Friend Murine Erythroleukemia Research Articles

The Transcription Factor Spi-1/PU.1 Interacts with the Potential Splicing Factor TLS

Spi-1/PU.1 is an Ets protein deregulated by insertional mutagenesis during the murine Friend erythroleukemia. The overexpression of the normal protein in a proerythroblastic cell prevents its terminal differentiation. In normal hematopoiesis Spi-1/PU.1 is a transcription factor that plays a key role in normal myeloid and B lymphoid differentiation. Moreover, Spi-1/PU.1 binds RNA and interferes in vitro with the splicing process. Here we report that Spi-1 interacts in vivo with TLS (translocated in liposarcoma), a RNA-binding protein involved in human tumor-specific chromosomal translocations. This interaction appears functionally relevant, since TLS is capable of reducing the abilities of Spi-1/PU.1 to bind DNA and to transactivate the expression of a reporter gene. In addition, we observe that TLS is potentially a splicing factor. It promotes the use of the distal 5' splice site during the E1A pre-mRNA splicing. This effect is counterpoised in vivo by Spi-1. These data suggest that alteration of pre-mRNA alternative splicing by Spi-1 could be involved in the transformation of an erythroblastic cell.

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells.

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT-PCR for the constitutive and inducible expression of megakaryocyte-specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor-4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid alpha- and beta-globin genes were not transcribed appreciably. However, MELC exposed to 5 mM hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA. and simultaneous induction of the globin genes. Megakaryocyte-related genes were expressed. in the absence of globin gene transcription, by MELC treated with either phorbol-12-myristate acetate (PMA; 100 ng/ml) or colcemid (40 nM), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c-mpl. PMA and colcemid did not affect high basal c-myb mRNA levels which, in turn, were down-regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo-R and IL-3R mRNAs, whereas no expression of granulocyte/macrophage-related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro-megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

NF-E2p18/mafK is required in DMSO-induced differentiation of Friend erythroleukemia cells by enhancing NF-E2 activity.

When Friend murine erythroleukemia (F-MEL) cells are induced to differentiate by dimethylsulfoxide (DMSO), erythroid-specific genes are transcriptionally activated. The erythroid transcription factor NF-E2 is essential for enhancer activity of the globin locus control regions. NF-E2 functions as a heterocomplex consisting of a 45-kDa subunit (NF-E2p45) and a 18-kDa subunit (NF-E2p18). The larger subunit NF-E2p45 is tissue-restricted and is believed to play a role in globin gene expression in F-MEL cells. The expression of the smaller subunit NF-E2p18, which is a Maf family member (MafK), is cell type- and developmental stage-specific. We have investigated the possible role of NF-E2p18 in Friend erythroid differentiation by stably transfecting either sense and antisense p18 constructs into differentiation-sensitive 745A and partially defective-differentiation TFP10 cell lines. Overexpression of NF-E2p18 induced expression of globin transcripts in both cell lines and increased their sensitivity to erythroid differentiation when exposed to DMSO. Conversely, inhibition of p18 expression by antisense transcripts resulted in the inhibition of DMSO-induced differentiation in both cell lines. These results indicate that NF-E2p18 is necessary for globin expression in F-MEL cells and that it is the predominant gene of the Maf family involved in DMSO-induced erythroid differentiation. Moreover F-MEL clones overexpressing NF-E2p18 showed an increase in specific NF-E2 DNA-binding activity whereas this activity was decreased in clones expressing antisense p18. Finally, studies using transient transfection assays showed that p18 activated NF-E2 site-dependent transcription in F-MEL cells. These data suggest that NF-E2p18 can participate in DMSO-induced erythroid differentiation of F-MEL cells by enhancing NF-E2 activity.

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture.

The authors established apoptosis resistant COS-1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS-1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl-2 gene. Both bcl-2 and mock transfected COS-1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl-2 transfected cells was ninefold of that of the mock transfectants. Both bcl-2 and mock transfectants were further transfected with the vector pcDNA-λ containing SV40 ori and immunoglobulin λ gene for transiently expressing λ protein. The bcl-2 expressing COS-1 cells produced more λ protein than the mock transfected COS-1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl-2 gene. Both bcl-2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl-2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag-1 with bcl-2 improved survival of hybridoma 2E3 cells more than bcl-2 expression alone. The bag-1 and bcl-2 coexpressing cells produced more IgG than the the cells expressing bcl-2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.

