Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BACKGROUND: Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. METHODS: Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-ß-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. RESULTS: Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. CONCLUSIONS: We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.

Acute lymphoid leukemia cells with greater stem cell antigen-1 (Ly6a/Sca-1) expression exhibit higher levels of metalloproteinase activity and are more aggressive in vivo.

Stem cell antigen-1 (Ly6a/Sca-1) is a gene that is expressed in activated lymphocytes, hematopoietic stem cells and stem cells of a variety of tissues in mice. Despite decades of study its functions remain poorly defined. These studies explored the impact of expression of this stem cell associated gene in acute lymphoid leukemia. Higher levels of Ly6a/Sca-1 expression led to more aggressive leukemia growth in vivo and earlier death of hosts. Leukemias expressing higher levels of Ly6a/Sca-1 exhibited higher levels of matrix metalloproteinases. The results suggest the hypothesis that the more aggressive behavior of Ly6a/Sca-1 expressing leukemias is due at least in part to greater capacity to degrade microenvironmental stroma and invade tissues.

Evidence of B cell immune responses to acute lymphoblastic leukemia in murine allogeneic hematopoietic stem cell transplantation recipients treated with donor lymphocyte infusion and/or vaccination.

These experiments explored mechanisms of control of acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation using a murine model of MHC-matched, minor histocompatibility antigen-mismatched transplantation. The central hypothesis examined was that addition of active vaccination against leukemia cells would substantially increase the effectiveness of allogeneic donor lymphocyte infusion (DLI) against ALL present in the host after transplantation. Although vaccination did increase the magnitude of type I T cell responses against leukemia cells associated with DLI, it did not lead to substantial improvement in long-term survival. Analysis of immunologic mechanisms of leukemia progression demonstrated that the failure of vaccination was not because of antigen loss in leukemia cells. However, analysis of survival provided surprising findings that, in addition to very modest type I T cell responses, a B cell response that produced antibodies that bind leukemia cells was found in long-term survivors. The risk of death from leukemia was significantly lower in recipients that had higher levels of such antibodies. These studies raise the hypothesis that stimulation of B cell responses after transplantation may provide a novel way to enhance allogeneic graft-versus-leukemia effects associated with transplantation.

Acute lymphoblastic leukemia cells that survive combination chemotherapy in vivo remain sensitive to allogeneic immune effects.

Allogeneic hematopoietic stem cell transplantation is often performed for patients with acute lymphoblastic leukemia (ALL) whose disease has relapsed after chemotherapy treatment. However, graft versus leukemia (GVL) effects in ALL are generally weak and the mechanisms of this weakness are unknown. These studies tested the hypothesis that ALL cells that have survived conventional chemotherapy in vivo acquire relative resistance to the allogeneic GVL effect. C57BL/6 mice were injected with murine pre-B ALL lines driven by human mutations and then were treated with combination chemotherapy. ALL cells surviving therapy were analysed in vitro and in vivo for acquisition of resistance to chemotherapy, radiation, cytolytic T cells, NK cells, LAK cells and cytokines. In vivo drug treatment did lead to leukemia population with more rapid proliferation and also decreased sensitivity to vincristine, doxorubicin and radiation. However, drug treatment did not produce ALL populations that were less sensitive to GVL effects in vitro or in vivo.

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant.

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.

Role of the renin-angiotensin system in ventilator-induced lung injury: an in vivo study in a rat model.

BACKGROUND: Injurious mechanical ventilation can cause a pro-inflammatory reaction in the lungs. Recent evidence suggests an association of the renin-angiotensin system (RAS) with lung inflammation. A study was undertaken to investigate the pathogenic role of the RAS in ventilator-induced lung injury (VILI) and to determine whether VILI can be attenuated by angiotensin converting enzyme (ACE) inhibition. METHODS: Male Sprague-Dawley rats were mechanically ventilated for 4 h with low (7 ml/kg) or high (40 ml/kg) tidal volumes; non-ventilated rats were used as controls. Lung injury and inflammation were measured by the lung injury score, protein leakage, myeloperoxidase activity, pro-inflammatory cytokine levels and nuclear factor (NF)-kappaB activity. Expression of the RAS components was also assessed. Some rats were pretreated with the ACE inhibitor captopril (10 mg/kg) for 3 days or received a concomitant infusion with losartan or PD123319 (type 1 or type 2 angiotensin II receptor antagonist) during mechanical ventilation to assess possible protective effects on VILI. RESULTS: In the high-volume group (n=6) the lung injury score, bronchoalveolar lavage fluid protein concentration, pro-inflammatory cytokines and NF-kappaB activities were significantly increased compared with controls (n=6). Lung tissue angiotensin II levels and mRNA levels of angiotensinogen and type 1 and type 2 angiotensin II receptors were also significantly increased in the high-volume group. Pretreatment with captopril or concomitant infusion with losartan or PD123319 in the high-volume group attenuated the lung injury and inflammation (n=6 for each group). CONCLUSIONS: The RAS is involved in the pathogenesis of ventilator-induced lung injury. ACE inhibitor or angiotensin receptor antagonists can attenuate VILI in this rat model.