Effects of controlled exposure of L cells to bromodeoxyuridine (BUdR)

Kasupski, George J.; Mukherjee, Barid B.

doi:10.1016/0014-4827(77)90178-1

Cited by 30 publications

(9 citation statements)

References 31 publications

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“…Studies of inducer-mediated MELC differentiation (34)(35)(36)(37)(38)(39) and a number of other differentiating cell systems (40)(41)(42)(43)(44), including normal erythropoiesis (45)(46)(47), suggest that DNA synthesis is required for the transition to expression of differentiated characteristics. For example, we found that inducermediated events during early S phase appear to be required for the expression of a-and ,3-globin genes in MELC cultured with HMBA (37,38).…”

Section: Discussionmentioning

confidence: 99%

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

Chen¹,

Banks²,

Rifkind³

et al. 1982

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

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There are a number ofagents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture with HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition ofhemin to cultures with HMBA and dexamethasone initiates accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumu-

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Section: Discussionmentioning

confidence: 99%

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

Chen¹,

Banks²,

Rifkind³

et al. 1982

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

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“…USA 76 (1979) 4515 culture. Fusion of myoblasts (29) and the synthesis of certain enzymes in mouse L cells (30) are inhibited under conditions of bromodeoxyuridine substitution into early, but not late, replicating DNA. Taken together, these studies collectively focus attention on events occuring during the late G1 or early S phase of the cell division cycle as a basis for further study of the mechanism of action of differentiation inducing agents.…”

Section: Methodsmentioning

confidence: 99%

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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“…The enzymes studied in Table I were chosen for their reported response to BUdR . Acid phosphatase is depressed in L cells (10), whereas alkaline phosphatase is induced in other cell lines (2,5) . In unsynchronized HTC cells, growth for over two generations in BUdR results in a decrease in alcohol dehydrogenase and an increase in malate dehydrogenase (15) .…”

Section: Table I Effect Of Budr On Various Parametersmentioning

confidence: 99%

“…The plating efficiency of HeLa cells (9), various enzymes in THE JOURNAL of CELL BIOLOGY -VOLUME 87 DECEMBER 1980 629-632 L cells (10), viral antigen production in rat embryo cultures (17), and mutagenesis in various hamster cell lines (4,22) have been found to respond to the incorporation of BUR into specific intervals of S phase . In HTC cells, however, TAT decreased whenever BUdR was incorporated into DNA during S phase (19) .…”

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confidence: 99%

Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells.

O'Brien¹

1980

The Journal of Cell Biology

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Synchronized hepatoma tissue culture (HTC) cells, accumulated at the G,/S boundary with aminopterin, were released into S phase with either thymidine or 5-bromodeoxyuridine (BUdR) . Tyrosine aminotransferase (TAT) activity was found to be unaffected by BUdR over the initial 3 h of S phase, but then to rapidly decline to a new basal level of 40% of control by 9 h . There was no corresponding response in the activities of alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and alkaline phosphatase, or in the rate of protein and RNA synthesis. If BUdR incorporation was restricted to limited periods of S phase, TAT was found to be maximally suppressed by incorporation into the initial 40% of the DNA . Incorporation of the analogue into the latter 60% of DNA synthesized during S phase had no effect on TAT. This is the first report that the effect of BUdR on TAT in HTC cells is associated with incorporation of the analog into DNA synthesized during a specific interval of S phase.The thymidine (dThd) analogue 5-bromodeoxyuridine (BUdR) has been found to be a unique modulator of eukaryotic gene expression . Its incorporation into DNA is associated with blocked differentiation, the induction of viral particles, and either the suppression or stimulation of specific functions in terminally differentiated cell types (for review, see Goz [7]) .In attempts to understand the mechanism(s) of action of BUdR, one of the most extensively studied systems is tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells . Stellwagen and Tomkins (19,20) reported that growth of HTC cells in BUdR led to a rapid decrease in TAT activity that could not be attributed to a soluble inhibitor, a defective enzyme, or a change in enzyme degradation . Within the time period studied, there was no effect on other enzymes, cell proliferation, or protein and RNA synthesis. The analogue affected TAT only if present during S phase, and O'Brien and Stellwagen (16) found the TAT decrease was proportional to the percent of dThd residues replaced by BUdR in each new strand of DNA . Stellwagen and Tomkins (19) proposed that BUdR blocked transcription of the TAT structural gene .The S phase of the eukaryotic cell is envisioned as being a synchronous period within itself during which genes replicate in repetitive sequence from one S phase to another (for review, see Hand [8]) . If the TAT gene exists as a unique, single copy of DNA, and BUdR exerts its effect only when in the structural gene, then the effect should be limited to the incorporation of BUdR during a specific interval of S phase . That interval should be the time during which the effected gene replicates .

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Effects of controlled exposure of L cells to bromodeoxyuridine (BUdR)

Cited by 30 publications

References 31 publications

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

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

Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells.

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