Electrorotational studies of the cytoplasmic dielectric properties of Friend murine erythroleukaemia cells

Electrorotation (ROT) spectra of Friend murine erythroleukaemia DS19 cells were measured in the frequency range 10 kHz-100 MHz as a function of suspension osmolality and cell differentiation treatment with hexamethylene bisacetamide (HMBA). A minimization program was employed to curve-fit the measured spectra using a single-shell dielectric model, allowing the derivation of cellular interior conductivity and permittivity (valid for the frequency range 10-100 MHz) and the cytoplasmic membrane capacitance (its dependence on the cell differentiation state and suspension osmolality having been reported earlier). Following HMBA treatment, DS19 cells exhibited a slight increment in average interior permittivity and a decrement in interior conductivity, although the changes were not statistically significant. For both untreated and HMBA treated samples, the average interior conductivity increased and permittivity decreased with increasing suspension osmolality. Of significance was that the average permittivity of cell interiors was larger than that of pure water. The electrorotation spectra of freshly prepared cell nuclei were measured, and the derived nuclear dielectric parameters were employed in numerical simulations to investigate the effects of nuclei on the ROT spectra of intact cells. Other cellular internal structures such as mitochondria were also analysed using theoretical simulations. It was concluded that the derived large permittivity values did not result from cell nuclei or mitochondria, and, instead, we suggest that they may arise from the combined effects of several cytoplasmic organelles.

Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins

The c- myb proto-oncogene encodes a transcription factor that has been implicated in the regulation of haemopoietic cell differentiation and appears also to be required for cell proliferation in a number of different lineages. Typically, transcription of c- myb is down-regulated during haemopoietic cell differentiation, and it has been found in several erythroid and myeloid cell lines that constitutive c- myb expression, from a transfected plasmid, blocks this differentiation process. To investigate further the activity of c- myb in haemopoietic cell differentiation, we have transfected Friend murine erythroleukaemia (F-MEL) cells with plasmids encoding conditionally active c-Myb/oestrogen receptor (Myb/ER) fusion proteins. Transcriptional activity of the Myb/ER fusion proteins was found to be strictly hormone-dependent, and this property was correlated with the ability of these proteins to inhibit erythroid differentiation. From analysis of a Myb/ER protein that lacks the c-Myb transactivation domain, it was apparent that the C-terminal ER transactivation domain could substitute for that of c-Myb in inhibition of differentiation. Activation of Myb/ER in F-MEL cells had no effect upon the early and transient inhibition of entry into S phase associated with dimethyl sulfoxide (DMSO) induction. Further analyses of α-globin and PU.1 gene transcription suggested that c-Myb is unable to influence gene expression immediately following DMSO-induction and that inhibition of F-MEL cell differentiation must therefore result from the function of c-Myb in the post-commitment period. Nonetheless, c-Myb had effects on the erythroid differentiation programme that were clearly dissociated from its role in cell proliferation. The potential use of this system to identify c-Myb target genes involved in F-MEL cell differentiation is discussed.

Identification and Conditions for Selective Expression of Megakaryocytic Markers in Friend Erythroleukemia Cells

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase C beta from Friend erythroleukemia cells is associated with chromatin and DNA.

Certain protein kinase C (PKC) isotypes are localized to the nucleus during cellular proliferation in murine erythroid cells, as well as in human promyelocytic leukemia and erythroleukemia cells. Because the structure of these PKC isotypes contains a conserved cysteine-rich region that contains the zinc finger DNA binding motif, we tested the hypothesis that selected PKC isotypes found in Friend erythroleukemia cells can bind to DNA. Cell lysates from murine Friend erythroleukemia cells, which express alpha, beta I, and beta II PKC, expressed greater amounts of the beta isoforms than the alpha isoform of PKC in their nuclei, and PKC beta I was found in the chromatin of these cells. Lysates of these cells were tested for their ability to bind to a DNA-cellulose column. Bound proteins were eluted with a step gradient of increasing KCl concentrations, and eluant fractions were then subjected to immunoblot analysis using isotype-specific antibodies to the alpha and beta I isotypes of PKC. DNA binding was detected for the PKC beta I isotype, which is present in the nucleus, but not for the more abundant PKC alpha isotype, which resides primarily in the cytoplasm. These results demonstrate that PKC can associate with DNA, and that this association is isotype specific in Friend erythroleukemia cells.

