Mouse deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.     

Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function

Formation of the dorsal-ventral axis of the Drosophila wing depends on activity of the LIM-homeodomain protein Apterous (Ap). Here we report that Ap activity levels are modulated by dLMO, the protein encoded by the Beadex (Bx) gene. Overexpression of dLMO in Bx mutants interferes with Apterous function. Conversely, Bx loss-of-function mutants fail to down-regulate Apterous activity at late stages of wing development. Biochemical analysis shows that dLMO protein competes for binding of Apterous to its cofactor Chip. These data suggest that Apterous activity depends on formation of a functional complex with Chip and that the relative levels of dLMO, Apterous, and Chip determine the level of Apterous activity. The dominant interference mechanism of dLMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells.     

The expression of CD26 and CD40 ligand is mutually exclusive in human T-cell non-Hodgkin's lymphomas/leukemias

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T-cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.     

Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: characterization and diagnostic utility

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1;19) (q23;p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1;19)-positive from t(1;19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1;19)-positive childhood ALL.     

Cyclin E overexpression in relapsed adult acute lymphoblastic leukemias of B-cell lineage

Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller-molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.     

Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain

A unique clone, isolated from a human pancreatic cDNA library, was sequenced and characterized. Northern blot analysis showed that the gene is active in a number of fetal and adult tissues, and immunoblots showed expression in nuclear and cytosolic cell fractions. The gene corresponding to the clone was localized to chromosome 13 by human/rodent somatic cell hybrid panels. The largest open reading frame contains a LIM domain, and the deduced peptide from the open reading frame appears to have the characteristics of a LIM-only protein, designated LMO7. RT-PCR and genomic sequence analyses indicate that expression of this gene product is subject to tissue-specific modulation by elimination of the LIM domain by alternative splicing in neural tissues.     

Antibodies TC-12 ("unique") and TH-111 (CD96) characterize T-cell acute lymphoblastic leukemia and a subgroup of acute myeloid leukemia

To extend the panel of monoclonal antibodies useful for immunophenotyping of acute leukemias, two new reagents, TC-12 and TH-111, were developed. TC-12 was found "unique," and TH-111 was assigned to the recently defined CD96 cluster. Both reagents show little reactivity with blood and bone marrow nucleated cells but define a major (TH-111: 78.3%) or an important (TC-12: 45.6%) subset of T-cell acute lymphoblastic leukemia (ALL). In addition, in acute myeloid leukemia (AML), the expression of TC-12 was found in 64 (20.2%) of 317 and TH-111 in 97 (29.1%) of 333 of these patients. TC-12 positivity in AML was virtually restricted to the Fab subtypes M0, M1, M2, and M6. In the group of immature AML characterized by the coexpression of CD7 as well as CD117 and CD34 positivity, leukemic blasts frequently disclosed the TC-12 and TH-111 antigen. Although the TC-12 antigen could not be determined, TH-111 immunoprecipitated the TACTILE (CD96) antigen and, when expressed, was found to be associated with the transferrin receptor. These reagents may help not only to define and dissect T-cell ALL, but also to characterize a subgroup of immature AML at the divergence of T-cell and myeloid lineage.     

DNA-PKcs: a T-cell tumour suppressor encoded at the mouse scid locus

Severe combined immunodeficiency (SCID) mice are defective in their ability to rearrange their variable (V), diversity (D) and joining (J) genetic elements to generate functional immunoglobulin (Ig) and T-cell receptor (TCR) molecules; as a result, they lack mature B and T cells. These mice are highly sensitive to ionizing radiation, suggesting that the product of the scid gene plays a critical role in both V(D)J recombination and DNA double-strand break repair. Recent studies suggest that the SCID defect lies in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PK; refs 6-8), a nuclear protein made up of the Ku 70 and Ku 86 subunits as well as the large catalytic subunit, DNA-PKcs. Other reports have implied that the SCID phenotype correlates with nonsense mutations at the extreme 3' end of Prkdc, the DNA-PKcs gene. The identity of the gene remains in doubt, however, because the consequences of genetic inactivation of Prkdc have not been determined. This study shows that complete inactivation of Prkdc in a novel insertional mouse mutant recapitulates the SCID phenotype and that Prkdc and scid are alleic. Significantly, DNA-PKcs null mice demonstrate complete penetrance of thymic lymphoblastic lymphomas, strongly suggesting that Prkdc functions in mice as a T-cell tumour suppressor and, by virtue of its association with DNA repair and recombination, belongs to the 'caretaker' class of tumour-suppressor genes that includes ATM, BRCA1 and BRCA2 (ref. 15).     

Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area

In Hoxa-2(-/- )embryos, the normal skeletal elements of the second branchial arch are replaced by a duplicated set of first arch elements. We show here that Hoxa-2 directs proper skeletal formation in the second arch by preventing chondrogenesis and intramembranous ossification. In normal embryos, Hoxa-2 is expressed throughout the second arch mesenchyme, but is excluded from the chondrogenic condensations. In the absence of Hoxa-2, chondrogenesis is activated ectopically within the rostral Hoxa-2 expression domain to form the mutant set of cartilages. In Hoxa-2(-/- )embryos the Sox9 expression domain is shifted into the normal Hoxa-2 domain. Misexpression of Sox9 in this area produces a phenotype resembling that of the Hoxa-2 mutants. These results indicate that Hoxa-2 acts at early stages of the chondrogenic pathway, upstream of Sox9 induction. We also show that Hoxa-2 inhibits dermal bone formation when misexpressed in its precursors. Furthermore, molecular analyses indicate that Cbfa1 is upregulated in the second branchial arches of Hoxa-2 mutant embryos suggesting that prevention of Cbfa1 induction might mediate Hoxa-2 inhibition of dermal bone formation during normal second arch development. The implications of these results on the patterning of the branchial area are discussed.     

The LMO1 and LDB1 proteins interact in human T cell acute leukaemia with the chromosomal translocation t(11;14)(p15;q11)

The ectopic expression of LMO1 or LMO2 in T cell acute leukaemias resulting from chromosomal translocations t(11;14)(p15;qll) or t(11;14)(p13;q11) respectively in a causal factor in tumorigenesis. LMO1 has been found as a heterodimer with a 46 Kd protein in a T cell line derived from a childhood T-acute leukaemia. This 46 Kd protein is the LIM-binding protein LDB1/NLI. The latter is a phosphoprotein and binds to LMO1 in its phosphorylated state and essentially all the LMO1 and LDB1 protein in the T cell line is part of the complex. Therefore, the LMO1-LDB1 interaction is likely to be involved in tumorigenesis after LMO1 is ectopically expressed following chromosomal translocation in T cells prior to development of acute leukaemias.