A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic Protein 1 in lymphopoiesis and innate immunity

Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4−CD8− (double negative) to CD4+CD8+ (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor κB (NF-κB)–dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-κB–dependent transcription proceeds independently of Hem1 and F-actin polymerization

Myc stimulates B lymphocyte differentiation and amplifies calcium signaling

Deregulated expression of the Myc family of transcription factors (c-, N-, and L-myc) contributes to the development of many cancers by a mechanism believed to involve the stimulation of cell proliferation and inhibition of differentiation. However, using B cell–specific c-/N-myc double-knockout mice and Eμ-myc transgenic mice bred onto genetic backgrounds (recombinase-activating gene 2−/− and Btk−/− Tec−/−) whereby B cell development is arrested, we show that Myc is necessary to stimulate both proliferation and differentiation in primary B cells. Moreover, Myc expression results in sustained increases in intracellular Ca2+ ([Ca2+]i), which is required for Myc to stimulate B cell proliferation and differentiation. The increase in [Ca2+]i correlates with constitutive nuclear factor of activated T cells (NFAT) nuclear translocation, reduced Ca2+ efflux, and decreased expression of the plasma membrane Ca2+–adenosine triphosphatase (PMCA) efflux pump. Our findings demonstrate a revised model whereby Myc promotes both proliferation and differentiation, in part by a remarkable mechanism whereby Myc amplifies Ca2+ signals, thereby enabling the concurrent expression of Myc- and Ca2+-regulated target genes

Dynamic contrast-enhanced magnetic resonance imaging of tumor-induced lymph flow.

The growth of metastatic tumors in mice can result in markedly increased lymph flow through tumor-draining lymph nodes (LNs), which is associated with LN lymphangiogenesis. A dynamic magnetic resonance imaging (MRI) assay was developed, which uses low-molecular weight gadolinium contrast agent to label the lymphatic drainage, to visualize and quantify tumor-draining lymph flow in vivo in mice bearing metastatic melanomas. Tumor-bearing mice showed greatly increased lymph flow into and through draining LNs and into the bloodstream. Quantitative analysis established that both the amount and the rate of lymph flow through draining LNs are significantly increased in melanoma-bearing mice. In addition, the rate of appearance of contrast media in the bloodstream was significantly increased in mice bearing melanomas. These results indicate that gadolinium-based contrast-enhanced MRI provides a noninvasive assay for high-resolution spatial identification and mapping of lymphatic drainage and for dynamic measurement of changes in lymph flow associated with cancer or lymphatic dysfunction in mice. Low-molecular weight gadolinium contrast is already used for 1.5-T MRI scanning in humans, which should facilitate translation of this imaging assay

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 (Eμ-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Eμ) 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

B Lymphocytes Promote Lymphogenous Metastasis of Lymphoma and Melanoma1

The prognosis of patients with many types of cancers correlates with the degree of metastasis to regional lymph nodes (LNs) and vital organs. However, the mechanisms and route of cancer cell metastasis are still unclear. Previous studies determined that B-cell accumulation in tumor-draining LNs (TDLNs) induces lymphatic sinus growth (lymphangiogenesis) and increases lymph flow, which could actively promote tumor dissemination through the lymphatic system. Using young Eµ-c-Myc mice that feature LN B-cell expansion as hosts for tumor transplants, we show that subcutaneously implanted lymphomas or melanomas preferentially spread to TDLNs over non-TDLNs, thus demonstrating that these tumors initially metastasize through lymphatic rather than through hematogenous routes. In addition, the rate and amount of tumor dissemination is greater in Eµ-c-Myc mice versus wild-type hosts, which correlates with LN B-cell accumulation and lymphangiogenesis in Eµ-c-Myc hosts. The increased lymphatic dissemination in Eµ-c-Myc hosts is further associated with rapid hematogenous tumor spread of subcutaneously implanted lymphomas, suggesting that TDLN metastasis secondarily drives lymphoma spread to distant organs. In contrast, after intravenous tumor cell injection, spleen metastasis of lymphoma cells or lung metastasis of melanoma cells is similar in Eµ-c-Myc and wild-type hosts. These studies demonstrate that the effect of Eµ-c-Myc hosts to promote metastasis is limited to the lymphatic route of dissemination. TDLN B-cell accumulation, in association with lymphangiogenesis and increased lymph flow, thus significantly contributes to dissemination of lymphomas and solid tumors, providing new targets for therapeutic intervention to block metastasis

Helicobacter infection is required for inflammation and colon cancer in Smad3-deficient mice

Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-β (TGF-β) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-β signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3-/- mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4+ T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1α (IL-1α), IL-1β, IL-6, IFN-γ, and tumor necrosis factor-α, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-β signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans

Modulation of T-lymphocyte development, growth and cell size by the Myc antagonist and transcriptional repressor Mad1

Activated lymphocytes must increase in size and duplicate their contents (cell growth) before they can divide. The molecular events that control cell growth in proliferating lymphocytes and other metazoan cells are still unclear. Here, we utilized transgenesis to provide evidence suggesting that the basic helix–loop– helix–zipper (bHLHZ) transcriptional repressor Mad1, considered to be an antagonist of Myc function, inhibits lymphocyte expansion, maturation and growth following pre-T-cell receptor (pre-TCR) and TCR stimulation. Furthermore, we utilized cDNA microarray technology to determine that, of the genes repressed by Mad1, the majority (77%) are involved in cell growth, which correlates with a decrease in size of Mad1 transgenic thymocytes. Over 80% of the genes repressed by Mad1 have previously been found to be induced by Myc. These results suggest that a balance between Myc and Mad levels may normally modulate lymphocyte proliferation and development in part by controlling expression of growth-regulating genes