Induction of differentiation in murine erythroleukemia cells by 1 alpha,25-dihydroxy vitamin D3.

The Friend murine erythroleukemia (MEL) cells can be stimulated to differentiate in response to a variety of chemical inducing agents. In the present study, the effect of 1 alpha,25-dihydroxyvitamin D3 on differentiation of MEL cells was investigated. Vitamin D3 induced differentiation of MEL cells in culture as determined by elevated hemoglobin content, a rise in the number of benzidine-positive cells and increase in acetylcholine esterase activity. The optimum concentration of the vitamin required to induce differentiation of MEL cells was found to be 750 nM. The pattern of induction of differentiation was similar to that observed with DMSO and the induction of differentiation by vitamin D3 was inhibited by dexamethasone.

Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation

We used electrorotation measurements to investigate alterations in the plasma membranes of DS19 murine erythroleukaemia cells that accompanied erythropoietic differentiation induced by hexamethylene bisacetamide (HMBA). Following 3 days of HMBA treatment, the mean cell membrane specific capacitance determined from electrorotation spectra of individual, viable cells at physiological tonicity (300 mosmol/kg) fell from 1.74 to 1.53 μF/cm 2, in agreement with trends observed earlier by dielectrophoretic measurements on bulk cell populations. Scanning and transmission electron microscopy revealed that the relatively high values found for cell membrane capacitance ( > 1 μ F/cm 2 ) reflected the large area of plasma membrane associated with complex surface morphology including numerous microvilli. Furthermore, it demonstrated that the fall in membrane capacitance during HMBA treatment correlated with a reduction in the density of these complex surface features. Differences in the mechanical characteristics of the cell membranes of untreated and treated cells were then examined by exposing cells to osmotic stress. The intricacy of membrane morphology intensified with increasing osmolality of the suspending medium and this was reflected in higher specific capacitance values. When the osmolality was increased from 210 to 450 mosmol/kg, the mean membrane capacitance of untreated DS19 cells changed from 1.58 to 2.05 μF/cm 2 while that for HMBA-treated cells changed from 1.47 to 1.72 μF/cm 2, a significantly smaller response. This demonstrated that cells exposed to 72 h of differentiation treatment had an enhanced mechanical resilience as compared with their untreated counterparts, evidencing the early stages of the development of the membrane skeleton which becomes fully developed in mature erythrocytes. Our findings demonstrate the value of electrorotation measurements as a method for the non-invasive characterisation of viable leukaemic cells and their responses to stimuli and show that the membrane capacitance values so derived reflect membrane morphology.

Putative Oncogenic Role of the Erythropoietin Receptor in Murine and Human Erythroleukemia Cells

Abstract To determine whether the erythropoietin receptor (Epo-R) plays a role in the course of malignant erythropoietic disorders, this gene was studied in murine and human erythroleukemia cells. An altered Epo-R gene was found in a murine Friend erythroleukemia cell line, FCL1, due to a spleen focus-forming virus (SFFV) long terminal repeat insertion within the noncoding region of the first exon, leading to Epo-R mRNA overexpression. A similar mechanism of Epo-R activation has previously been described in the T3CL-2 Friend erythroleukemia cell line. An elevated number of Epo-binding sites has been observed in two human erythroleukemia cell lines, TF-1 and UT7. In UT7 cells, homogeneously staining region of the short arm of chromosome 19 [hsr (19)] was evidenced, which contained an amplification of the Epo-R gene. This Epo- R gene amplification was confirmed by the quantification of Southern blots in which the intensity of the Epo-R signal was compared in UT7 DNA and in DNA from normal cells. The Epo-R gene was present in UT7 at a mean number of seven to eight copies per cell. Interestingly, the Epo- R gene was rearranged; the breakpoint region was located near the 3′ end of the gene, 3 kb downstream from the end of the last exon. Taken together, these results suggest that, in both murine and human systems, genetic alterations of the Epo-R gene are not rare events and could be involved in the occurrence of the erythroleukemic process.

