Extinction of globin gene expression in human fibroblast × mouse erythroleukemia cell hybrids

We have chosen human fibroblast x mouse erythroleukemia hybrid cells as a model system to examine regulation of unique genes. The globin genes were studied as a marker of erythroid differentiation. Three separate hybrid cell lines were incubated in 2% dimethylsulfoxide, an agent which induces erythroid differentiation of the parental erythroleukemia cells. Neither human nor mouse globin mRNA sequences could be detected by a sensitive molecular hybridization assay which utilized globin complementary DNA. However, DNA from one of the cell lines was shown to contain both the mouse and human globin genes. Thus, loss of the genes by chromosomal segregation did not account for their failure to be expressed. Cocultivation of the mouse erythroleukemia cells with excess human fibroblasts did not prevent erythroid differentiation of the erythroleukemia cells in the presence of dimethylsulfoxide. Similarly globin gene expression was preserved in tetraploid cells generated by fusion of two erythroleukemia lines. Thus, extinction of globin gene expression in the human fibroblast x erythroleukemia hybrids occurred at the level of mRNA production and appeared to be due to the presence of the fibroblast genome within the hybrid cell.     

Chicken transferrin receptor expression during erythroid differentiation and by retrovirus transformed cells

Antiserum prepared against sucrose gradient purified reticuloendotheliosis virus (REV) recognized the chicken transferrin receptor. Molecules immunoprecipitated from red blood cells (RBC) obtained from embryonic chickens with either the anti-REV reagent or a chicken transferrin immunomatrix were demonstrated to be identical by co-migration in both reducing and nonreducing SDS-polyacrylamide gels and in two-dimensional isoelectric focusing analyses, reciprocal immunodepletion analyses and by peptide mapping. The chicken transferrin receptor was shown to be a 190,000 dalton cell surface membrane molecule consisting of two similar disulfide-bonded subunits of approximately 95,000 daltons. The chicken transferrin receptor was expressed on erythroid cell surface membranes as 95,000 dalton monomers as well as 190,000 dalton dimers. The chicken transferrin receptor was expressed on all differentiation/maturation stages, including mature RBC, of both the primitive and definitive type I erythroid cell series. In adult chickens, the transferrin receptor was expressed by immature erythroid cells in the bone marrow, but not by mature circulating RBC. REV-transformed immature lymphoid cells and avian erythroblastosis virus (AEV)-transformed erythroid cells expressed dimers composed of 95,000 and 110,000 dalton subunits. Comparisons among V8 protease derived peptides from 95,000 dalton transferrin receptors obtained from RBC and REV-transformed lymphoid cells revealed a high degree of homology; however, the 95,000 dalton molecules isolated from REV-transformed lymphoid cells exhibited a 56,000 dalton peptide that was unique. Cloned AEV-transformed erythroleukemia cells induced to differentiate by supplementation of the media with 1 mM butyric acid expressed elevated transferrin receptor levels. Both serological and peptide mapping studies demonstrated the human transferrin receptor on K562 cells and the chicken transferrin receptor to be distinct. However, chicken transferrin was shown to be capable of reacting with the human transferrin receptors on K562 cells.     

Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells

Somatic cell hybrids between hepatoma and Friend erythroleukemia parental cells were studied for the expression of liver-specific and erythroid properties. Several independent clones were isolated using HAT selection and were shown to be true hybrids by isozyme and chromosome analysis. All displayed a complete extinction of hemoglobin and globin mRNA production, but a retention of albumin and transferrin secretion. The data suggest that erythroid differentiation is being actively inhibited by the hepatoma genome. Possible mechanisms that might explain these results are discussed in the light of current hypotheses regarding the mechanism of cell differentiation.     

