Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,ß1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Congenital immature pure erythroid leukemia with E-cadherin expression.

Congenital pure erythroid leukemia is exceedingly rare and poses a diagnostic challenge. We report an atypical case of congenital pure erythroid leukemia that did not express typical erythroid markers. The patient presented with a high white blood cell count with blastic cells at birth. Although flow cytometric analyses of peripheral blood and bone marrow showed a large CD45-negative cell population, we did not identify any evidence of monoclonality. While the circulating blasts decreased with only supportive care, hepatomegaly with multiple nodules was accompanied by liver failure, disseminated intravascular coagulation, and development of hemophagocytic lymphohistiocytosis. Pathological examination of the liver biopsy specimen revealed a small round cell tumor that was negative for nearly all hematopoietic cell markers, including classical erythroid cell markers, and positive for CD43, CD71, and E-cadherin, an early erythroid marker epithelial calcium-dependent adhesion protein, suggesting that these tumor cells originated from an immature erythroblast. We found high ß-catenin and c-Myc protein expression, which were not previously described in pure erythroid leukemia. Cytosine arabinoside temporarily alleviated clinical symptoms; however, the patient died of progressive disease at 8 months of age. This case indicates that E-cadherin is useful for diagnosing pure erythroid leukemia, even in immature cases.

Two cases of acute erythroid leukemia presenting with marked macrocytic anemia, reticulocytosis and hemolysis.

Case 1. The laboratory findings of a hematological analysis of a 53-year-old woman with palpitations and dyspnea revealed the following: red blood cell (RBC) count: 9.4×10(5)/µL with 60.0‰ reticulocytes; Hb: 3.7 g/dL; mean corpuscular volume (MCV): 124.5 fL; white blood cell (WBC) count: 2,800/µL with 10.0% myeloblasts. Case 2. Similarly, a 42-year-old man with dizziness had a RBC count of 1.63×10(6)/µL with 24.0% reticulocytes, an Hb level of 6.0 g/dL, an MCV of 120.2 fL and a WBC count of 3,100/µL with 4.0% myeloblasts. Bone marrow aspirates in both patients confirmed a diagnosis of acute erythroid leukemia (AEL), which can present as marked macrocytic anemia with an MCV in excess of 120 fL and hemolysis.

Acute myeloid leukaemia (M6B: pure acute erythroid leukaemia) in a Thoroughbred foal.

A 10-week-old Thoroughbred filly was referred for anaemia of 4 weeks' duration. Haematology revealed severe anaemia and panleucopenia. Cytological examination of bone marrow smears revealed a myeloid to erythroid ratio <0.02:1 (reference range 0.5-2.4:1.0) and an abundance of erythroid precursor cells. The erythroid cell population included rubriblasts, prorubricytes and rubricytes, with only scant numbers of metarubricytes present. There were numerous mitotic erythroid cells, some of which were atypical and megaloblastic. These cytomorphological changes are consistent with pure acute erythroid leukaemia. No treatment was instituted and the filly died three days after presentation. This case illustrates the need to consider both haematology and bone marrow findings to establish a diagnosis of pure erythroid leukaemia. To our knowledge, there is no documented case of acute myeloproliferative disease in horses involving cells of erythroid lineage, but this condition should be considered a differential diagnosis for horses presenting with anaemia.

Precise quantification of haemoglobin in erythroid precursors and plasma.

INTRODUCTION: Haemoglobin (Hb) quantification in whole blood is possible by various spectrophotometric methods. However, determination of low-level Hb in erythroid precursors or haemolytic plasma is inaccurate because of contribution from light scatter and/or nonhaemoglobin components with overlapping absorbance. Therefore, this study developed a sole method allowing accurate spectrophotometric quantification of Hb at a low concentration range. METHODS: Advantage was taken of the unique absorption spectra of carbon monoxide-Hb complex (COHb) as compared to oxyHb. The visible absorption spectra of samples were recorded prior and following carbon monoxide exposure. A difference extinction coefficient at the maximal difference absorption was used to calculate Hb concentrations. RESULTS: Known amounts of Hb were added to mouse erythroleukaemia (MEL) cells lysate or plasma to yield known 'theoretical' concentrations. The concentrations were measured by the current and known methods. The current method was found much more accurate compared with previous methods specifically at low concentrations. CONCLUSION: The method is valid for accurate quantification of Hb at a wide concentration range (>0.1 µm/L) in erythroid precursors or plasma and is optional for other biological fluids.