Putative oncogenic role of the erythropoietin receptor in murine and human erythroleukemia cells

To determine whether the erythropoietin receptor (Epo-R) plays a role in the course of malignant erythropoietic disorders, this gene was studied in murine and human erythroleukemia cells. An altered Epo-R gene was found in a murine Friend erythroleukemia cell line, FCL1, due to a spleen focus-forming virus (SFFV) long terminal repeat insertion within the noncoding region of the first exon, leading to Epo-R mRNA overexpression. A similar mechanism of Epo-R activation has previously been described in the T3CL-2 Friend erythroleukemia cell line. An elevated number of Epo-binding sites has been observed in two human erythroleukemia cell lines, TF-1 and UT7. In UT7 cells, homogeneously staining region of the short arm of chromosome 19 [hsr (19)] was evidenced, which contained an amplification of the Epo-R gene. This Epo-R gene amplification was confirmed by the quantification of Southern blots in which the intensity of the Epo-R signal was compared in UT7 DNA and in DNA from normal cells. The Epo-R gene was present in UT7 at a mean number of seven to eight copies per cell. Interestingly, the Epo-R gene was rearranged; the breakpoint region was located near the 3' end of the gene, 3 kb downstream from the end of the last exon. Taken together, these results suggest that, in both murine and human systems, genetic alterations of the Epo-R gene are not rare events and could be involved in the occurrence of the erythroleukemic process.

Distribution of globin genes and histone variants in micrococcal nuclease‐generated subfractions of chromatin from Friend erythroleukemia cells at different malignant states

The distribution of the alpha and beta-globin genes and histone variants was examined in micrococcal nuclease-generated chromatin fractions of three Friend murine erythroleukemia cell types differing in malignant potential and inducibility to erythroid differentiation. A preferential concentration of globin gene sequences, as compared to satellite DNA, was noted in a physiological salt-soluble, histone H1-depleted, mononucleosomal chromatin fraction (Sup 120) in all Friend cell types, even those in which the globin gene was not capable of transcriptional activation by chemical induction. The level of globin gene enrichment in the Sup 120 fraction was highest in the most malignant and inducible cell type. The chemical induction of erythroid differentiation in this cell line did not change the distribution of globin genes in the chromatin fractions. The Sup 120 chromatin fraction prepared from mouse brain nuclei was not enriched in globin genes. Besides the previously reported low H2A. 1/H2A.2 ratio [Blankstein and Levy: Nature 260:638-640, 1976], chromatin from the most tumorigenic cell type showed the lowest H2B.2 to H2B.1 ratio, highest levels of histone H4 acetylation, and the most pronounced change in relative amounts of two major electrophoretic bands of histone H1 variants as compared to the less malignant cell types. The histone variant content of the micrococcal nuclease-generated chromatin fractions from the three Friend cell types reflected the core histone variant differences for the respective intact nuclei. However, the electrophoretic separation of mononucleosomes by size revealed several classes with different H2A variant ratios. The results demonstrate the existence of structural differences in globin gene and histone variants in erythroleukemia cell chromatin associated with distinguishable phenotypes during malignant cell progression.

The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types.

Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca2+]i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca2+]i was measured in cells loaded with the Ca(2+)-specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca2+]i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca2+]i spike in all of the cell types was not prevented by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca(2+)-channel blockers methoxyverapamil (D600; 100 microM), nifedipine (20 microM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La3+; 1 mM), which blocks both Ca2+ channels and membrane Ca2+ pumps, there was a sustained increase in [Ca2+]i in response to 1% DMSO. By contrast, pretreating P19 cells with La3+ (1 mM) did not prolong the DMSO-triggered [Ca2+]i transient. In all cases, the DMSO-induced [Ca2+]i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 microM). These results suggest that DMSO almost instantaneously triggers the release of Ca2+ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca2+ spike may play a role in the induction of cell differentiation by DMSO.

