Erythropoietin-induced acute erythroid leukemia-like picture: a potential pitfall.

A 31-year-old male patient presented with fever and pancytopenia. He was diagnosed as a case of chronic anemia since early childhood. The etiology of the anemia was not known. The patient was transfusion dependent, and he had been maintained on erythropoietin for three years prior to admission. A bone marrow examination revealed prominent proliferation of immature and dysplastic erythroid precursors. Acute erythroid leukemia of the pure erythroid subtype was suspected. However, because of the history of erythropoietin therapy a definite diagnosis was not made. On follow-up one month later, the marrow changes had reversed to normal.

Combined inhibition of PI3K and activation of MAPK p38 signaling pathways trigger erythroid alternative splicing switch of 4.1R pre-mRNA in DMSO-induced erythroleukemia cells.

There is increasing evidence showing that many extracellular cues modulate pre-mRNA alternative splicing, through different signaling pathways. We here show that 4.1R exon 16 splicing is altered in response to specific signals. The switch from erythroblastic isoform lacking exon 16 to mature erythrocytic isoform containing this exon is tightly regulated during late erythroid differentiation, and blocage of this splicing switch in erythroleukemia cells is seen as a consequence of the deregulation of important regulatory pathways. We support that combined inhibition of PI3K and activation of p38 signaling pathways impinge on erythroid 4.1R pre-mRNA alternative splicing switch, and on cell differentiation as witnessed by hemoglobin production. By contrast, MEK/ERK signaling appeared not to affect neither cell hemoglobin production nor erythroid 4.1R pre-mRNA splicing. We also found that the signal-induced alternative splicing is not typically distinctive of EPO-non-responsive cells, but operates in EPO-responsive cells as well. Pre-mRNA splicing is a major regulatory mechanism at the crossroad between transcription and translation. We here provide evidence that inhibition of PI3K activates the splicing switch in a promoter-dependent manner, whereas p38 activation induces this event in a promoter-independent fashion. Our data further support that constitutive activation of EPO-R by the viral protein gp55 and the short form of the tyrosine kinase receptor Stk, transduces PI3K proliferation signal, but not MAPK p38 differentiation signal. Concurrently, this work lend credence to the concept that DMSO triggers transient activation of p38 signaling and irreversible inhibition of PI3K/AKT signaling pathway, hence uncovering an old conundrum regarding the mechanism by which DMSO induces erythroleukemia cell differentiation.

Epistaxis and severe weakness in a patient with multiple myeloma. Therapy-related acute myeloid leukemia, pure erythroid leukemia.

Therapy-related acute myeloid leukemias arise as a result of cytotoxic chemotherapy and/or radiation therapy. The most common types of acute myeloid leukemia arising in this setting are acute myeloid leukemia with maturation, and lesser numbers of acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, or acute megakaryocytic leukemia. We present a patient with multiple myeloma who was treated with melphalan and 4 years later developed acute erythroid leukemia. The morphologic diagnosis of pure erythroid leukemia developing in the setting of multiple myeloma may be challenging.

Secondary leukemia after treatment with paclitaxel and carboplatin in a patient with recurrent ovarian cancer.

The occurrence of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) has been reported after treatment with cytotoxic alkylating agent-based chemotherapy for solid tumors. We report a patient with metastatic ovarian carcinoma treated with carboplatin and paclitaxel, who developed secondary acute erythroid leukemia. The overall survival of patients with stage III and IV ovarian cancer has increased in the past decade. Monitoring of the long-term outcome of paclitaxel- and platinum-based regimens is warranted, particularly with regard to monitoring the development of secondary MDS and/or AML. The incidence and outcome of secondary leukemia in the setting of active ovarian carcinoma is reviewed.

[The induction of high-level expression of a novel VL-30 like gene during the chemical-inducing erythroid differentiation of murine erythroleukemia cells (MEL)].

