Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Gazitt, Yair; Deitch, Arline D.; Marks, Paul A.; Rifkind, Richard A.

doi:10.1016/0014-4827(78)90154-4

Cited by 38 publications

(23 citation statements)

References 8 publications

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“…The total cell cycle time of these MELC is about [11][12] hr (10,15,16). Cells that were in GC after 12 hr of culture were largely in GC at the time of onset of culture with HMBA and proceeded through S, G2, and M and then back into the GC during which accelerated globin mRNA synthesis was initiated.…”

Section: Methodsmentioning

confidence: 99%

“…RNA was purified from cytoplasmic and nuclear fractions according to the method of Bastos and Aviv (19 Cell Cycle Fractionation and Synchronization. The Elutriator Rotor (Beckman) was used to fractionate MELC, by countercurrent centrifugation, according to cell volume which is a measure of position in the cell division cycle, as reported (16). A maximum of 5 X 108 logarithmic phase MELC in Hanks' balanced salt solution without protein were introduced into the spinning rotor, and elutriation was accomplished at 5YC at a constant rotor speed of 2000 rpm by stepwise (2.5 ml/min) increments in flow rate from 10 to 35 ml/min.…”

Section: Methodsmentioning

confidence: 99%

“…Geller et al (10) have demonstrated that MELC may be fractionated according to their position in the cell cycle by unit gravity sedimentation, which sorts the cells according to their volume, and is directly proportional to their position between GI and G2 (16). In the present studies, centrifugal elutriation has been used to achieve fractionation of MELC according to their size and cell cycle position.…”

mentioning

confidence: 99%

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Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Gambari¹,

Marks²,

Rifkind³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Murine erythroleukemia cells (MELC) are induced to erythroid differentiation by culture with 4 mM hexamethylenebisacetamide. The relationship between onset of accumulation of globin mRNA and cell cycle phase has been examined in MELC fractionated with respect to cell cycle (GI, (1). The cellular and molecular target(s) of inducer effect on MELC have yet to be elucidated although data have been adduced to support putative targets at the plasma membrane (2, 3) as well as at nuclear (chromatin) sites (4-6). A number of studies have suggested that the action of inducers of MELC differentiation may be a cell cycle-related phenomenon. Levy et al. (7) and McClintock and Papaconstantinou (8) demonstrated a requirement for exposure to inducer during at least one S phase in order to achieve induction of differentiation; Harrison (9) has demonstrated that MELC arrested as single cells in the presence of inducer fail to differentiate. More recently, Geller et al. (10) have shown that commitment to differentiate, the ability to express differentiation despite removal of the inducing agent (11,12), is more rapidly achieved in cultures of MELC initiated with cells synchronized in the G2 or GI phase of the cell cycle than in mid or late S. Previous studies in this laboratory revealed an inducer-mediated prolongation of the GI phase of MELC undergoing differentiation induced by HMBA or dimethyl sulfoxide (13). We have also found (14) that the initial detectable HMBA-induced increase in the rate of accumulation of newly synthesized globin mRNA coincides with the first (prolonged) GI phase after MELC synchronized at the GI/S interface by the method of sequential treatment with thymidine and hydroxyurea (15) proceed through S. G2, and M.The present investigation was designed to evaluate the relationship between the cell cycle-related effects of inducers and the initiation of accelerated globin mRNA synthesis and of alteration in cell cycle transit, without the use of agents such as thymidine or hydroxyurea that may in themselves perturb the processes of cell differentiation (9). Geller et al. (10) have demonstrated that MELC may be fractionated according to their position in the cell cycle by unit gravity sedimentation, which sorts the cells according to their volume, and is directly proportional to their position between GI and G2 (16). In the present studies, centrifugal elutriation has been used to achieve fractionation of MELC according to their size and cell cycle position. We have found that the initial increment in induced globin mRNA synthesis is confined to the first G1 phase after a complete S phase traversed in the presence of HMBA. Once an accelerated rate of globin mRNA synthesis is initiated, synthesis continues through the S and GI phases of subsequent cell cycles. The data suggest that there is an effect of HMBA that occurs during early S phase and is critical for the initiation of globin mRNA synthesis and for the prolongation of the GI phase of the cell cycle. MATERIALS AND METHODSCell Culture. MELC...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Gambari¹,

