Massive splenomegaly in acute erythroid leukaemia (FAB Class-M6): an unusual presentation.

AML-M6 has a peak incidence in the seventh decade with slight male preponderance, and can also present at a younger age. The usual features are anaemia, thrombocytopenia, malaise, fatigue, easy bruising, epistaxis and petechiae. Splenomegaly may occur in 20-40 % of the cases but massive splenomegaly is rare presentation and have been only reported once in humans and once in animals. A 22 year Asian female, presented with fatigue, pallor, mild jaundice, exertional dyspnoea, epigastric pain, tender right hypochondrium and massive splenomegaly. Investigations revealed anaemia and thrombocytopenia, tear drop cells, basophilic stippling, piokilocytosis and anisochromia; increased uric acid and LDH. Abdominal ultrasound showed enlarged liver (22cm) and spleen (20cm). Bone marrow aspiration revealed 51% erythroid and 24% non-erythroid precursors, depressed leukopoeisis and megakarypoeisis. Erythroblasts were PAS and CD71 positive and also reacted to Antihaemoglobin-Antibody. This report highlights characteristic features and diagnostic criteria of erythroleukaemia, differential diagnosis of massive splenomegaly and their rare association.

[TF-1 cell apoptosis-inducing effect of matrine and its effect on SALL4 expression].

OBJECTIVE: To explore the mechanism of matrine (Mat) induced human erythroleukemia TF-1 cell apoptosis and its effect on SALL4 expression. METHOD: Different concentrations of the Mat (0.5, 1.0, 1.5, 2.0 g x L(-1) ) were cultured in vitro in TF-1 cells at different time (24, 48, 72 h). Cell proliferation was assayed by MTT. Cell cycle was determined by flow cytometry (FCM). Cell apoptosis was detected by Annexin V and PI double staining method. SALL4 mRNA expression was detected by reverse transcription RT-PCR (RTT-PCR). RESULT: Administrated with Mat (0.5-2.0 g x L(-1)) after 24, 48, 72 h, the proliferation of TF-1 cells were inhibited (P < 0.01) , and in dose- and time-dependent manner. Half inhibitory concentration (IC50 ) was 1.0 g L(-1) at 48 h. After 48 h that the Mat acted on TF-1 cells, the proportion of G0/G1 phase cells increased while compared with the control group, and S phase cells decreased (P < 0.01). Apoptosis were 8.6% , 11.21%, 15.26% , 17.63%, which showed statistically significant difference (P < 0.01) compared with the control group (5.05%). RT-PCR results showed the ratio between SALL4 mRNA expression and beta-actin (internal reference) expression significantly decreased (P < 0.01) with Mat dose increased. CONCLUSION: In a certain range of concentration and time, Mat can inhibit TFT-1 cells proliferation. The mechanism is to make the cells G0/G1 phase blocked, to inhibit SALL4 gene expression and induce cell apoptosis.

Host humoral and cellular immune mechanisms in the continued suppression of Friend erythroleukemia metastases after interferon alpha/beta treatment in mice.

DBA/2 mice were injected intravenously with 2 x 10(6) 3C18 Friend erythroleukemia cells (FLC), a cell line resistant to interferon alpha/beta (IFN-alpha/beta). Although daily administration of mouse IFN-alpha/beta markedly increased the mean survival time, most IFN-treated mice continued to harbor FLC in different organs. To investigate the mechanisms responsible for this persistent suppression of FLC growth in IFN-treated mice, we undertook a series of adoptive transfer experiments with sera and spleen cells. Sera from FLC-injected, IFN-treated mice were very effective in conferring protection on DBA/2 mice even when injected systemically (intravenously) 18-24 h before intravenous challenge with FLC. These sera also exhibited antitumor activity when injected subcutaneously or intraperitoneally together with FLC. The protective factor in serum was shown to be an immunoglobulin. FLC-injected, IFN-treated mice developed antibodies to FLC demonstrable by radioimmunoassay and complement-dependent cytotoxicity. Sera from these mice recognized a specific 65-kD FLC membrane antigen(s) not detectable on membrane extracts from RBL-5 or ESb tumor cells, or on normal spleen cells. FLC-injected, IFN-treated mice also developed a specific cellular response demonstrable by transfer of protection with spleen cells injected intravenously or subcutaneously. Analysis of the responsible spleen cell populations indicated that the effector cells were neither T nor B cells. These results demonstrating the importance of host humoral and cellular immune mechanisms in the persistent suppression of FLC in IFN-treated mice may be relevant to the use of IFN-alpha/beta in patients in whom tumors may regress and tumor cells may then remain latent for extended periods of time.

Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex.

The complement system augments the humoral immune response to low concentrations of antigen. This effect may be partly mediated by complement receptors on the surface of B lymphocytes that bind immunogenic complexes bearing fragments of C3 and C4. We have shown by immunoprecipitation analysis that the two complement receptors expressed by B lymphocytes, complement receptor 1 (CR1) and CR2, form a detergent-sensitive complex on the surface of tonsillar B lymphocytes and on K562 erythroleukemia cells that were co-transfected with cDNAs encoding CR1 and CR2. The CR1/CR2 complex is distinct from the CR2/CD19 complex and may assist B cell activation by efficiently capturing C3b-containing immunogens and maintaining such immunogens on the B cell after CR1 and factor I-mediated cleavage to iC3b and C3dg. The complement activating immunogen may then trigger signal transduction by the CR1/CR2 complex, the CR2/CD19 complex, or membrane immunoglobulin.

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells.

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.

Loss of adhesion of murine erythroleukemia cells to fibronectin during erythroid differentiation.

Uninduced murine erythroleukemia cells specifically attached to fibronectin-coated dishes but not to dishes coated with laminin or type I or IV collagen. Dimethyl sulfoxide-induced differentiation of these cells caused a dramatic decrease in adhesion to fibronectin that was correlated with synthesis of the erythrocyte glycoprotein "band III," a membrane marker of the differentiated erythrocyte. Loss or modification of fibronectin binding sites on the cell surface during erythroid differentiation may cause the release of reticulocytes from the interstitial matrix of bone marrow into the blood.

Phosphatidylinositol 4-phosphatase type II is an erythropoietin-responsive gene.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistep process. At disease onset, preleukemic cells are arrested in differentiation at the proerythroblast stage (HS1 stage) and their survival and growth are under the tight control of erythropoietin (Epo). During disease progression, malignant proerythroblasts characterized by Epo autonomous growth and in vivo tumorigenicity can be isolated (HS2 stage). During analysis of transcriptional profiling representive of discrete stages of leukemic progression, we found that the phosphatidylinositol 4-phosphatase type II gene was turned off in malignant cells. PI-4-phosphatase II is an enzyme that hydrolyses the 4-phosphate position of phosphatidylinositol-3-4-bisphosphate (PtdIns(3,4)P(2)) to form PtdIns(3)P. Using malignant cells engineered to stably express PI-4-phosphatase II, we showed that PI-4-phosphatase II reduced Akt activation level. Moreover, stimulation of malignant cells with Epo-induced PI-4-phosphatase II transcription pointing this gene as an Epo-responsive gene. This study provides first insight for a physiological role of PI-4-phosphatase II in the proerythroblast by controlling Epo responsiveness through a negative regulation of the PI3K/Akt pathway.

Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells.

Matrine is a major component of Sophora Flavescens and has been reported to stimulate differentiation of erythroleukemia cells. Here we show that matrine inhibits cell proliferation or induces apoptosis in a cell type-specific manner. The latter effect was investigated in more detail in the p53 deficient erythroleukemia cell line, K562. Matrine exposure induced apoptosis in a time- and dose-dependent manner in these cells. Interestingly, co-treatment with etoposide potentiated apoptosis. Further analysis of matrine-induced apoptotic changes revealed that E2F-1 and Apaf-1 were upregulated, whereas Rb was downregulated after 24 h of exposure. This was followed by Bax translocation, cytochrome c release, and caspase-9 and -3 activation. These results demonstrate that matrine triggers apoptosis of K562 cells primarily through the mitochondrial pathway and that matrine is a potential anti-tumor drug.

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation.

