Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis.

The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. We show here that during murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, we disrupted the mad3 gene by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage.

Spontaneous loss of B lineage transcription factors leads to pre-B leukemia in Ebf1+/-Bcl-xLTg mice.

Early B-cell factor 1 (EBF1) plays a central role in B-cell lineage specification and commitment. Loss of this critical transcription factor is strongly associated with high-risk, relapsed and therapy-resistant B-cell-acute lymphoblastic leukemia, especially in children. However, Ebf1 haploinsufficient mice exhibit a normal lifespan. To determine whether prolonged survival of B cells would enable tumorigenesis in Ebf1 haploinsufficient animals, we generated Ebf1+/-Bcl-xLTg mice, which express the anti-apoptotic factor Bcl-xL in B cells. Approximately half of Ebf1+/-Bcl-xLTg mice develop aggressive oligoclonal leukemia as they age, which engrafts in congenic wild-type recipients without prior conditioning. The neoplastic cells display a pre-B phenotype and express early developmental- and natural killer cell/myeloid-markers inappropriately. In addition, we found tumor cell-specific loss of several transcription factors critical for maintaining differentiation: EBF1, TCF3 and RUNX1. However, in the majority of tumors, loss of Ebf1 expression was not due to loss of heterozygosity. This is the first spontaneous mouse model of pre-B leukemia to demonstrate inappropriate expression of non-B-cell-specific genes associated with loss of Ebf1, Tcf3 and Runx1 expression.

c-Myc enhances protein synthesis and cell size during B lymphocyte development.

Members of the myc family of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-myc genes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Emu-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Emu) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-myc expression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.

Control of B cell development by Ras-mediated activation of Raf.

Cell fate commitment in a variety of lineages requires signals conveyed via p21ras. To examine the role of p21ras in the development of B lymphocytes, we generated transgenic mice expressing a dominant-negative form of Ras in B lymphocyte progenitors, using a novel transcriptional element consisting of the Emu enhancer and the lck proximal promoter. Expression of dominant-negative Ras arrests B cell development at a very early stage, prior to formation of the pre-B cell receptor. Furthermore, an activated form of Raf expressed in the same experimental system could both drive the maturation of normal pro-B cells and rescue development of progenitors expressing dominant-negative Ras. Hence p21ras normally regulates early development of B lymphocytes by a mechanism that involves activation of the serine/threonine kinase Raf.

Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors.

Successful in-frame rearrangement of immunoglobulin heavy chain genes or T cell antigen receptor (TCR) beta chain genes in lymphocyte progenitors results in formation of pre-BCR and pre-TCR complexes. These complexes signal progenitor cells to mature, expand in cell number, and suppress further rearrangements at the immunoglobulin heavy chain or TCRbeta chain loci, thereby ensuring allelic exclusion. We used transgenic expression of a constitutively active form of c-Raf-1 (Raf-CAAX) to demonstrate that activation of the Map kinase pathway can stimulate both maturation and expansion of B and T lymphocytes, even in the absence of pre-TCR or pre-BCR formation. However, the same Raf signal did not mediate allelic exclusion. We conclude that maturation of lymphocyte progenitors and allelic exclusion require distinct signals.

Control of B cell production by the adaptor protein lnk. Definition Of a conserved family of signal-modulating proteins.

Lnk is an SH2 domain-containing adaptor protein expressed preferentially in lymphocytes. To illuminate the importance of Lnk, we generated lnk(-/-) mice. Whereas T cell development was unaffected, pre-B and immature B cells accumulated in the spleens. In the bone marrow, B-lineage cells were proportionately increased, reflecting enhanced production of pro-B cells that resulted in part from hypersensitivity of precursors to SCF, the ligand for c-kit. Hence, Lnk ordinarily acts to regulate B cell production. Further characterization of lnk(-/-) mice also revealed that full-length Lnk is a 68 kDa protein containing a conserved proline-rich region and a PH domain. Lnk is a representative of a multigene adaptor protein family whose members act, by analogy with Lnk, to modulate intracellular signaling.

Restoration of thymopoiesis in pT alpha-/- mice by anti-CD3epsilon antibody treatment or with transgenes encoding activated Lck or tailless pT alpha.

Mice deficient for the pre-TCR alpha (pT alpha) chain cannot form a pre-T cell receptor (TCR) and exhibit a severe defect in early T cell development, characterized by lack of "beta selection" and impaired generation of double-positive (DP) thymocytes. Here, we demonstrate that intraperitoneal injection of CD3epsilon-specific antibodies into pT alpha-/- x RAG-/- mice or introduction of an activated p56(lck) transgene in pT alpha-/- mice fully restores the number of DP thymocytes, and that expression of a transgenic pT alpha chain lacking its cytoplasmic portion can overcome all developmental defects associated with pT alpha deficiency. These results allow a better definition of the role of pT alpha in pre-TCR signal transduction and provide conclusive evidence that the cytoplasmic tail of pT alpha is not essential for pre-TCR signaling.