p210(BCR-ABL) reprograms transformed and normal human megakaryocytic progenitor cells into erythroid cells and suppresses FLI-1 transcription.

The BCR-ABL oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias. Here, we report that ectopic expression of p210(BCR-ABL) in the megakaryoblastic Mo7e cell line and in primary human CD34(+) progenitors trigger erythroid differentiation at the expense of megakaryocyte (MK) differentiation. Clonal culture of purified CD41(+)CD42(-) cells, a population highly enriched in MK progenitors, combined with the conditional expression of p210(BCR-ABL) tyrosine kinase activity by imatinib identified a true lineage reprogramming. In both Mo7e or CD41(+)CD42(-) cells transduced with p210(BCR-ABL), lineage switching was associated with a downregulation of the friend leukemia Integration 1 (FLI-1) transcription factor. Re-expression of FLI-1 in p210(BCR-ABL)-transduced Mo7e cells rescued the megakaryoblastic phenotype. Altogether, these results demonstrate that alteration of signal transduction via p210(BCR-ABL) reprograms MK cells into erythroid cells by a downregulation of FLI-1. In addition, our findings underscore the role of kinases in lineage choice and infidelity in pathology and suggest that downregulation of FLI-1 may have important implications in CML pathogenesis.

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA.

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5' upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5' uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5' uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5' uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons.

Spi-1/PU.1 is a positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines.

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the -200 region instead of position -400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the -200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the -270/-41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV.

Identification of two critical base pairings in 5' untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs.

We observed a marked difference between the in vitro translation efficiency of two uncapped synthetic mRNAs, displaying the entire human alpha or beta globin mRNA sequences and some additional non-globin sequences in 5'. The comparison of the translation efficiencies of chimeric mRNAs indicated that the alpha 5' untranslated region (5' UTR) is responsible for a low translation efficiency that cannot be explained neither by primary sequence nor by the overall stability of 5' UTR secondary structures only. By point mutations in this alpha 5' UTR, we identified two base pairings at position -1 and -2 preceding the initiation codon which are associated with a negative effect on translation efficiency.

Coactivation of human alpha1- and alpha2-globin genes in single induced MEL cells containing one human alpha-globin locus.

We developed a reverse-transcription polymerase chain reaction assay, performed on single isolated cells, to demonstrate the coexpression of human alpha1- and alpha2-globin mRNA in induced mouse erythroleukemic cells containing a single human alpha-globin locus. These results indicate that both alpha1 and alpha2 genes are activated from the same alpha-globin gene locus implying that HS-40-dependent transcriptional activation is mediated, either by a simultaneous interaction of HS-40 with both a alpha1 and alpha2-globin gene promoters, or by a dynamic process characterized by alternative, but short-lived, interactions with each alpha-globin gene promoter.

3' non-translated region of human alpha-globin messenger RNA does not affect translation efficiency.

The relative translation efficiency of three synthetic alpha-globin mRNAs differing by their 3' non-translated end was measured in vitro in a rabbit reticulocyte lysate. Results showed that substituting the 3' non-translated end of human alpha 2 globin mRNA by the 3' non-translated end of chimpanzee alpha 1 or alpha 2 mRNAs has no effect on translation efficiency. In contrast, the introduction of the alpha-Quong-Sze mutation (alpha 125, Leu----Pro) in human alpha 2 mRNA led to a 50% apparent reduction in globin synthesis due to the instability of the alpha-Quong-Sze globin chain. We conclude that human alpha 1 and alpha 2 globin mRNAs have the same translation efficiency, and that the reduction, previously reported, in the kinetics of alpha-globin synthesis by alpha 2 mRNA carrying the alpha-Quong-Sze mutation is due to the instability of the alpha-Quong-Sze globin chain only.

Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen.

Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes.

Human chronic myeloid leukemic cell line with positive Philadelphia chromosome exhibits megakaryocytic and erythroid characteristics.

A cell line (LAMA-84) has been established from the blood of a patient with chronic myeloid leukemia in acute phase. LAMA-84 cells retained the patient's chromosome abnormalities, i.e., triplication of all chromosomes except chromosome 18, the presence of Philadelphia (Ph) chromosome in 4-5 copies, and the presence of chromosome markers. LAMA-84 cells have morphological features of undifferentiated blast cells, but analyses have indicated that they belong to the megakaryocytic lineage; platelet peroxidase (PPO) was found in 8.5% of cells; LAMA-84 cells reacted spontaneously with poly- and monoclonal antibodies against the platelet glycoproteins (GP) IIb, IIIa, and the GPIIb/IIIa complex, whose presence was confirmed by crossed immunoelectrophoresis. LAMA-84 cells lack the membrane characteristics of lymphoid and mature granulocytic cells but do, however, react with certain antibodies to immature myeloid cells. Furthermore, they are positive with an antiglycophorin antibody, and contain alpha- and gamma-globin mRNA, thus demonstrating erythroid marker expression. Thus LAMA-84 is a tripotent, megakaryocytic, erythroid, and granulocytic cell line. The megakaryocytic and erythroid markers were enhanced by the addition of DMSO, butyrate, TPA, and hemin. The LAMA-84 cell line represents an interesting tool for the study of megakaryocytic and erythroid differentiation and the mechanisms of neoplastic growth.