Chromosome condensation is essential for proper segregation of duplicated sister chromatids in mitosis. Mammalian erythroid maturation is also associated with gradual nuclear condensation. However, few proteins that are directly involved in chromosome condensation during erythropoiesis have been identified. In this report, we show that MTB (more than blood), which was initially isolated in a yeast two-hybrid screen for proteins that interact with the basic helix-loop-helix (bHLH) protein stem cell leukemia (SCL), and later identified as the murine homolog of the condensin II subunit CAP-G2, participates in erythroid cell development. MTB interacts with SCL and another hematopoietic bHLH protein, E12, and is recruited to the nucleus by SCL and E12. In addition, MTB can repress SCL/E12-mediated transcriptional activation. Consistent with the model that MTB may function together with SCL/E12 heterodimer during erythroid cell development, MTB is highly expressed in the erythroid lineage and is upregulated upon erythroid differentiation. Moreover, overexpression of MTB promotes the terminal differentiation of the murine erythroleukemia erythroid cell line. Together, these findings demonstrate that the condensin II subunit MTB/mCAP-G2 plays a novel function during erythropoiesis and suggest that key hematopoietic transcription factors such as SCL and E12 may regulate the terminal differentiation of hematopoietic cells through the interaction with condensin complexes.

Friend leukemia: rapid development of erythropoietin-independent hematopoietic precursors.

Infection of mice with the polycythemia-inducing strain of Friend leukemia virus caused a rapid emergence of new erythroid precursor cells. These cells which, in the absence of erythropoietin, proliferated in vitro to form colonies and even differnetiated, quickly out-numbered the usual erythropoietin-dependent hematopoietic elements in bone marrow and spleen. Ultimately, the marrow and spleen were probably totally repopulated with this erythropoietin-independent cell.

Stimulation of normal erythropoiesis in vitro by macrophages from erythroleukemic mice.

The erythroleukemia induced in mice by the Friend virus complex is caused to regress by macrophages. To determine whether the effect of macrophages on leukemia is due to their role in the regulation of erythropoiesis, investigators examined the influence of macrophages from normal and leukemic mice on in vitro colony formation by erythroid progenitor cells [colony-forming units of erythroid progenitor cells (CFU-E)]. Plasma clot cultures of CFU-E were grown over monolayers of macrophages separated from the CFU-E cultures by a layer of agar. Macrophages from progressors (those leukemic mice that will not undergo leukemia regression) significantly stimulated CFU-E, whereas macrophages from regressors (those that will undergo leukemia regression) had no effect on colony formation. Monolayers of normal resident macrophages did not affect in vitro erythropoiesis, while less mature macrophages (peripheral blood mononuclear cells) or nonspecifically activated macrophages (exudate induced) in monolayer stimulated CFU-E. Stimulation by macrophages from leukemic mice was dependent on the presence of virus-producing cells. CFU-E from erythroleukemic mice were resistant to stimulation by leukemic macrophages. These results demonstrated that macrophages from progressively leukemic mice influenced normal colony formation of CFU-E in vitro and that these effects could be related to the ability of macrophages to cause leukemia regression.

Activation of ion channels in tumor cells by leukoregulin, a cytostatic lymphokine.

We used the whole-cell patch-clamp recording technique to study the effects of leukoregulin (LR), a cytostatic lymphokine produced by activated human peripheral blood lymphocytes, on the electrical properties of K562 tumor cells. LR induced changes in the membrane excitability in 33 of 55 cells studied. A minute or more after application, LR elicited a complex and reversible electrical response. The response lasted for several more minutes in the continued presence of LR. It consisted of (a) moderate-conductance (50 pS), cation-selective, ion-channel activity, (b) a shift of the zero-current membrane potential close to 0 mV, and (c) a suppression of a depolarization-activated K+ conductance. All of these changes in tumor cell excitability act coordinately to depolarize the tumor cells and may be important in the cytostatic actions of LR.

Growth factor(s) produced by a human leukemic cell line growing in a protein-free chemically defined medium.