Marks²,

Rifkind³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore it is shown that benzidine fluorescent cells are mainly accumulated in small cell size channels. This change which have already been reported was postulated to be the result of an accumulation of GI cells in the early induction (10,30). In the present work we demonstrate that treating cells with dexamethazone prevents the decrease in larger cells.…”

Section: Discussionmentioning

confidence: 52%

The effect of hexamethylene‐bis‐acetamide on multiparameter analysis of friend leukemia cells

Mishal¹,

Fourcade²,

Tapiero³

1981

Cytometry

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Friend leukemia cells were induced to differentiate by hexamethylene-bis-acetamide (HMBA). The relationship between cell growth cell volume distribution, fluorescence intensity and fluorescence anisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) was analyzed in differentiated and undifferentiated cells with the aid of the fluorescence activated cell sorter (FACS II). The induction of a differentiated state was associated with a decrease in cell volume and an increase of fluorescence anFriend leukemia cells (FLC) have been used as a model for the study of erythroid differentiation (7). They can be induced to differentiate and synthesize hemoglobin in vitro when grown in the presence of a variety of structurally unrelated agents (2, 3, 11,14,18,19,22). Among the agents particularly active, hexamethylene-bis-acetamide (HMBA) is one of the most potent inducers (18). The expression of globin genes during cell differentiation can be suppressed by other unrelated agents (16,20, 23, 29). The mechanism whereby these inducers and inhibitors operate is still not known. Studies on the events which occur in induced FLC have shown various changes in membrane properties (4,5,9,12,15,21, 28), synthesis of erythrocyte membrane antigens and restriction of certain other proteins (7,17), whereas, very little work was done on the effect of the inhibitors. In the present work, the effects of an inducer (HMBA) and an inhibitor (dexamethazone) were analyzed. Changes of cell volume and fluorescence Key terms: Cell sorter, hexamethylene-bisacetamide induced Friend leukemia cells, dexamethazoneanisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) were observed and their relation to the differentiated state of the cell was analyzed. Materials and MethodsCell culture and benzidine staining: FLC which were derived from a clone of Friend virus transformed cells 745 A were grown as previously described (5). Stimulation of globin synthesis was obtained by growing cells for 4 days in medium supplemerted with 5 mM HMBA kindly provided by Dr. Y. Gazitt.Benzidine staining: Benzidine dihydrochloride (Sigma Chem. Co., St. Louis, MO) 0.1 g was dissolved in 50 ml dilute acetic acid (1.5 ml glacial acetic acid diluted to 50 ml with water) and stored at 4°C in the dark. Just before use, 1 ml of benzidine solution was dispensed into a test tube at 4'C and 20 p1 of 30% H202 were added followed by immediate mixing. To this mixture, 1 ml of a culture containing approximately 3 X lo6 cells/ml was added and swirled rapidly during the addition. After 2 min incubation at 4"C, cells were centrifuged, washed and resuspended in phosphate buffer saline (PBS). The percentage of benzidine reactive cells was dekrmined in a Thoma hemocytometer (Precis, Paris, France) with the aid of the FACS by determining the fluorescence intensity after an excitation with a light source a t 488 nm. As a control the percentage of B+ cells was determined from noninduced cultures.Fluorescence labeling and measurements: The fluorescent hydrocarbon DPH was ...

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“…Cell cycle parameters are modified early in the induction process (17,2,3,4). It was shown that a transient accumulation of the cells in the GI phase of the cell cycle occurs early after the input of the inducer (from 6 to 16 h), and during this period the progression of the cells in the S phase is not affected.…”

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confidence: 99%

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation

et al. 1992

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Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G , phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G, accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G , block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of bemidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the GI block. The analysis of two molecular markers of cell differentiation confirmed these results. 0 1992 Wiley-Liss, Inc.

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Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Cited by 38 publications

References 8 publications

Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

The effect of hexamethylene‐bis‐acetamide on multiparameter analysis of friend leukemia cells

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation

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