Transient inhibition of initiation of S-phase associated with dimethyl sulfoxide induction of murine erythroleukemia cells to erythroid differentiation.

Abstract

The murine erythroleukemia cell (MELC) line in suspension culture can be induced to differentiate to erythroid cells by various compounds, including dimethyl sulfoxide (Me2SO). Analysis of the cell cycle, during differentiation induced by Me2SO, using thymidine incorporation, thymidine labeling index, and relative DNA content per cell as measured by flow microfluorometry, demonstrates a transient inhibition of entry of (2), accumulation of globin mRNAs (3-6), a and f globin synthesis (7), increase in heme synthesis (8), synthesis of erythrocyte-specific proteins (9), loss in capacity for cell division (2, 10), and the appearance of erythrocytespecific membrane proteins (11).The rate of DNA synthesis, proportion of cells in S-phase, and the pattern of DNA accumulation have been measured and compared in MELC grown in the presence and absence of Me2SO and other inducing agents, e.g., butyric acid (12) and dimethylacetamide (13). DNA synthesis has been measured using incorporation of isotopically labeled thymidine in pulse-labeling experiments; cells in S-phase were assayed by autoradiography of thymidine pulse-labeled cells; DNA accumulation per cell was determined using propidium iodide staining with flow microfluorometric analysis. By MATERIALS AND METHODS Cells. Strain 745-A, which is infected with Friend virus complex, was kindly provided by Charlotte Friend and has been maintained in culture in our laboratory as described elsewhere (14). Isolation of the Me2SO-sensitive cell line (DS19) and resistant cell line (DR10) was described by Ohta et al. (15). Cell concentration and proportion of hemoglobin-containing (benzidine-reactive) cells were determined as previously described (14).Pulse Labeling with Thymidine. DNA synthesis was determined as incorporation of [3Hjthymidine (20 Ci/mmol, New England NuclearCorp.) into cells (2 X 105 cells/per ml) incubated for 20 min at 370 at a concentration of 20 ,Ci/ml of culture medium. Thymidine pulse labeling in this manner was performed on aliquots removed from culture at the times indicated for each experiment. Following incubation with radioisotope, cells were washed with phosphate-buffered saline, pH 7.4, containing 2 mM thymidine, and precipitated with 10% trichloroacetic acid. Precipitates were collected on Millipore filters and their radioactivity was measured in Aquasol. A separate aliquot of cell suspension was removed from the culture and, washed with the same phosphate-buffered saline, and smears were prepared employing the cytocentrifuge. These slides were processed for radioautography (16

Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.

“…An early manifestation of termination of cell division in MELC appears to be a transient prolongation of the GI phase of the cell cycle, which can be detected after cell transit through one complete S phase in the presence of inducer (8)(9)(10). After up to 4-5 terminal cell divisions, induced MELC are arrested in a GI-like phase of the cell cycle.…”

Continued withdrawal from the cell cycle and regulation of cellular genes in mouse erythroleukemia cells blocked in differentiation by the c-myc oncogene.

“…c-K-ras, a 600-bp StuI-EcoRI fragment from the mouse c-K-ras first exon (18) DNA synthesis and growth curves. DNA synthesis was measured as described previously (46) Run-on transcription assays. Transcription was measured by performing run-on transcription assays (27).…”

“…MEL cells respond to induction with DMSO by undergoing a temporary withdrawal from the cell cycle (46). This transitory decrease in DNA synthesis occurs roughly during the time of suppression of c-myc expression.…”

“…A final, irreversible cessation of DNA synthesis occurs 3 to 4 days postinduction as the cells undergo terminal differentiation, which is also accompanied by suppression of c-myc expression (26). The correlation between the number of cells that undergo temporary withdrawal from the cell cycle (14) and the number that ultimately differentiate has led to the hypothesis that the temporary withdrawal is an important, perhaps necessary event in the process of DMSO-induced differentiation (30,46).…”

Inhibition of dimethyl sulfoxide-stimulated Friend cell erythrodifferentiation by hydrocortisone and other steroids.

“…HC treatment in the absence of dimethy( sulfoxide doubled the production of virus. Alterations in DNA metabolism (1,2) and structure (3) are among the many changes that occur in Friend leukemia (FL) cells undergoing dimethyl sulfoxide (Me2SO)-induced erythrodifferentiation (4). The increase in single-stranded breaks in DNA, detected early after Me2SO treatment (5,6), before globin mRNA appeared (Scher, unpublished), suggested that these breaks might play a role in differentiation in this system as postulated in lens differentiation (7,8) and in transcription (9,10).…”