To examine whether human leukemic cells produce growth factor(s), a protein-free culture line of human erythroleukemic cells (K-562T1) has been established. This unique cell line has been continuously propagated in protein-free Ham's F-10 medium without any supplement for 5 yr. Growth-promoting activity was determined by measuring [3H]thymidine incorporation into DNA in serum-deprived chick embryo fibroblasts. The conditioned medium of K-562T1 contained the growth-promoting activity against chick embryo fibroblasts, mouse 3T3-L1 cells, and K-562 human leukemic cells. This leukemia-derived growth-promoting activity was heat and acid stable and trypsin sensitive. The activity was destroyed by dithiothreitol. Size exclusion chromatography revealed three peaks of activity, with apparent molecular weights of 13,500, 6,300, and 2,400, respectively.

Resistance to erythroleukemia in immunodeficient xid- CBA/N mice with susceptibility after immune stimulation.

CBA/N (xid-) mice failed to develop erythroleukemia when inoculated with an NB-tropic, anemia-causing Friend virus stock (FVA), while Fv-2ss mouse strains succumbed rapidly to the characteristic Friend disease, even after a virus dose 30-fold lower than that given to CBA/N mice. Immunization with bacterial antigens or with spleen cell allografts prior to FVA inoculation rendered CBA/N mice highly susceptible to FVA. Transplantation studies confirmed that non-immunized CBA/N mice were able to both support erythroleukemic cells and permit erythroleukemic transformation, thus arguing against host defense mechanisms as a cause of resistance. On the basis of early erythroid progenitor cell sensitivity to hydroxyurea in vivo, the CBA/N strain appeared to have the FVA sensitive genotype (Fv-2ss). These results imply that CBA/N mice are not intrinsically resistant to FVA and that an as yet unknown type of immunological activity, evoked both by various immunizations and allogeneic transplantation, is required for Friend leukemogenesis in this immunodeficient inbred strain. These findings further suggest that the erythroid target cells transformed by Friend viruses are influenced by immunological activity.

Mechanism of macrophage reversal of Friend erythroleukemia: macrophage regulation of normal and leukemic erythropoiesis.

The acute erythroleukemia induced in mice by the anemia-inducing strain of the Friend virus complex is caused to regress by normal macrophages. We examined the possibility that reversal of leukemia is related to a macrophage regulatory function in erythropoiesis. We found that the ability of macrophages to induce leukemia regression correlates with nonimmunological, in vivo suppression of normal and susceptible leukemic erythroid progenitors. The macrophage effect on erythropoiesis appears to be due to changes in a humoral regulator, related to but independent of erythropoietin. The results suggest a novel regulatory system for erythropoiesis, operative in vivo, and involving macrophages as accessory or suppressor cells. This regulation appears to be disrupted in erythroleukemic mice, but can be restored, and the disease can be made to regress by treatment with normal macrophages.

Changes in DNA associated with induction of erythroid differentiation by dimethyl sulfoxide in murine erythroleukemia cells.

The Friend virus-infected murine erythroleukemia cell can be induced to differentiate along erythroid cells in culture with various compounds, including dimethyl sulfoxide. DNA from murine erythroleukemia cells cultured with dimethyl sulfoxide shows a decrease in sedimentation rate in alkaline sucrose gradients after alkali lysis of the cells. These changes can be detected as early as 27 hr after the beginning of culture. Similar results are observed with DNA of the cells cultured with other inducers, butyric acid and dimethylacetamide, but not with DNA from a variant cell line resistant to induction with dimethyl sulfoxide. Ultraviolet irradiation, which is known to cause similar changes in the sedimentation rate of DNA in alkaline sucrose gradients, induces differentiation of the murine erythroleukemia cells. These studies suggest that alterations in DNA may be related to events involved in the induction of differentiation of murine erythroleukemia cells by dimethyl sulfoxide.

The human homologue of the putative proto-oncogene Spi-1: characterization and expression in tumors.

Spi-1 is a putative proto-oncogene involved in murine virus-induced acute erythroleukemias. We report here the identification of the human homologue of Spi-1 and its expression in normal and tumorigenic human tissues. Characterization of cDNA clones revealed that the human Spi-1 gene encodes a 216 amino acids protein showing 85% identity with the murine counterpart. By sequencing genomic clones, five exons were identified. To investigate the possible role of Spi-1 gene in human cancers, we studied its expression in a panel of human tumors by Northern blot analysis. Spi-1 expression was detected in all the tumors examined. There was no noticeable evidence of messenger RNA alteration as compared to normal tissues.

Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage.

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.