Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model.

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.

The pachytene checkpoint in Saccharomyces cerevisiae requires the Sum1 transcriptional repressor.

Saccharomyces cerevisiae mutants that fail to complete meiotic recombination are blocked by the RAD17/RAD24/MEC1 checkpoint signaling pathway in pachytene when early sporulation genes are expressed. Middle genes are not activated in checkpoint-arrested cells because the Ndt80 transcription factor is inhibited. We find that the pachytene checkpoint requires Sum1, a transcriptional repressor that recognizes a subset of Ndt80-binding sites. Mutants lacking Sum1 or Rad17 partially bypass the block to the nuclear divisions but do not form spores, while mutants lacking both Sum1 and Rad17 completely bypass the block and form morphologically normal spores. The level of Sum1 protein decreases as middle genes are expressed, and this decrease is blocked in checkpoint-arrested cells. These data suggest that Sum1 levels are regulated by the checkpoint and that progression of the meiotic divisions and spore differentiation can be differentially controlled by competition of the Sum1 repressor and Ndt80 activator for occupancy at key middle promoters.

Pretransplant chemotherapy reduces inflammatory cytokine production and acute graft-versus-host disease after allogeneic bone marrow transplantation.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known to be a critical effector molecule in the pathogenesis of graft-versus-host disease (GVHD), and elevated levels during bone marrow transplantation (BMT) conditioning are associated with more severe GVHD. Many patients receive chemotherapy prior to BMT, but its effect on subsequent toxicities is controversial. METHODS: We studied the effect of prior chemotherapy on GVHD severity and inflammatory cytokine generation in a well-established murine model of allogeneic BMT (B6-->B6D2F1). RESULTS: Three weeks after a single dose of cyclophosphamide, bone marrow and splenic cellularity was reduced by 50% and the production of TNF-alpha to LPS stimulation by macrophages was also markedly impaired (both before and after total body irradiation). Allogeneic BMT recipients previously treated with cyclophosphamide had significantly less GVHD and improved survival relative to recipients previously pretreated with diluent only. This survival advantage was associated with reduced systemic levels of both TNF-alpha and interleukin-1beta 7 days after BMT. This reduction occurred despite equivalent serum levels of lipopolysaccharide, consistent with the reductions in TNF-alpha and interleukin-1beta production by host macrophages after cyclophosphamide pretreatment. CONCLUSIONS: These data support the notion that patients entering BMT conditioning without prior cytotoxic treatment (e.g., patients with chronic myeloid leukemia) may be at increased risk of posttransplant complications associated with excessive inflammatory cytokine production.

Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforin-dependent pathway while preventing graft-versus-host disease.

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)-mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF-mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 --> B6D2F1). B6 mice (H-2(b)) were injected subcutaneously with human G-CSF (100 micrograms/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v 0%, P <.001). Systemic levels of lipopolysaccharide and tumor necrosis factor-alpha were markedly reduced in recipients of allogeneic G-CSF-mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2(d)) at the time of transplantation, all surviving recipients of G-CSF-mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell-depleted (TCD) splenocytes from G-CSF-mobilized B6 donors died of leukemia. When splenocytes from G-CSF-mobilized perforin-deficient (pfp-/-) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF-mobilized PBSCT. These data illustrate that G-CSF-mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.

Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease.

Donor T cell responses to host alloantigen are known predictors for graft-versus-host disease (GVHD); however, the effect of donor responsiveness to an inflammatory stimulus such as lipopolysaccharide (LPS) on GVHD severity has not been investigated. To examine this, we used mouse strains that differ in their sensitivity to LPS as donors in an experimental bone marrow transplant (BMT) system. Lethally irradiated (C3FeB6)F1 hosts received BMT from either LPS-sensitive (LPS-s) C3Heb/Fej, or LPS-resistant (LPS-r) C3H/ Hej donors. Mice receiving LPS-r BMT developed significantly less GVHD as measured by mortality and clinical score compared with recipients of LPS-s BMT, a finding that was associated with significant decreases in intestinal histopathology and serum LPS and TNF-alpha levels. When donor T cell responses to host antigens were measured, no differences in proliferation, serum IFN-gamma levels, splenic T cell expansion, or CTL activity were observed after LPS-r or LPS-s BMT. Systemic neutralization of TNF-alpha from day -2 to +6 resulted in decreased intestinal pathology, and serum LPS levels and increased survival after BMT compared with control mice receiving Ig. We conclude that donor resistance to endotoxin reduces the development of acute GVHD by attenuating early intestinal damage mediated by TNFalpha. These data suggest that the responsiveness of donor accessory cells to LPS may be an important risk factor for acute GVHD severity independent of T cell responses to host antigens.

Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation.

Administration of IL-11 prevented lethal graft-versus-host disease (GVHD) in a murine bone marrow transplant (BMT) model (B6 --> B6D2F1) across MHC and minor H antigen barriers (survival at day 50: 90 vs 20%, P < 0.001). Surpisingly, IL-11 administration polarized the donor T cell cytokine responses to host antigen after BMT with a 50% reduction in IFNgamma and IL-2 secretion and a 10-fold increase in IL-4. This polarization of T cell responses was associated with reduced IFNgamma serum levels and decreased IL-12 production in mixed lymphocyte cultures (MLC). In addition, IL-11 prevented small bowel damage and reduced serum endotoxin levels by 80%. Treatment with IL-11 also reduced TNFalpha serum levels and suppressed TNFalpha secretion by macrophages to LPS stimulation in vitro. IL-11 thus decreased GVHD morbidity and mortality by three mechanisms: (a) polarization of donor T cells; (b) protection of the small bowel; and (c) suppression of inflammatory cytokines such as TNFalpha. We conclude that brief treatment with IL-11 may represent a novel strategy to prevent T cell-mediated inflammatory processes such as GVHD.

Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain.

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.

Persistence of pulmonary pathology and abnormal lung function in IL-3/GM-CSF/IL-5 beta c receptor-deficient mice despite correction of alveolar proteinosis after BMT.

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor (beta cR KO) develop lung disease similar to that seen in human pulmonary alveolar proteinosis (PAP) which includes lymphocytic infiltration around airways and vessels and the progressive accumulation of surfactant and macrophages within the alveolar space. We investigated bone marrow transplantation (BMT) as a curative treatment of PAP in beta cR KO mice by semiquantitative histologic analysis and evaluation of pulmonary function. BMT from wild-type (WT) donors into lethally irradiated beta cR KO recipients (WT --> KO) led to the complete resolution of alveolar protein accumulation and to normalization of BAL fluid cellularity and macrophage morphology. However, detailed microscopic analysis of lung tissue revealed the persistence of significant cellular infiltrates in WT --> KO recipients which were equivalent to those seen in KO --> KO animals. Evaluation of pulmonary function demonstrated that only dynamic compliance (Cdyn) and not airway conductance (G[L]) was significantly improved in the WT --> KO group compared to KO --> KO animals and that both of these measurements remained significantly abnormal when compared to WT --> WT controls. We conclude, that although BMT for PAP reverses alveolar macrophage and protein accumulation, it does not decrease the interstitial inflammatory component of this disease. The importance of this residual pathology is demonstrated by the incomplete correction of alveolar function (Cdyn) and lack of improvement in increased airway resistance (G[L]). These findings may have important implications with regard to the extent that BMT can be considered a potential curative procedure for this clinical disorder.

Localization and quantification of endoplasmic reticulum Ca(2+)-ATPase isoform transcripts.

The Ca(2+)-adenosinetriphosphatase pump of the sarcoplasmic or endoplasmic reticulum (SERCA) plays a critical role in Ca2+ signaling and homeostasis in all cells and is encoded by a family of homologous and alternatively spliced genes. To understand more clearly the role the different isoforms play in cell physiology, we have undertaken a quantitative and qualitative assessment of the tissue distribution of transcripts encoding each SERCA isoform. SERCA1 expression is restricted to fast-twitch striated muscles, SERCA2a to cardiac and slow-twitch striated muscles, whereas SERCA2b is ubiquitously expressed. SERCA3 is expressed most abundantly in large and small intestine, thymus, and cerebellum and at lower levels in spleen, lymph node, and lung. In situ hybridization analyses revealed SERCA3 transcripts in cells of the intestinal crypt, the thymic cortex, and Purkinje cells in cerebellum. In addition, SERCA3 was expressed abundantly in isolated rat spleen lymphocytes, in various murine lymphoid cell lines, and in primary cultured microvascular endothelial cells. This analysis demonstrates that SERCA3 is expressed selectively in cells in which Ca2+ signaling plays a critical and sensitive role in regulating physiological processes.