Differentiation Of Friend Leukemia Cells Research Articles

Control of globin gene expression by steroid hormones in differentiating friend leukemia cells

Low levels of dexamethasone and related gluco-corticoid hormones suppress the expression of globin genes during the DMSO-induced differentiation of Friend leukemia cells. In this response, the glucocorticoids appear to act at both the transcriptional and post-transcriptional levels in that 10 −8 M dexamethasone prevents the accumulation of both globin mRNA and globin protein, whereas 10 −9 M dexamethasone allows the accumulation of normal levels of hybridizable globin mRNA but prevents the accumulation of globin protein. This suppressive action of dexamethasone is more effective with DMSO as the inducer of globin gene expression than with hemin as the inducer. In contrast to the situation with glucocorticoids, certain sex steroids (etiocholanolone, testosterone and estradiol) facilitate the expression of globin genes in DMSO-treated Friend leukemia cells. The modulation of globin gene expression by steroid hormones is achieved in DMSO-treated cells without altering the growth and morphological changes which characteristically attend the differentiation of these cells.

Pyridine derivatives as potent inducers of erythroid differentiation in Friend leukemia cells

Pyridine derivatives as potent inducers of erythroid differentiation in Friend leukemia cells

Disappearance of a 32 000 D chromatin polypeptide during early erythroid differentiation of Friend leukemia cells

The patterns of non-histone chromatin proteins have been investigated in dimethyl sulphoxide (DMSO)-stimulated Friend leukemia cells (FLC) undergoing the “early” events of erythroid differentiation. Sucrose-purified whole nuclei were lysed and proteins extracted with 6 M urea and 4 M guanidium hydrochloride. The proteins were analysed in SDS-and in SDS-urea-polyacrylamide gel electrophoresis. The disappearance of a 32 000 D chromatin protein component was observed in cells of the 745A “inducible” line harvested as early as 24 h after DMSO treatment, as compared with untreated 745A cells and to cells harvested 6 and 12 h after DMSO addition to the cultures. By contrast, a 32 000 D chromatin protein component is always present in untreated as well as in DMSO-treated cells of the “uninducible” Fw line of FLC.

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine.

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.

Nuclear chromatin changes during erythroid differentiation of friend virus induced leukemic cells

Friend leukemia (FL) cells grown in the presence of dimethylsulfoxide (DMSO) undergo erythroid differentiation. Acridine orange (AO) binding to DNA and thermal denaturation of DNA in situ were studied in differentiated and non-differentiated FL cells using flow-through cytofluorometry. The differentiated cells bind less AO than do the non-differentiated ones. The difference in AO binding is higher in the spectrum of emission characteristic for AO intercalation (at 530 nm) than for AO stacking (>600 nm) and depends on AO concentration. The difference is abolished after extraction of acid-soluble macromolecules from cells. During stepwise extraction by lowering pH, there is a progressive increase of AO binding to DNA. Most of the increase in AO binding of the non-differentiated cells occurs at pH 2.5-2.0; of the differentiated cells at pH 1.75-1.50. There are differences in sensitivity of DNA in situ to heat-denaturation between differentiated and non-differentiated cells, as evidenced by the variation in height and in position of the melting bands on derivative melting profiles. The changes described suggest that a profound modulation of chromatin structure, perhaps involving altered DNA-histone interactions, occurs during the DMSO-induced erythroid differentiation of FL cells.

Cryoprotective Agents as Inducers of Erythroleukemic Cell Differentiation In Vitro

The ability of families of compounds with known and potential cryoprotective properties to induce the differentiation of Friend leukemia cells in vitro was studied. For each agent, both the proportion of differentiated cells in the culture and the total amount of heme/10(7) cells were determined. Within each family of compounds there was a direct correlation between a compound's cryoprotective ability, its ability to donate electron pairs for hydrogen bonding (basicity), and its ability to induce differentiation. While individual agents differed with respect to the proportion of cells which were induced to differentiate, the biology of the process of differentiation appeared to be similar, regardless of the agent used. A cell line which was unresponsive to DMSO was responsive to other inducers, suggesting that this DMSO-resistant cell line differed from its parent DMSO-responsive cell line either in its metabolism of the inducers or in the ability of the inducers to enter the cell. Alternatively, there may be more than one mechanism involved in the chemical induction of differentiation.