Inhibition of viral carcinogenesis by Phyllanthus amarus.

Friend murine leukemia virus (FMuLv) is an acutely oncogenic retrovirus, and its infection leads to erythroblastosis and leukemia in mice. This infection model is used in the search for new antiviral agents. In the present study, the authors have evaluated the potential of an extract of Phyllanthus amarus against FMuLv-induced erythroleukemia in BALB/c mice. Injection of newborn mice with FMuLv resulted in leukemia and animals died due to splenomegaly. Oral administration of P.amarus was found to enhance the life span of leukemia-harboring animals and decrease the incidence of anemia. The authors also performed a series of hematological, biochemical, histopathological, and gene expression analyses to evaluate the effect of P.amarus administration on erythroleukemia initiation and progression. The data obtained indicate that P.amarus administration could significantly decrease the progression of erythroleukemia. Treatment with P.amarus induced the expression of p53 and p45NFE2 and decreased the expression of Bcl-2 in the spleen of infected mice. Histopathological evaluations of the spleen demonstrated that administration of P.amarus decreased the infiltration of leukemic cells into the sinusoidal space when compared with the vehicle treated group. P.amarus is known to inhibit chemically induced neoplasm in different rodent models.The current results indicate that P.amarus has the ability to suppress virally induced cancers as well.

siRNA-mediated reduction of alpha-globin results in phenotypic improvements in beta-thalassemic cells.

beta-thalassemia is an inherited hemoglobinopathy caused by defective synthesis of the beta-globin chain of hemoglobin, leading to imbalanced globin chain synthesis. Excess alpha-globin precipitates in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anemia. Decreased alpha-globin synthesis leads to milder symptoms, exemplified in individuals who co-inherit alpha- and beta-thalassemia. In this study, we investigated the feasibility of utilizing short-interfering RNA (siRNA) to mediate reductions in alpha-globin expression. A number of siRNA sequences targeting murine alpha-globin were tested in hemoglobinized murine erythroleukemic cells. One highly effective siRNA sequence (si-alpha 4) was identified and reduced alpha-globin by approximately 65% at both the RNA and the protein level. Electroporation of si-alpha 4 into murine thalassemic primary erythroid cultures restored alpha :beta-globin ratios to balanced wild-type levels and resulted in detectable phenotypic correction. These results indicate that siRNA-mediated reduction of alpha-globin has potential therapeutic applications in the treatment of beta-thalassemia.

[Erythroleukemia - a subtype of myelodysplastic syndrome?].

In order to study whether erythroleukemia was really a subtype of acute leukemia, the clinical laboratory characteristics and development of disease in 21 cases of erythroleukemia were analyzed. The results indicated that the percentage of patients with leucocytopenia, anemia and thrombocytopenia were 42.9%, 81% and 81% respectively at the time of diagnosis. These were 85.7% of patients with myelocytes and premonocyte, 52.4% of patients with erythroblast in their blood smear respectively. All of the bone marrow showed active or significantly active proliferation. The median percentage of erythro-lineage, myeloblast of NEC and displasia were (58.3 +/- 8.0)%, (58.0 +/- 18.4)% and 66.7% respectively, that is different from typical AML. 52.4% of M(6) patients transferred to RAEB/RAEB-T and AML-M(2) subtype in the disease progression. 11/19 cases (57.4%) achieved remission (CR 10; PR 1) after chemotherapy. The median remission length were 6 months for CR patients and 2 months for PR patients, but most of CR patients displayed obvious displasia of bone marrow and cytopenia of blood in the period of CR. The median survival length of M(6) and MDS-->M(6) from time of diagnosis were 13.0 +/- 13.2 and 2.3 +/- 1.3 months respectively. It is concluded that there are differences between M(6) and typical AML. Most of M(6) patients would rather be classified MDS RAEB and RAEB-t with over-hyperplasia of erythron lineage than a subtype of AML.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL.