Membrane changes accompanying the induced differentiation of Friend murine erythroleukemia cells studied by dielectrophoresis

Dielectrophoresis measurements obtained using an image processing technique are reported over the frequency range 1 Hz to 100 kHz for the Friend murine erythroleukemia cell lines DS19 and R1 before and after treatment with hexamethylene bisacetamide and dimethylsulfoxide, agents that induce terminal differentiation in DS19 but not in R1 cells. Data are analyzed according to the single shell dielectric model of the cell. The membrane capacitance was found to fall by 30% and membrane conductivity by a factor of at least 5 when DS19 cells were induced to differentiate. R1 cells showed no such response. While the theoretical model was found to be useful for comparing differences in data for the different cell lines, several significant discrepancies between its predictions and the experimental data were observed, including positive dielectrophoretic collection at frequencies below 20 Hz and a smaller than predicted response to the membrane permeabilizing agents saponin and valinomycin. Factors that may have accounted for these discrepancies include surface charge effects, conduction parallel to the plasma membrane surface, and intracellular compartments.

Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.

Acetylcholinesterase in Murine Erythroleukemia (Friend) Cells: Evidence for Megakaryocyte-like Expression and Potential Growth-Regulatory Role of Enzyme Activity

Features of true acetylcholinesterase (AChE) regulation during growth and differentiation of Friend murine erythroleukemia cells (MELC) have been investigated with respect to other erythroid and nonerythroid murine elements. Enzyme levels of uninduced MELC were in between the very low AChE contents of erythroid cells and the huge amounts of activity exhibited by megakaryocytes and platelets. After MELC commitment to terminal division, the enzyme-specific activity increased largely, approaching values that were much closer to those of thrombocytic than of normal erythroid elements. The bulk of AChE activity in MELC, megakaryocytes, and platelets was found to be located in the cytosol as a free-soluble form. Moreover, during incubation, MELC actively released large amounts of AChE into the medium, like it occurs in murine thrombocytes. Conversely, the enzyme of the erythroid elements was mainly associated with the membranes and was not released extracellularly. Experiments with inducers showed that changes in AChE-specific activity of MELC correlated directly with the arrest of cell proliferation rather than with the activation of differentiated erythroid functions. The inverse relationship existing between MELC growth rates and AChE levels was further supported by the relative enzyme activities of the slow- and fast-growing subclones. We conclude that uninduced MELC potentially share properties of both the erythroid and megakaryoblastic phenotype. The latter might be revealed by typical regulation of AChE activity according to a thrombocytic-like program activated upon MELC commitment to terminal division. Eventually, the inhibition of MELC growth by exogenous pure bovine AChE suggested that the secreted murine enzyme might serve as a potential negative signal of cellular replication.

Acetylcholinesterase in murine erythroleukemia (Friend) cells: evidence for megakaryocyte-like expression and potential growth-regulatory role of enzyme activity

Features of true acetylcholinesterase (AChE) regulation during growth and differentiation of Friend murine erythroleukemia cells (MELC) have been investigated with respect to other erythroid and nonerythroid murine elements. Enzyme levels of uninduced MELC were in between the very low AChE contents of erythroid cells and the huge amounts of activity exhibited by megakaryocytes and platelets. After MELC commitment to terminal division, the enzyme-specific activity increased largely, approaching values that were much closer to those of thrombocytic than of normal erythroid elements. The bulk of AChE activity in MELC, megakaryocytes, and platelets was found to be located in the cytosol as a free-soluble form. Moreover, during incubation, MELC actively released large amounts of AChE into the medium, like it occurs in murine thrombocytes. Conversely, the enzyme of the erythroid elements was mainly associated with the membranes and was not released extracellularly. Experiments with inducers showed that changes in AChE- specific activity of MELC correlated directly with the arrest of cell proliferation rather than with the activation of differentiated erythroid functions. The inverse relationship existing between MELC growth rates and AChE levels was further supported by the relative enzyme activities of the slow- and fast-growing subclones. We conclude that uninduced MELC potentially share properties of both the erythroid and megakaryoblastic phenotype. The latter might be revealed by typical regulation of AChE activity according to a thrombocytic-like program activated upon MELC commitment to terminal division. Eventually, the inhibition of MELC growth by exogenous pure bovine AChE suggested that the secreted murine enzyme might serve as a potential negative signal of cellular replication.

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine friend erythroleukemia cells

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.