Cryoprotective agents as inducers of erythroleukemic cell differentiation in vitro

The ability of families of compounds with known and potential cryoprotective properties to induce the differentiation of Friend leukemia cells in vitro was studied. For each agent, both the proportion of differentiated cells in the culture and the total amount of heme/10(7) cells were determined. Within each family of compounds there was a direct correlation between a compound's cryoprotective ability, its ability to donate electron pairs for hydrogen bonding (basicity), and its ability to induce differentiation. While individual agents differed with respect to the proportion of cells which were induced to differentiate, the biology of the process of differentiation appeared to be similar, regardless of the agent used. A cell line which was unresponsive to DMSO was responsive to other inducers, suggesting that this DMSO-resistant cell line differed from its parent DMSO- responsive cell line either in its metabolism of the inducers or in the ability of the inducers to enter the cell. Alternatively, there may be more than one mechanism involved in the chemical induction of differentiation.

Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells.

The process of erythroid differentiation in mouse Friend leukemia virus transformed cells (T3-C1-2) was examined by following changes in several enzyme activities of the heme biosynthetic pathway and in heme concentration while the cells were undergoing erythroid differentiation after treatment with dimethylsulfoxide. Untreated cells on the one hand, have a limited capacity for spontaneous differentiation. On the other hand, dimethylsulfoxide(DMSO)-treated cells showed an increase in the activities of delta-aminolevulinic acid (ALA) synthetase, ALA dehydratase, uroporphyrinogen-I synthetase, ferrochelatase, and heme concentration by days 1, 1.5, 2, and 4, respectively. The increase of the heme pathway enzymes and heme concentration followed the order of these enzymes or products as they are arranged in the heme biosynthetic pathway. These changes induced by DMSO were effectively inhibited by treatment with actinomycin D, suggesting that continued RNA synthesis is required for the differentiation process. 5-bromo-2'-deoxyuridine (BrdU) (10(-5) M) inhibited the DMSO-induced changes of the heme pathway enzymes. BrdU was most effective when it was present during the first 2 days of cell culture. It gradually lost its inhibitory effect when added after the 3rd day or later. The BrdU-mediated inhibition was completely overcome by the addition of thymidine (7 x 10(-5) M), but not by uridine (7 x 10(-5) M). All these data suggest that a sequential induction of the heme pathway enzyme takes place during erythroid differentiation of Friend leukemia cells, and that the sequential induction of the enzymes may be due to a sequential activation of genes coding for these enzyme activities.

In vitro and preliminary in vivo studies of compounds which induce the differentiation of Friend leukemia cells.

The progeny of normal hematopoietic stem cells ultimately become mature functional cells through a process involving both cell replication and differentiation. The proliferative aspect is limited and in fact as full maturation is approached the cells lose their proliferative potentital. Built into the process of differentiation is an inherent limitation of the life span of the maturing cells. In essence, acute myelogenous leukemia (AML) results from a disruption of the normal maturation process. Leukemic stem cells proliferate without normal constraints and their progeny for the most part remain at the stem cell level. This latter phenomenon results in the cells retaining their proliferative potential and endows the cells with a life span which is longer than that of normal cells. The resulting increasing mass of leukemic cells, through an as yet undefined process, interferes with the proliferation and maturation of normal myeloid elements ultimately causing the pancytopenia which is directly responsible for the morbidity and mortality which accompanies AML.

Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. 3. Effects of 5-bromo-2'-deoxyuridine, dimethylformamide and dimethylsulfoxide.

AbstractErythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5‐Bromo‐2′‐deoxyuridine (BUdR) (10−5M) inhibits both DMF‐ and DMSO‐stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect.Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO‐stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR‐inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.