Genetic requirement for Ras in the transformation of fibroblasts and hematopoietic cells by the Bcr-Abl oncogene.

To determine the functional importance of Ras in transformation by Abl oncogenes, we used a genetic approach to measure the effect of impaired Ras activity on the ability of Bcr-Abl or v-Abl to transform cells. Expression of the catalytic domain of the GTPase activating protein for Ras (Gap C terminus) impaired soft agar colony formation by fibroblasts expressing v-Abl or Bcr-Abl by 70-80%. To test Ras function in a model that more closely resembles clinical diseases involving Bcr-Abl, double gene retroviruses expressing Bcr-Abl paired with the Gap C terminus or dominant negative Ras were introduced into naive mouse bone marrow cells. Transformation by Bcr-Abl was completely blocked in both situations. Coexpression of normal c-H-Ras accelerated the transforming activity of Bcr-Abl. These findings show that Ras activation is essential for the leukemogenic activity of Abl oncogenes in two distinct model systems. The results genetically define a connection between the Bcr-Abl cytoplasmic tyrosine kinase and Ras and add to the accumulating evidence that deregulation of Ras is a central event in the genesis of a number of molecularly distinct forms of human myeloid leukemia

Regulated progression of B lymphocyte differentiation from cultured fetal liver.

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner

Long-term cultures of murine fetal liver retain very early B lymphoid phenotype.

Long-term cultures of murine fetal liver have been successfully established using a modification of our in vitro bone marrow culture system (14, 15). Fetal liver cells from midgestation BALB/c embryos were plated onto BAB-14 bone marrow stromal cell-adherent layers. After a 3-5 wk period, cell growth began to increase and these cells were expanded in number on fresh feeder layers. The cultured fetal liver cells were lymphoid in morphology, 5-20% cytoplasmic Ig-positive, but less than 1% surface Ig-positive. Southern blot analysis of the cultured fetal liver cells, as well as cultured bone marrow-derived B cells, demonstrated a population with germline Ig heavy chain loci, possibly representing very early B cell precursors. Abelson murine leukemia virus (A-MuLV) clonal transformants of such cultured fetal liver cells had a phenotypic distribution similar to that seen with fresh fetal liver transformants but distinct from those obtained with the transformation of either cultured or fresh bone marrow. All A-MuLV transformants isolated had rearrangements at the mu heavy chain locus of both chromosomes, irrespective of Ig production. In addition, most mu heavy chain producers had at least one rearranged kappa gene locus. These long-term fetal liver cultures provide large numbers of cells for studying events early in the B lymphocyte lineage. The cultured fetal liver cells retained phenotypic traits similar to fresh fetal liver B cells and distinctive from bone marrow cells cultured under similar conditions

Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells.

Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression

Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells

Extracts from lymphoid and fibroblast cell lines transformed by Abelson murine leukemia virus (A-MuLV) contain a protein of molecular weight 120,000 (P120). Immunoprecipitation with specific sera shows that P120 contains regions homologous to the 5'-terminal segment of the MULV gag gene complex--p15, p12, and at least part of p30--but lacks detectable determinants of p10, reverse transcriptase, and the envelope glycoprotein. P120 is phosphorylated and has an intracellular half-life of 3--6 hr. In vitro translation of virion RNA from A-MuLV, with Moloney MuLV as helper, yields a product of molecular weight 120,000 with serological reactivity similar to that of the cellular P120. Translation of the RNA from the helper gave no P120. P120 is expressed in all lymphoid and fibroblastic cell lines we have tested that were transformed by A-MuLV but is not detectable in a lymphoid line in which the A-MuLV genome was established by infection but was not responsible for the transformation. Expression of P120 is selectively retained in clones of A-MuLV-transformed lymphocytes that convert to a nonproducer state after loss of expression of helper MuLV intracellular precursors. These results suggest that the P120 product of the A-MuLV genome may be responsible for maintenance of the transformed phenotype of lymphoid and fibroblast cells transformed by the virus

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation

Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells

Diagnosis and Treatment of Chronic Myelomonocytic Leukemias in Adults: Recommendations From the European Hematology Association and the European LeukemiaNet

Chronic myelomonocytic leukemia (CMML) is a disease of the elderly, and by far the most frequent overlap myelodysplastic/myeloproliferative neoplasm in adults. Aside from the chronic monocytosis that remains the cornerstone of its diagnosis, the clinical presentation of CMML includes dysplastic features, cytopenias, excess of blasts, or myeloproliferative features including high white blood cell count or splenomegaly. Prognosis is variable, with several prognostic scoring systems reported in recent years, and treatment is poorly defined, with options ranging from watchful waiting to allogeneic stem cell transplantation, which remains the only curative therapy for CMML. Here, we present on behalf of the European Hematology Association and the European LeukemiaNet, evidence- and consensus-based guidelines, established by an international group of experts, from Europe and the United States, for standardized diagnostic and prognostic procedures and for an appropriate choice of therapeutic interventions in adult patients with CMML

“God is Hidden in the Earthly Kingdom:” The Lutheran Two-Kingdoms Theory as Foundation of Scandavanian Secularity

Martin Luther’s signature “two kingdoms” teaching of the sixteenth century was an early and innovative theory of secularization that lies at the heart of historical Scandinavian culture. Defying the organic medieval models of Western Christendom, Luther separated the heavenly and earthly kingdoms, the saint and the sinner, faith and reason, church and the state, Gospel and the Law, as well as the spiritual and secular uses of law, government and authority. Though God is separated from day-to-day life, Luther wrote, God is still hidden in the earthly kingdom” and can be seen through various “masks,” “mists,” and “mimes.” Though the visible church is separated from the state and other institutions, religion remains pervasive in the common callings of every person to be God’s prophet, priest and king in every vocation and location of life. Luther’s two kingdoms theory is a complicated and controversial part of this thinking, but it is worth re-exploring today as pluralistic Scandinavia faces strong new pressures of both sacralization and secularization and seeks to discern anew “the hidden sacraliity of the secular.