OBJECTIVE: To study the gene expression during the chemical inducing erythroid differentiation of murine erythroleukemia cells (MEL). METHODS: Differential display assay was used to analyze the gene expression before and after the induction differentiation of MEL cells by DMSO and hemin; and then, the flanking sequences of one of the cDNA fragment was amplified by modified rapid amplification of cDNA end (RACE) method, Northern blot analysis was adopted to characterize the expression of this gene. RESULT: The expression of a new transcript similar to VL-30 retrotransposon family increased significantly during the chemical inducing erythroid differentiation in MEL cells. The cloned cDNA is 1883bp, and it is highly homologous to the internal region of murine BVL-1 (1,955-4,000 nt). At least three transcripts in MEL cells were detected by Northern hybridization and all of them increased after the induction. CONCLUSION: A new VL-30-like gene is identified for the first time in MEL cells during chemical inducing erythroid differentiation.

ras and p53 genes are infrequently involved in N-nitroso-N-butylurea (NBU)-induced rat leukemia.

Chemically-induced rodent tumor models help us to understand a series of genetic changes during carcinogenesis. In this study, we present N-nitroso-N-butylurea (NBU)-induced rat leukemia and compare it with the genetic alterations found in 7,12-dimethylbenz[a]anthracene (DMBA)-induced erythroblastic leukemias which consistently have an A to T transversion at the second base of codon 61 in N-ras. By continuous NBU treatment for 120-150 days, 14 primary leukemias were induced in Long-Evans rats. Myeloblastic leukemia cells predominantly increased in all rats except in one case which predominantly had erythroblastic leukemia cells. Point mutations of Ha-, Ki-, N-ras and p53 were determined after RNA was transcribed into cDNA and this cDNA was used as a substrate for polymerase chain reaction (PCR) which was eventually sequenced. No abnormalities in exons 1 and 2 of Ha-, Ki- and N-ras were detected in all leukemias. In the p53 gene, an A to C transition was found at the second base of codon 198 (Asn-Thr) in one leukemia, but others had no mutation. These results suggest that ras and p53 genes are infrequently involved in NBU-induced leukemias. The genetic target of NBU during leukemogenesis seemed to be different from that of DMBA.

[Establishment and biological characterization of a transplantable BALB/c mouse erythroblastic leukemia model].

OBJECTIVE: Biological characterization of a BALB/c mouse transplantable erythroblastic leukemia model EL9611. METHODS AND RESULTS: The original erythroblastic leukemia (EL) mouse was obtained by 7,12-DMBA induction. The spleen cell suspension from the original EL mouse was injected into the same inbred BALB/c strain mice. After successive transplantation for 20 generations, the incidence of EL was 100% without spontaneous remission. There was a linear relationship between the survival time of the EL mice and the number of leukemic cells inoculated. When 10(6) leukemic cells were inoculated intravenously, the EL mice survived 10.2 +/- 1.3 days. Invasion of leukemic cells involved mainly in the bone marrow, the spleen and the liver. There was no obvious infiltration of leukemic cells in lymph-nodes and thymus. In peripheral blood, there were a large number of leukemic cells and most of them were basophilic or polychromatophilic erythroblasts. At the advanced stage, the mice developed anemia and thrombocytopenia. The reaction of the leukemic cells for hemoglobin staining was positive or weakly positive. While the peroxydase reaction was negative, Thy-1 antigen and Fc-recepter were not presented. No virus-like particles was found in the cells under electron microscope. EL9611 leukemia model was sensitive to some antitumor drugs used in clinical therapy. CONCLUSION: The establishment of EL9611 leukemia model offered a useful tool for studying proliferation and differentiation of erythroid cells, as well as pathogenesis and experimental treatment of non-virus erythroid malignancy.

WT1 contributes to leukemogenesis: expression patterns in 7,12-dimethylbenz[a]anthracene (DMBA)-induced leukemia.