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorheological indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

[Haematological characteristics, FAB and WHO classification of 153 cases of myeloid acute leukaemia in Tunisia]. / Caractéristiques hématologiques et classification FAB et OMS de 153 cas de leucémies aiguës myéloïdes en Tunisie.

A complete blood analysis with a careful morphologic examination of peripheral blood and bone morrow smears completed by cytochemical reaction will help to classify the most acute myeloid leukaemia (AML). Actually, the study of other cytogenetis and immunophenotypic markers are now necessary to confirm diagnosis. The World Health Organisation WHO classification (2001) incorporates theses approaches. The purpose of this study is a bio-clinical review according to the WHO recommendations in 153 cases of LAM diagnosed between January 1998 and December 2003. The patients were aged 2 months to 90 years with sex ratio (M/F) of 1,22. The morphologic conclusion was difficult in 12% cases. Presence of dysplasia is noted in 50% of cases with multilineage dysplasia in 42% of cases. Our results showed cloned chromosomal abnormalities in 57% of cases (t(8;21): 12%, t(15;17) : 10%, Inv16: 1,3%, 11q23: 2,6% et complex karyotype: 14,3%). In 69% of cases with multilineage dysplasia, the karyotype was normal. 3 cases of LAM were noted at patients treated for breast cancer with chirurgic chemotherapy and radiotherapy 3, 4 et 5 years after treatment (LAM3 with t(15;17), LAM4 with genetic abnormalities of chromosomes 3, 5, 7, 8, 9, 14 et 16 et LAM 6 with genetic abnormalities of chromosomes 4, 7, 12, 14, 19 et 21). In WHO classification, cytology is essential in diagnosis of LAM even if the karytype have an important prognostic value. Research of signs of dysplasia lineage after lineage constitutes an important microscopic work and it is difficult to quantify dysplasia when the lineage is poor.

Alterations in hemorheological properties of erythrocytes in nude mice with erythroleukemia and the treatment effects of etoposide.

The purpose of this study was to examine the changes of hemorheological properties of erythrocytes in the nude mice with erythroleukemia and the treatment effects of etoposide (VP16). Thirty mice were randomly divided into three groups: the control group (C group), injected with 1 ml saline solution, the MEL group (M group) injected with 1 ml MEL (murine erythroleukemia cell line) and the MEL + VP16 group (V group) injected with 1 ml MEL and from the 8th day after injection, 20 microl VP16 (1 microg/microl) was injected intraperitoneally every five days. One week after MEL injection, erythroblastic cells increased in the bone marrow and proerythroblasts were found in the peripheral blood, suggesting that erythroleukemia was induced. Abnormalities were also found in spleens and livers later. At around twenty days after injection, the mice in M group died and about four weeks after injection, the mice in V group also died. Compared with C group, the hemorheological indexes [the deformation index DI, orientation index (DI(or)), and the small deformation index (DI(d))], electrophoretic mobility, membrane fluidity as well as osmotic fragility of red blood cells (RBC) in M and V groups changed significantly. But after VP16 administration, the changes of above parameters in V group were less significant than those of M group. The results above suggested that intraperitoneal injection of MEL cells could cause erythroleukemia in nude mice, VP16 could alleviate the erythroleukemia symptom and improve the hemorheological properties, and could prolong V group nude mice survival.

Tumorigenesis of murine erythroleukemia cell line transfected with exogenous p53 gene.

Mutant p53 gene and wild-type p53 gene were introduced into murine erythroleukemia cell line (MEL). The MEL cells transfected with mutant p53 gene (MEL-M) and with wild-type p53 gene (MEL-W) were obtained by G418 selection. MEL, MEL-W and MEL-M were injected intraperitoneally into BALB/C mice. In the first week after injection, the signs of erythroleukemia were induced in all three groups. Abnormalities were mainly found in the spleen, bone marrow, liver and peripheral blood. There was an increase of proerythroblasts in the bone marrow. A large amount of normoblasts (early and intermediate erythroblasts) appeared in the spleen. In the peripheral blood, the white blood cells, reticulocyte and platelet counts increased and RBC count and hematocrit decreased. The degree of abnormalities in the MEL-W group was significantly lower than that in other two groups. Hemorheological measurements indicated that the deformability and orientation of RBCs in MEL and MEL-M groups were impaired, whereas those in MEL-W group did not change significantly. Micropipette aspiration measurement revealed that MEL-W had higher elastic modulus than MEL and MEL-M, indicating that it was more difficult for MEL-W to deform and migrate in vivo. The results of animal test and micropipette suggest that exogenous wild-type p53 gene could reduce the tumorigenesis of murine erythroleukemia cells.