SLC30A3促进人髓性白血病细胞K562向早期红系分化

 目的 研究SLC30A3在红系分化中的作用。 方法 在人髓性白血病K562细胞系中用质粒载体过表达SLC30A3后，检测细胞红系分化指标CD235、ε-、γ-和β-珠蛋白的表达量及细胞增殖速度。流式细胞术检测CD235的表达，荧光实时定量PCR检测珠蛋白mRNA水平，Western blot法检测转录因子GATA-2蛋白水平，MTS法检测细胞增殖速度。结果 过表达SLC30A3使K562细胞的红系分化特异标志CD235的表达升高，CD235阳性细胞率由对照组的34.25%±16.89%增加至95.7%±0.14%，ε-、γ-和β-珠蛋白的表达明显升高，红系分化相关转录因子GATA-2的表达降低，细胞增殖速度加快。结论 SLC30A3促进人髓性白血病K562细胞系向早期红系分化。     

Enhancement of polyethylene glycol-mediated cell hybridization by inducers of erythroleukemia cell differentiation

Dimethyl sulfoxide (DMSO) has many biological effects, which include enhancement of polyethylene glycol (PEG) -mediated cell fusion, induction of cell differentiation in erythroleukemia and other cell systems, and cryoprotection of cells from freezing damage. In this study, compounds which induce erythroleukemia cell differentiation were tested for their ability to enhance PEG-mediated cell fusion. It was found that many compounds which induce erythroleukemia cell differentiation also promote cell membrane fusion as well as protect cells against freezing damage. Hence, many inducers of erythroleukemia cell differentiation have direct and similar effects on cell membranes. This study also demonstrates previously unrecognized effects of cryoprotective agents and cell fusogens on the differentiated state of cultured cells.     

NonO蛋白在丁酸钠诱导小鼠红白血病细胞分化过程中的表达

目的探讨NonO蛋白在丁酸钠诱导小鼠红白血病(MEL)细胞向红细胞系分化过程中的表达变化。方法利用联苯胺染色观察丁酸钠诱导MEL细胞红细胞系分化情况,通过Western blotting、免疫细胞化学检测NonO蛋白在丁酸钠诱导MEL细胞分化的过程中表达量的变化,PCR技术检测NonO基因在RNA水平上的表达变化,免疫细胞化学技术检测NonO蛋白在MEL细胞中的定位。结果发现NonO在丁酸钠诱导MEL细胞向红细胞系分化过程中在基因水平和蛋白水平上均上调表达,NonO蛋白在MEL细胞中定位于细胞质和细胞核。结论NonO蛋白与丁酸钠诱导MEL细胞分化密切相关。     

Expression and capping of a proliferation-associated surface membrane p34 kDa antigen on different human hematopoietic cell lines

A novel surface membrane nonglycosylated acidic polypeptide (34 kDa), encoded by a structural gene on chromosome 11, has been identified using murine monoclonal antibody (MoAb) 53.6 (IgG2a). MoAb 53.6, raised against uninduced cells of a human erythroleukemia line (HEL), recognizes a surface membrane antigen that is displayed on proliferating (cell cycle phase G1, S, and M + G2 phase) human leukocytes. The expression and redistribution (i.e., patching and capping) of the p34 kDa antigen on 27 different long-term human hematopoietic cell (HHC) lines was defined by fluorescence microscopy. These lines had been established from patients with leukemia or healthy donors and included phenotypically defined populations of T cells, B cells, and myelomonocytic cells. Almost all (greater than 95%) of the leukocytes of the 27 lines reacted strongly with MoAb 53.6. The majority of the leukocytes displayed p34 kDa antigen patching (26/27 lines; patched cells, 96-100%); moreover, 20 of 27 lines exhibited p34 kDa antigen capping (capped cells, 8-96%). Presentation of the p34 kDa antigen on surface membrane ultrastructures, imaged with immunogold using an indirect antibody labeling procedure, was illustrated by scanning electron microscopy, and endocytosis of the gold-tagged antigen-antibody complex was studied by transmission electron microscopy. The HHC lines are thought to represent immortalized populations of different human leukocyte subsets that are in different stages of maturation and/or differentiation; thus these lines should prove useful as models for further characterizing this unique p34 kDa proliferation-associated antigen and for defining the mechanisms and significance of surface membrane antigen redistribution and modulation that has been associated with leukocyte activation and propagation.     