The Wilms'-tumor gene WT1 may have a different function from a tumor-suppressor gene in some leukemias. Using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia system, we examined whether WT1 expression was involved during leukemogenesis, since this model enabled us to analyze cells altered by DMBA at various stages of leukemogenesis. By the semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) method, WT1 expression was detected in 15 (71%) of 21 DMBA-induced erythroblastic leukemias. Among 15 WT1-expressing leukemias, GATA-1, which is an erythroid-specific transcription factor and might regulate WT1 expression, was also expressed in 13 cases (p < 0.05). On the other hand, WT1 expression was not detected in any normal or early pre-leukemic rats and was detected in 1 of 8 rats in late pre-leukemic stages. These results showed that cells with a high expression level of WT1 tended to develop into leukemia and that WT1 contributed to leukemogenesis in the late stage, suggesting that the expression of WT1 plays an important role in cell proliferation and in maintaining the viability of some leukemia cells.

The specific N-ras mutation in rat 7,12-dimethylbenz[a]anthracene (DMBA)-induced leukemia.

Intravenous injections of 7,12-dimethylbenz[a]anthracene (DMBA) induce erythroblastic leukemia (erythroleukemia) with #2 trisomy and Long #2 in Long-Evans rats. Recently, a consistent type of mutation, A to T transversion in codon 61 of N-ras gene, was found in all of 6 cultured leukemia cell lines and 13 primary leukemias induced by DMBA using polymerase chain reaction (PCR) and direct sequencing. On the contrary, no mutation was observed in Ha- and Ki-ras genes in these leukemias. The consistent occurrence of the above N-ras mutation in DMBA-induced leukemias indicates that N-ras gene plays an important role in DMBA-leukemogenesis. Mutations in ras genes generally takes place during the initiation stage of carcinogenesis because they often appear in the premalignant stage of tumors. In order to detect the N-ras mutation in an early stage of leukemogenesis, we designed the mutant-allele-specific amplification (MASA) method to detect the mutation in bone marrow (BM) cells of DMBA-treated rats. The MASA method was sensitive enough to detect one mutant cell mixed in 10(6) normal cells. Using this method, the N-ras mutation was found in BM cells 2 days after single DMBA injection and thereafter throughout the preleukemic stage. These results suggest that the N-ras mutation is an earliest event in DMBA-induced leukemogenesis.

Establishment and characterization of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat erythroleukemia cell lines.

To investigate molecular mechanisms and biological behaviors of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat erythroleukemia, we established 8 new culture cell lines from 7 primary erythroleukemias. We designated them KYD-10, 12, 17, 32, 38, 44A, 44B, and 49. Representative clones isolated from each cell line in early passage were analyzed cytogenetically and genetically. All cell lines except KYD-12 possessed the specific N-ras mutation at the 2nd base of codon 61. Four of them showed #2 trisomy (KYD-10, 32, 38, 44B), and the rest normal diploid karyotype (2n). KYD-32 cells showed Robertson type II #2 trisomy which had never been clonally isolated in vitro although it was reported in some DMBA leukemias in vivo. We further studied the genomic imbalance related to the N-ras allele using mutant-allele-specific amplification (MASA) method. Deletion of normal N-ras allele was found in 5 of 8 cell lines. KYD-32 and 38 retained the normal N-ras allele. The specific N-ras mutation and allelic loss of wild type N-ras were correlated with advanced cell proliferation in culture probably independent of #2 trisomy.

A third human tissue transglutaminase homologue as a result of alternative gene transcripts.