Acute erythroid neoplastic proliferations. A biological study based on 62 patients.

BACKGROUND AND OBJECTIVES: The terms acute erythroleukemia and AML-M6 are defined in the FAB classification as proliferations of dysplastic erythroid elements mixed with blasts of myeloid origin, but pure erythroid leukemias are not included. The recent WHO classification has a category of acute myeloid leukemia not otherwise categorized, which includes acute erythroid leukemia (M6) of two subtypes: M6a-erythroleukemia (erythroid/myeloid) and M6b-pure erythroid leukemia. The aims of this co-operative study were to discover the incidences of these different subtypes, and pay special attention to the morphology of these entities. DESIGN AND METHODS: We reviewed a series of 62 patients with erythroid neoplastic proliferations. Previous medical history, age, sex, peripheral blood and bone marrow cell counts, cytochemical stains, immunophenotype, and cytogenetics were evaluated at presentation. We analyzed the incidence of erythrocyte, leukocyte and platelet abnormalities in the peripheral blood. In bone marrow we analyzed dysplastic features of erythroblasts, granulocytic elements and the megakaryocytic lineage. RESULTS: Fifty-three patients met the criteria of M6a subtype of the WHO classification, and 2 were classified as having pure erythremia (M6b); 7 cases could not be classified according to the WHO criteria. Fifty-five patients presented with de novo acute leukemia, and seven patients had secondary acute leukemia. The most frequent dysplastic features in blood smears were: schistocytes, tear-drop and pincered cells in erythrocytes; hypogranulation and hyposegmentation in leukocytes; gigantism and hypogranulation in platelets. In bone marrow, megaloblastic changes, multinuclearity, karyorrhexis and basophilic stippling in erythroblasts; hypogranulation and gigantism in granulocytic series, and micromegakaryocytes and unconnected nuclei in megakarocytes were the most dysplastic features. A positive PAS reaction and increase of bone marrow iron with ring sideroblasts were common features. Trilineage dysplasia was present in 54% of cases. Dysplastic features in granulocytic elements were absent in 26% of patients and minimal erythroblastic dysplasia was observed in seven patients. A complex karyotype was seen in 27% of patients; chromosomes 5 and 7 were the most frequently involved. INTERPRETATION AND CONCLUSIONS: De novo acute erythroid leukemia was more frequent than secondary cases in our series. The most frequent type of acute erythroid proliferation was the WHO M6a subtype and the least the pure erythroid leukemia. We found a group of seven patients (11%) who could not be classified according to the WHO criteria. Morphologic findings of erythrocytes in peripheral blood, such as schistocytes, tear-drop and pincered cells, were outstanding features. Morphologic aspects remain one of the most important tools for diagnosing these entities.

[Presentation of a case of hematological malignancy in reversed C.P.C].

We report a 68-year old male who was admitted to our hospital with the chief complaint of fatigue. Laboratory examinations revealed: 1) macrocytic anemia; 2) leukopenia; 3) reticulocytosis; 4) reduction in serum folate level; and 5) erythroid hyperplasia in bone marrow. One year later, the patient was admitted again to our hospital. At that time, laboratory data showed: 1) leukocytosis; 2) appearance of blast cells in peripheral blood; 3) normocytic anemia; 4) thrombocytopenia; and 5) predominant proliferation of blast cell(91%) in bone marrow. Blasts showed prominent nucleoli, markedly basophilic cytoplasma with vacuolation and some blebs on cell surface. The cells were negative for peroxidase stain but positive for PAS stain. We describe the evaluation of hematological laboratory data and the diagnosis in this patient.