Alpha-thalassemia due to the deletion of nucleotides -2 and -3 preceding the AUG initiation codon affects translation efficiency both in vitro and in vivo.

We previously hypothesized that a 2 nucleotide deletion, causing a A-greater than C change at position -3 preceding the ATG initiation codon of alpha globin gene, reduced translation efficiency of alpha globin mRNA and was responsible for a form of alpha + thalassemia displayed by an Algerian patient. We presently show that this deletion leads to a 30-45% reduction in translation efficiency of synthetic alpha globin mRNA in rabbit reticulocyte lysate. In other experiments, we constructed alpha/G gamma hybrid globin genes in which the 3' end of normal or mutated alpha globin genes downstream to the ATG initiation codon was substituted by the 3' part of a G gamma globin gene. COS cells transfected with either of these 2 hybrid genes were shown to synthesize a similar amount of alpha/G gamma hybrid mRNAs but 50% less G gamma globin when transfected with the alpha/G gamma hybrid gene carrying the deletion. These results definitively establish that the 2 nucleotide deletion reduces translation efficiency by 30-50%. This contrasts with the 93% reduction induced by a similar A-greater than C change at position -3 in the different nucleotide context preceding the ATG codon of the rat preproinsulin gene.

Functional analysis of the 4 bp deletion identified in the 5' untranslated region of one of the beta-globin genes from a Chinese beta-thalassaemic heterozygote.

Two plasmids have been constructed in which a beta-galactosidase/phleomycin-resistance fusion gene reporter is placed under the control of the human beta-globin gene promoter and 5' untranslated region including or not including nucleotides 40-43 previously found deleted in one Chinese beta-thalassaemic allele. Transient expression assays of these two plasmids failed to reveal any significative effect of this 4 bp deletion either on the level of the beta-galactosidase activity produced in HeLa cells transfected in standard conditions, or on the rate of synthesis of the beta-galactosidase protein in transfected HeLa cells submitted to increasing osmotic shocks. These results suggest that this 4 bp deletion is not responsible for the beta-thalassaemic phenotype in vivo.

Heterogeneity in beta 0 thalassemia from Algeria: genetic, clinical and molecular studies.

Six Algerian patients with beta 0 thalassemia are presented, in addition to the two patients already reported (Godet et al., 1977). Family studies indicate that all the patients had homozygous beta thalassemia characterized by absence of beta globin chain synthesis in peripheral blood. The clinical severity varies from one family to the other and within the same family, from typical Cooley's anemia to thalassemia intermedia and appears to be related to the child death rate observed in each family. The gamma/alpha biosynthetic ratio was 0.36-0.40 in seven patients and 0.2 in the most seriously affected patient. The mRNA beta content in peripheral reticulocytes was less than 1.5% of mRNA alpha in seven patients and 13.3% in one patient. These results indicate that Algerians homozygous for beta 0 thalassemia are heterozygous at the clinical, biochemical and molecular levels.

Inducible expression of a neo gene integrated into the human alpha-globin gene cluster.

We replaced the 3' flanking region of the human alpha 1-globin gene that binds in vitro the specific transcription factors GATA-1 and AP1/NF-E2, by a neo marker gene using homologous recombination in a MEL (mouse erythroleukemia line) hybrid cell line harbouring a single human chromosome 16. Using an improved method of the neo-positive and HSV-tk negative selection, one correctly targeted clone was obtained out of 164 clones analyzed. In contrast to non-targeted clones, the expression of teh neo gene in the targeted clone acquired the erythroid differentiation-dependent inducibility normally characteristic of the alpha-globin genes. No difference was observed in the expression of the human zeta, alpha 2, alpha 1, or theta-globin genes before and after induction of differentiation between the targeted clone and parental cells. These results indicate that, at least in the experimental system used, the 3' flanking region of the human alpha 1-globin gene can be replaced by an exogenous non-erythroid gene without affecting the regulation of the globin genes contained in the alpha-globin cluster.

Extent and high frequency of a short conversion between the human A gamma and G gamma fetal globin genes.

Southern blotting and DNA sequencing after polymerase chain reaction (PCR) amplification provide evidence for the frequent occurrence (in 7 out of 24 chromosomes) of a short conversion G gamma----A gamma in the 3' end of the human fetal A gamma globin gene. This short conversion is characterized by the presence, 3 nucleotides downstream from the termination codon of the A gamma gene, of the TCAC sequence that is normally present at the equivalent position at the 3' end of the G gamma gene; it is therefore identical to a conversion already described. Interestingly, we have found that this conversion is associated with the presence of the HindIII polymorphic restriction site in the A gamma IVS2, occupying an equivalent position in both the G gamma and A gamma genes. Our observations strengthen the hypothesis that the presence of the HindIII polymorphic restriction site in A gamma IVS2 and the presence of the sequence TCAC at the 3' end of the A gamma gene might be the result of a single conversion event.

alpha-Thalassaemia associated with the deletion of two nucleotides at position -2 and -3 preceding the AUG codon.

