Roberta C. Reuben

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide.

Abstract Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.

Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation.

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6-8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.

REGULATION OF DIFFERENTIATION IN NORMAL AND TRANSFORMED ERYTHROID CELLS

SummaryStudies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.

DNA polymerase activities during induced differentiation in murine erythroleukemia cells.

Abstract DNA polymerase activities with properties similar to those described for other eukaryotic cells were measured in murine erythroleukemia cells (MELC). Counter-current centrifugation was employed to obtain populations of cells synchronized in different stages of the cell cycle. Alpha DNA polymerase activity, the major DNA polymerase activity in the cell, varies with stage of cell cycle, attaining the highest values in S (DNA synthesis) phase both in differentiating and non-differentiating MELC. When MELC are induced to differentiate by culture with hexamethylene bisacetamide (HMBA) the observed alteration in progression through the cell cycle is reflected in α DNA polymerase activity. Induced cells, delayed in G1, have a low level of α DNA polymerase activity, characteristic of G1 cells, but α activity increases when cells re-enter S phase. MELC induced to differentiate undergo a limited number of cell divisions and then lose the ability to divide. Terminal cell division is accompanied both by a loss of the ability to incorporate labelled thymidine into DNA and by a marked decrease in α DNA polymerase activity. No significant differences in β DNA polymerase activity were detected either in different stages of the cell cycle or during terminal differentiation.