Inflammatory myofibroblastic tumor of the thigh: presentation of a rare case and review of the literature.

Inflammatory myofibroblastic tumors are uncommon neoplasms; presentation of these tumors in the lower extremities is extremely rare. We present a case of a 47-year-old male with fever, fatigue, and a slow-growing thigh mass. The inflammatory markers were elevated and the MR images showed a well-defined intermuscular lesion with mild heterogeneous enhancement. The lesion was excised and histologic examination was consistent with an inflammatory myofibroblastic tumor. No adjuvant therapy was needed and the patient remained asymptomatic with no evidence of tumor recurrence during the 2 years of follow-up.

Cloning and characterization of a potential transcriptional activator of human gamma-globin genes.

Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human gamma-globin and switch to human beta globin expression as time in culture advances. One explanation for the initially predominant expression of gamma-globin gene in these hybrids is the presence of trans-acting factors that activate gamma-globin gene transcription. We used differential display of hybrids before and after the gamma to beta switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential gamma gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only gamma-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to beta globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of gamma mRNA in HFE x MEL cell hybrids that undergo the gamma to beta switch, suggesting that this new gene encodes a protein that may function as gamma gene activator.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin.

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human gamma and beta globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific gamma globin gene inducers are recognized by their ability to increase gamma-firefly luciferase (gamma(F)) gene activity significantly more than beta-renilla luciferase (beta(R)) gene activity, identified by an increased ratio of gamma-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase gamma-globin gene expression. It can therefore be used for the screening of chemical agents that may have gamma-globin gene inducibility.

Correct function of the locus control region may require passage through a nonerythroid cellular environment.

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice.

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.

Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion.

The human beta-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the beta-globin locus chromatin domain and globin gene expression. Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences. To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a beta-globin locus residing on a 248-kb beta-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice. Human epsilon-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c. In contrast, gamma-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver. These results indicate that the core element of HS3 is necessary for epsilon-globin gene transcription in embryonic cells and for gamma-globin gene transcription in definitive cells. Normal gamma-globin gene expression in embryonic cells and the absence of gamma-globin gene expression in definitive cells show that different HSs interact with gamma-globin gene promoters in these two stages of development. Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.