Nrf2通过调控EZH2的表达促进DMSO诱导的MEL细胞红系分化

目的 探讨Nrf2/EZH2通路参与DMSO诱导MEL细胞红系分化的作用及相关分子机制.方法 以MEL细胞为靶细胞,DMSO为刺激源,联苯胺染色法检测细胞红系分化情况;免疫印迹检测EZH2和Nrf2蛋白表达水平.结果 DMSO可显著诱导MEL细胞红系分化,同时EZH2和Nrf2表达水平也明显升高.通过使用Nrf2诱导剂TBHQ及Nrf2 shRNA,证明Nrf2调控MEL细胞中EZH2的表达.敲低Nrf2和EZH2的表达能够抑制DMSO诱导的MEL细胞红系分化.结论 Nrf2可通过调控EZH2的蛋白水平从而发挥促进MEL细胞红系分化的作用.     

Transformation of erythroid cells by rous sarcoma virus (RSV)

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive (ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42 degrees), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type (wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37 degrees) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60src kinase is involved in erythroid cell transformation. Since the src and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.     

Lipid changes associated with erythroid differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells were induced to differentiate by dimethyl sulfoxide (DMSO) and hexamethylene-bis-acetamide (HBMA) in order to investigate whether their lipid characteristics, common to other systems of transformed cells, revert to a normal differentiation pattern. DBA/2 mouse erythrocytes were examined as a model of terminal differentiation in erythroid lineage. Variants of erythroleukemia cells not inducible to erythroid differentiation by DMSO and HMBA were also used in this study, in order to test whether lipid modifications occurring in differentiated erythroleukemia cells were related to the differentiation process or caused by specific effects of the inducers. Friend erythroleukemia cells showed the same lipid characteristics as those found in other transformed cell types. That is, a high level of ether-linked lipids and low percentages of long chain polyunsaturated fatty acids along with an accumulation of monoenoic fatty acids in phospholipids. These lipid characteristics remained unchanged when erythroleukemia cells were induced to differentiation by either DMSO or HMBA. However, other lipid components of erythroleukemia cells, e.g., phosphatidylethanolamine and triglycerides, were affected by erythroid differentiation. There were also changes of some lipid components of erythroleukemia cells, such as cholesteryl esters, which were related to specific effects of the inducers. Both DMSO- and HMBA-resistant variants differed from the inducible erythroleukemia cells, mainly in their ether-linked phospholipid pattern.     

Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.     

Phenotypic heterogeneity among temperature-sensitive mutants of rous sarcoma virus. Studies with inhibitors of protein synthesis

Fourteen temperature-sensitive (ts), transformation-defective mutants have been isolated from mutagenized Schmidt-Ruppin Rous sarcoma virus. We report that, while in cells infected with most of the mutants all parameters of cell transformation were coordinately suppressed, certain ts mutants induced the ability of infected cells to multiply in soft agarose and to express the tumor-specific surface antigen (TSSA), in the absence of morphological conversion. Studies with inhibitors of protein synthesis have shown that cycloheximide (Ch) and pactamycin (Pac) act differently on focus formation from Pu and that the heat-labile src gene product of these mutants may be spontaneously reactivated following a downshift to permissive temperature. Our data also indicate the existence of two classes of mutants regarding the response of infected cells to Pu. In most cases, focus formation is inhibited by Pu when infected cells are shifted to 37°. However, cells infected with three ts mutants resume morphological transformation at permissive temperature in the presence of this inhibitor. In addition, the fact that the same mutants induce high levels of TSSA expression at restrictive temperatures suggests that the two properties may be linked.     

Wnt-10a在氯高铁血红素诱导K562细胞分化过程中的作用

目的 探讨Wnt-10a在氯高铁血红素（hemin）诱导K562细胞分化过程中的变化及功能。 方法 通过联苯胺染色检测K562细胞被hemin诱导的阳性率,利用Western blotting和免疫细胞化学分析K562细胞诱导分化过程中Wnt-10a表达水平和细胞定位的变化情况,通过半定量PCR与Real-time PCR检测K562细胞诱导分化过程中Wnt信号途径关键因子的表达水平及其变化,通过Wnt信号途径激活剂与构建Wnt-10a高表达K562细胞株的方法探讨Wnt途径改变后对K562细胞诱导分化的影响。 结果 在hemin诱导K562细胞分化过程中,细胞中的Wnt-10a蛋白表达量和mRNA量都出现了短暂下降后回复的趋势,同时Wnt-10a向胞膜迁移,Wnt蛋白所参与的3条信号途径中关键因子的表达水平均有着不同程度的变化。使用Wnt信号通路抑制剂后,诱导分化过程中的细胞增殖活力加强,Wnt-10a高表达的K562细胞被hemin诱导分化的能力开始提高,并且Wnt经典信号途径的关键因子表达水平也有上升。 结论 Wnt-10a与hemin诱导K562细胞红系分化的过程密切相关。     

Different sequences of expression of band 3, spectrin, and ankyrin during normal erythropoiesis and erythroleukemia.

