at the G1/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.

A general method for heterokaryon detection using resonance energy transfer and a fluorescence-activated cell sorter.

A simple and general labeling method has been adapted for the rapid detection and isolation of heterokaryons soon after somatic cell fusion. The method involves prelabeling each parental cell type by adding one of two hydrophobic fluorescent probes, F18 or R16, to the culture medium. These probes are nontoxic, nonmutagenic, and do not inhibit either cellular replication or the efficiency of fusion. Following polyethylene glycol (PEG)-induced fusion, heterokaryons are identified on a cell sorter as cells showing fluorescence as a result of nonradiative resonance energy transfer (RET) between the two fluorochromes. Optimal conditions are described for the unambiguous detection of heterokaryons by fusion-induced RET. The value of this method lies in the universal applicability of F18 and R16 as simple and direct membrane labels for any pair of parental cells, even those lacking selectable genetic markers or detectable antigenic differences. This potential for heterokaryon selection in any cell cross should significantly expand the range of cell types amenable to investigation through somatic cell genetics, while the rapidity of the method should facilitate the study of early events following fusion.

Inositol lipid metabolism accompanies erythroid differentiation of K562 human leukemic cells.

The role of hemin induction of K562 in inositol phospholipid metabolism has not been previously studied. K562 cells were induced to synthesize hemoglobin upon addition of bovine hemin to the culture media. The phospholipid content of K562 was determined before and after the addition of hemin. The results of this study demonstrated significant differences in the phosphoinositides between induced and non-induced cells. Phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-inositol-4,5-bisphosphate (PIP2) levels increased upon induction, and remained above control levels. Phosphatidylinositol (PI) levels decreased 15 min after hemin addition, then increased to control levels by 1 h. From 2-8 h PI levels then remained depressed below control levels. These data suggest that hemoglobin induction in K562 cells occurs concomitantly with inositol phospholipid turnover.

Hemoglobin induction by Ara-C in human erythroleukemic cells (K562) is cell-cycle dependent.

This study was undertaken to answer the question of whether chemical induction of hemoglobin in the human erythroleukemic line K562 is cell-cycle dependent. Cells were synchronized with respect to the cell cycle by manipulation of culture conditions. S phase was detected microscopically by immunofluorescent staining. Hemin and arabinofuranosylcytosine (Ara-C) were used to induce hemoglobin synthesis in K562 cells. The action of hemin causes induction without specificity in regard to cell cycle phase, while Ara-C is dependent upon cell cycle phase, with cells within late G1 and early S phases being sensitive to the effect of the agent. These studies also confirm the results that the action of hemin is reversible but the induction caused by Ara-C is irreversible. The synergistic effects of these two inducing agents resulted in a sharper rise in the percentage of induced cells.

The effect of 3-aminobenzamide on thymidine incorporation in ultra-violet irradiated chick pigment epithelium cells.

The addition of 3-aminobenzamide (3-AB) to cultures of chick embryo pigmented epithelium rescues these cells after high doses of ultraviolet treatment. The addition of 3-AB prevents cells from losing pre-formed protein and DNA and stimulates thymidine incorporation by the cells after ultraviolet irradiation. Since 3-AB is an inhibitor of poly (ADP) ribosylation, these observations support the conclusion that death of these cells after ultra-violet irradiation depends upon poly (ADP) ribosylation and may be an apoptotic response.