A 2.4 kilobase (kb) cDNA encoding a new form of human tissue transglutaminase homologue (TGH2) was isolated from retinoic acid-induced human erythroleukemia cell (HEL) library. Full-length cDNA analysis gives an open reading frame coding for a polypeptide of 349 amino acid residues with a molecular mass of 38,700 Da. This variant differs from the previously reported homologue TGH in that it is 199 amino acids shorter and has an alternative, 63 amino acid COOH-terminal peptide. The 3'-untranslated region of the cDNA also differs from the previously reported sequences for both TGH and human tissue transglutaminase. The region coding for the first 286 amino acids of TGH2, which contains the active site is identical to TGH. Immunoprecipitation of the in vitro translation product from a synthetic TGH2 mRNA and immunoprecipitation of total protein of human heart, liver, kidney and cultured erythroleukemia HEL cell, revealed a protein with a molecular mass of 37,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the cDNA sequence for the previously known tissue transglutaminases with genomic DNA and the TGH2 cDNA described here indicate that the sequence divergence points correlate with known intron-exon boundaries. The smaller RNA species encode for truncated proteins with novel carboxyl termini. The TGH cDNA and the TGH2 cDNA both produce transcripts which start with the regular coding sequence for TGase and then fail to splice at specific donor sites, resulting in the use of an alternative exon that contains a stop codon.

N-ras mutation in 7,12-dimethylbenz[a]anthracene (DMBA)-induced erythroleukemia in Long-Evans rats.

Intravenous injections of 7,12-dimethylbenz[a]anthracene (DMBA) induce erythroblastic leukemia (erythroleukemia) with No.2 trisomy in Long-Evans rats. Activation of some oncogenes such as abl and Ha-ras has been reported to occur in relation to the secondary chromosomal translocations. In the present studies, a consistent type of mutation, A to T transversion in codon 61 of N-ras gene, was found in all of 6 cultured leukemia cell lines and 5 primary leukemias induced by DMBA. The N-ras mutation was also found in bone marrow cells of 2 out of 8 preleukemias. On the contrary, no mutation was observed in Ha- and Ki-ras genes in all leukemias and preleukemias. The consistent occurrence of above N-ras mutation in leukemias indicates that it plays an important role in DMBA-leukemogenesis.

ras oncogene activation and occupational exposures in acute myeloid leukemia.

BACKGROUND: Epidemiologic studies of acute myeloid leukemias (AMLs) show small increases in risk of disease associated with certain occupations and chemical exposures. PURPOSE: This study was designed to determine whether the presence of mutationally activated ras oncogenes in AML are associated with occupational and chemical exposures. METHODS: We interviewed 62 patients with newly diagnosed AML (or their next-of-kin), all of whom were enrolled in a national multicenter clinical trial, and 630 healthy control subjects. DNA extracted from patients' pretreatment bone marrow samples was amplified by using the polymerase chain reaction and probed with allele-specific oligonucleotides for activating point mutations at the 12th, 13th, and 61st codons of three protooncogenes: H-ras (also known as HRAS), K-ras (also known as KRAS2), and N-ras (also known as NRAS). RESULTS: Patients with ras mutation-positive AML had a higher frequency (six of 10 patients) of working 5 or more years in an a priori high-risk occupation than did patients with ras mutation-negative AML (eight of 52; odds ratio [OR] = 6.8; 95% confidence interval [CI] = 1.3-36). Patients with ras mutation-positive AML were more likely than patients with ras mutation-negative AML to have breathed chemical vapor on the job (OR = 9.1; 95% CI = 1.3-64) or to have had skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37). When ras-positive patients were compared with healthy control subjects, the ORs for occupation and occupational exposures remained elevated, while patients with ras mutation-negative AML showed no increased risk when compared with control subjects. CONCLUSION: Activation of ras proto-oncogenes may identify an etiologic subgroup of AML caused by occupation and chemical exposure. IMPLICATION: Disease etiology may be better understood if epidemiologic measures of exposure are integrated with molecular assays of the genetic defects responsible for cancer initiation and promotion.

Distinct cytogenetic and clinicopathologic features in acute myeloid leukemia after occupational exposure to pesticides and organic solvents.