Expression of the erythrocyte anion exchanger band 3, and ankyrin and spectrin, two cytoskeletal proteins of the red blood cell membrane, was studied by immunofluorescence using: 1) smears of human bone marrow from healthy donors and from a patient with erythroleukemia, 2) human red blood cell precursors grown in cell culture, and 3) murine erythroleukemia cells grown in cell culture. Double immunostaining with antibodies to band 3 in combination with spectrin or ankyrin revealed that these proteins become expressed synchronously during normal human erythropoiesis. In contrast, both murine erythroleukemia cells (induced by fibronectin and dimethyl sulfoxide to differentiate in vitro) and erythroblasts from a patient suffering from erythroleukemia displayed distinct asynchronicity in expression of these proteins, ie, ankyrin and spectrin were synthesized first, followed by band 3 at a later stage of erythroid development. After the onset of band 3 expression in human erythroleukemia cells, an increase of membrane-associated fluorescence was detectable for both ankyrin and spectrin, supporting the general view that band 3 promotes assembly of the membrane cytoskeleton. These findings indicate that the current concept of a sequential expression of spectrin/ankyrin and band 3 is valid only for erythroleukemia cells or transformed erythropoietic cell lines but does not occur in normal erythropoiesis, during which these proteins become expressed simultaneously.     

Differential induction of tumour antigens by transformation-defective virus mutants.

Normal rat kidney cells infected by a variety of transformation-defective temperature-sensitive avian leukosis sarcoma virus mutants were tested for the expression of transformation characteristics at permissive and restrictive temperature. Morphology, growth behaviour and agglutinability by concanavalin A corresponded fully to the phenotype of the infected cells: at permissive temperature the cells resembled wild type virus transformed cells, whereas when grown under restrictive conditions they became virtually indistinguishable from normal cells. The quantitative expression of allo- or xenogeneic cell surface antigens was not significantly affected by the phenotype of the cells. Two out of the five tested mutants induced tumour antigens in the expected temperature-dependent manner, whereas the other three mutants were able to induce tumour-specific cell surface antigens even in the revertant cells cultured at the restrictive temperature. These findings extend previous results about tumour antigen induction in mutant-infected cells of the natural host, the chicken embryo fibroblasts. The value of transformation-defective tumour antigen-positive mutants for vaccination purposes will be discussed.     

Characterization and induction of a cell line established from a patient with erythroleukemia (FAB M6)

A human cell line, designated as NISI, was established from a patient with erythroleukemia (FAB M6). The presence of a chromosome marker in NISI line indicates that it is derived from the leukemic clone which bears the common marker. The cell line shows morphology of immature erythroblast and has myelocytic properties from surface immunophenotyping. The differentiation capacities of NISI cells by three inducers (hemin, phorbor 12-myristate 13-acetate and 1-25-(OH)2D3) were evaluated. Hemin treated cells showed a significant increase in erythroid antigen expression as well as an increase in number of benzidine positive cells. Phorbor 12-myristate 13-acetate treated cells demonstrated a significant increase in megakaryocytic antigen expression, and a slightly diminished CD36 antigen expression. The surface markers of 1-25-(OH)2D3 treated cells did not demonstrate significant changes. NISI cell line, a human erythroleukemia cell line, still retained the tendency for differentiation to megakaryocytic lineage.     