The nucleotide sequence of three single alpha-globin genes resulting from a rightward 3.7-kb deletion is described. The alpha genes were isolated from the DNA of three subjects homozygous for this deletion, the first being in addition homozygous for the structural mutation alpha G Philadelphia (genotype -alpha G/-alpha G), the second, heterozygous for this structural mutation (genotype -alpha A/-alpha G) and the third homozygous for an alpha + -thalassaemic gene (genotype -alpha +thal/-alpha +thal). The latter subject produced HbH in contrast to the two others. Whereas the two alpha A and alpha G genes are identical to the normal alpha 1-globin gene (except for the alpha G point mutation), the alpha +thal gene has (i) a deletion of the two nucleotides at position -2 and -3 preceding the ATG codon, and (ii) a fusion between the 5' part of the normal alpha 2 gene and the 3' part of the normal alpha 1 gene. Using a dot-blot assay, we show that reticulocytes from the HbH subject contain at least as much alpha mRNA as reticulocytes from the two other subjects. In a transient expression system, the alpha +thal gene leads to normally spliced transcripts. We conclude from these data that the defective output of alpha chains by the alpha +thal gene, as evidenced by HbH production, results from a decreased efficiency of alpha-mRNA translation due to the two nucleotides deletion preceding the AUG codon.

Alpha-thalassemia haplotypes in the Algerian population.

DNA mapping was performed in seven unrelated Hb H patients and nine carriers for alpha-thalassemia trait originating from Algeria. This study has allowed us to identify four alpha-thalassemia haplotypes: the (-alpha 3.7) haplotype, which is the most frequent (18 of 23 alpha-thalassemic chromosomes), the (-(alpha)20.5) haplotype, a (--) haplotype, and an (alpha alpha)T haplotype. Our results also show that the (-alpha 3.7) haplotypes encountered in the Algerian population are heterogeneous and differ by the site of the unequal crossover responsible for the 3.7-kb deletion and the size of the interzeta fragment. In addition, during this survey we observed that normal chromosomes bearing a polymorphic BglII site are associated with different interzeta fragments.

Genetic factors involved in human G gamma and A gamma globin gene expression.

G gamma to A gamma globin ratios, haplotypes at the beta globin gene cluster and the C----T substitution at -158 5' to the G gamma globin gene were studied in three Algerian families that include SS or S-beta(0) thal patients. G gamma to A gamma ratios were found similar, within a family, in subjects displaying the same combination of chromosomes 11, the ratio observed for a given combination depending on the chromosome haplotypes. Our data can be explained by the existence of several alleles of a genetic factor closely linked to the beta globin gene cluster and involved in the determination of G gamma to A gamma globin ratio.

Expression of human colony-stimulating factor-1 (CSF-1) receptor in murine pluripotent hematopoietic NFS-60 cells induces long-term proliferation in response to CSF-1 without loss of erythroid differentiation potential.

NFS-60 and FDCP-Mix cells are interleukin-3--dependent multipotent hematopoietic cells that can differentiate in vitro into mature myeloid and erythroid cells. Retrovirus-mediated transfer of the human colony-stimulating factor-1 (CSF-1) receptor gene (c-fms) enabled NFS-60 cells but not FDCP-Mix cells to proliferate in response to CSF-1. The phenotype of NFS-60 cells expressing the human CSF-1 receptor (CSF-1R) grown in CSF-1 did not grossly differ from that of original NFS-60 as assessed by cytochemical and surface markers. Importantly, these cells retained their erythroid potentiality. In contrast, a CSF-1-dependent variant of NFS-60, strongly expressing murine CSF-1R, differentiated into monocyte/macrophages upon CSF-1 stimulation and almost totally lost its erythroid potentiality. We also observed that NFS-60 but not FDCP-Mix cells could grow in response to stem cell factor, (SCF), although both cell lines express relatively high amounts of SCF receptors. This suggests that SCF-R and CSF-1R signalling pathways share at least one component that may be missing or insufficiently expressed in FDCP-Mix cells. Taken together, these results suggest that human CSF-1R can use the SCF-R signalling pathway in murine multipotent cells and thereby favor self-renewal versus differentiation.

Identification of GATA-1 and NF-E2 binding sites in the flanking regions of the human alpha-globin genes.

Some of the elements involved in the erythroid-specific transcriptional regulation of the human gamma- and beta-globin genes and located inside or in the immediate proximity of these genes have been identified as sequences which bind erythroid-specific factors. In the present study, we found two regions located within 1 kb in 5' to the alpha 2- and in 3' to the alpha 1-globin genes which contribute to the induction of human alpha-globin genes following erythroid differentiation in stable MEL transformants. By DNAse I footprinting and gel mobility shift assays, we identified several GATA-1 and one AP-1/NF-E2-binding sites located inside these regions. These results strengthen the idea that, like for all other globin genes, flanking regions contribute in vivo to the regulation of human alpha-globin gene expression.