BACKGROUND: To study the correlation of environmental exposure to potentially mutagenic agents and the clinicopathologic picture in acute myeloid leukemia (AML), clinical features, morphologic characteristics, immunophenotype, and cytogenetics were studied in 59 patients with newly diagnosed AML. METHODS: Based on interviews on occupational hazards and hobbies showing prolonged contact with pesticides (18 patients) and organic solvents (7 patients), 25 patients were categorized as "exposed". Thirty-four patients were categorized as "unexposed,", based on anamnestic findings. RESULTS: Light microscopic studies showed myelodysplasia involving multiple cell lineages in all assessable patients with professional exposure to pesticides and organic solvents, whereas morphologic aberrations of the non-blast cell population were confined to a minority of cells in unexposed patients. These findings were confirmed by electron microscopic studies in 31 patients. Immunologic analysis showed the presence of a minor megakaryoblastic component in six exposed patients and showed positive findings for the CD34 stem cell marker in 85% of exposed patients, a figure significantly higher as compared with that for unexposed subjects. Cytogenetic studies confirmed the frequent occurrence of 5q and/or 7q aberrations in patients occupationally exposed (10 of 25 cases). Other recurring chromosome aberrations in the exposed group were 17p-, trisomy 11q, and translocation of 16q, 6p, 7p, and 11p, whereas the classic AML-specific translocations (i.e., t[15;17]; t[8;21]) were detected only in unexposed subjects. Conventional chemotherapy achieved complete remission in 1 of 19 exposed patients, as opposed to 14 of 29 unexposed patients, with a median survival of 2 months in the former group and 8 months in the latter. CONCLUSIONS: Taken together, these findings document that AML in patients professionally exposed to toxic substances may represent a distinct cytogenetic and clinicopathologic entity. The clinicobiologic characteristics in these exposed patients are similar to the features of AML arising in patients with prior chemotherapy for another tumor, thus suggesting that similar transformation pathways may underlie leukemogenesis induced by cytotoxic drugs and by environmental exposure to some pesticides or organic solvents.

Structure of transcriptionally active chromatin: radiological evidence for requirement of torsionally constrained DNA.

Synthesis of alpha- and beta-globin RNA in DMSO-induced Friend's erythroleukemia cells and synthesis of immunoglobulin gamma- and kappa-chain RNA, total RNA, 5S RNA, and tRNA in mouse myeloma cells (MPC-11) was inhibited by gamma-irradiation. For all RNA species, synthesis decreased nearly exponentially as a function of radiation dose, whereas RNA size distributions, turnover rates, and specific activities of radioactively labeled RNA were affected only insignificantly. D37 values for the loss of synthesis of various RNA species correspond to target sizes ranging from 21,000 to 53,000 kd, or 30-80 kbp of DNA. These target sizes are several-fold larger than the structural genes in question; however, they correspond well with the size of DNA loops, or "domains" constrained by the nuclear matrix. The data suggest that the eukaryotic transcription unit is the torsionally constrained chromatin loop, transcription of which may be inactivated, or significantly reduced by a DNA single-strand break.

[Successful treatment of secondary erythroleukemia with androgen].

A case of secondary erythroleukemia treated with apparent success with androgen is reported. The patient is 63-year-old Japanese female. She had a history of multiple myeloma and had been treated with melphalan, vincristine and prednisolone. She developed erythroleukemia 88 months after the initiation of chemotherapy, while her myeloma was a complete remission. She was treated first with vitamin D3 with no beneficial effect and subsequently with 0.5 mg/kg of mepitiostane. A hematologic improvement began two months from the initiation of androgen therapy, and a complete remission of erythroleukemia was attained thereafter. A chromosomal abnormality of bone marrow cells, which was observed at the time of developing erythroleukemia, also disappeared after the treatment. She remained in good condition and hematologic remission under the androgen therapy at the latest follow-up, 1-year after the development of erythroleukemia. Androgen therapy may be considered as a useful treatment for secondary erythroleukemia.