The relationship between the formation of inclusions and viral DNA synthesis in adenovirus 12-infected cells

The relationship between the formation of inclusions and viral DNA synthesis was examined in cells infected with human adenovirus type 12 (H12) DNA-minus temperature-sensitive (ts) mutants and with H12 wild type (WT) in the presence of inhibitors of DNA synthesis. Three groups of H12 ts mutants (tsA, tsB, and tsC) are defective in initiation of viral DNA synthesis at the restrictive temperature (40°C). Inclusions were either absent or abortively formed in cells infected with the ts mutants at 40°, although inclusions comparable to those in WT-infected cells were observed in cells infected with the ts mutants at the permissive temperature (34°). The temperature shift-up experiment showed that the inclusion formed in cells infected with tsA at 34° disintegrated at 40°, while those in cells infected with tsB and tsC were retained at 40°. This observation suggests that the morphological integrity of the inclusions is dependent on gene A and the function of the inclusions on genes A, B, and C. The temperature shift-down of tsA-infected cells also showed the formation of inclusions and the recovery in viral DNA synthesis. The inclusions were not formed in WT-infected cells, when viral DNA synthesis was inhibited with either cytosine arabinoside or hydroxyurea. Viral DNA synthesis was not induced and the inclusions were not formed in infected cells after removal of cytosine arabinoside. In contrast, viral DNA synthesis was induced rapidly and the inclusions were formed concomitantly in cells after removal of hydroxyurea. It was shown that the type II inclusions were formed in H12-infected monkey cells, although neither the type IV inclusions nor virions were formed. These observations show the close relationship between the formation of inclusions and viral DNA synthesis, suggesting an important role of DNA-binding protein, the gene-A product, in the formation and the function of inclusions.     

A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors

Anaemia is an important global health problem. Therefore, it is crucial to understand its pathophysiology in various genetic or infectious diseases where dyserythropoiesis is a key pathological feature. To this effect, reproducible and reliable models of erythropoiesis in vitro are much needed as investigative tools. We have developed a serum-free liquid culture model of erythropoiesis using human umbilical cord blood CD34(+) cells cultured in the cytokine combination, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) and erythropoietin (Epo), over 14 days. We found that these culture conditions favored erythroid differentiation over the expansion of the more primitive erythroid precursors. With an initiating culture density of 5x10(4) cells per ml, the nucleated cell fold expansion increased from 7.9+/-3.9 (range 4.5 to 11.1) after 4 days to 2990.2+/-1936.1 (range 626.6 to 6912.0) after 14 days in culture. Day-14 burst-forming unit-erythroid (BFU-E) frequencies peaked at day 4 (24.0+/-8.9%), with a marked decrease in BFU-E burst size as the cultures progressed. Time-course immunophenotypical profiles were characteristically erythroid with a decrease in CD34 expression (from 96.8+/-3.0% at day 0 to 0.8+/-0.8% at day 14), and a concomitant increase in the expression of erythroid-specific markers, CD36, glycophorin A (GpA) and CD71 (from 14.8+/-5.0%, 1.7+/-1.0% and 37.9+/-18.0% to 93.0+/-7.0%, 82.1+/-14.0% and 95.7+/-3.0%, respectively). Morphological studies revealed the presence of normoblasts with the complete absence of reticulocytes and mature erythrocytes after 14 days in culture. Once the culture conditions were optimized, we scaled down our culture model from 24-well plate (large-scale) to 96-well plate cultures (small-scale). We found that the small-scale cultures compared favorably with their large-scale counterpart in terms of erythroid progenitor cell proliferation and differentiation, particularly at low CD34(+) initiating cell doses. By using tumor necrosis factor-alpha (TNF-alpha), a known inhibitor of erythropoiesis, we validated our model system and showed a dose-dependent inhibition of erythroid differentiation with TNF-alpha in our cultures. Therefore, our results demonstrate a small-scale serum-free liquid culture model of erythropoiesis that is comparable with and complements our well-defined large-scale model. Our system would prove useful for screening the effects of exogenous factors on erythropoiesis in vitro.     

EDDF诱导产生与红细胞终末分化相关的HS2特异结合蛋白

以珠蛋白基因增强子为探针，研究从ＦＶＡ病毒诱导的小鼠脾脏中提取的ＥＤＤＦ诱导小鼠红白血病细胞（ＭＥＬ）及人红白血病细胞（Ｋ５６２）后的表达产物与ＨＳ２特异结合的可能性。结果表明，经ＥＤＤＦ诱导后的ＭＥＬ及Ｋ５６２细胞核中均可检测到两个与ＨＳ２结合的蛋白，其分子量约为１２ｋＤ和１４ｋＤ。提示ＥＤＤＦ有可能通过诱导产生与ＨＳ２特异结合的反式调节因子而导致红系细胞分化。