Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

AIMS: Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. METHODS AND RESULTS: Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. CONCLUSIONS: The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease.

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.

A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity.

Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91(phox) and p22(phox), which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47(phox) and p67(phox). A fifth subunit, p40(phox), plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40(phox), in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40(phox)R105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40(phox)-deficient granulocytes, with premature loss of p40(phox)R105Q from phagosomes. Thus, p40(phox) binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

Aberrant regulation of hematopoiesis by T cells in BAZF-deficient mice.

The BAZF (BCL-6b) protein is highly similar to the BCL-6 transcriptional repressor. While BCL-6 has been characterized extensively, relatively little is known about the normal function of BAZF. In order to understand the physiological role of BAZF, we created BAZF-deficient mice. Unlike BCL-6-deficient mice, BAZF-deficient mice are healthy and normal in size. However, BAZF-deficient mice have a hematopoietic progenitor phenotype that is almost identical to that of BCL-6-deficient mice. Compared to wild-type mice, both BAZF-deficient and BCL-6-deficient mice have greatly reduced numbers of cycling hematopoietic progenitor cells (HPC) in the BM and greatly increased numbers of cycling HPC in the spleen. In contrast to HPC from wild-type mice, HPC from BAZF-deficient and BCL-6-deficient mice are resistant to chemokine-induced myelosuppression and do not show a synergistic growth response to granulocyte-macrophage colony-stimulating factor plus stem cell factor. Depletion of CD8 T cells in BAZF-deficient mice reverses several of the hematopoietic defects in these mice. Since both BAZF- and BCL-6-deficient mice have defects in CD8 T-cell differentiation, we hypothesize that both BCL-6 and BAZF regulate HPC homeostasis by an indirect pathway involving CD8 T cells.

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity.

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.

Rac2 concentrations in umbilical cord neutrophils.

BACKGROUND: Human newborn infants display a variety of immunodeficiencies of immaturity, including diminished neutrophil adhesion, chemotaxis, and migration. Rac2, a guanosine triphosphate-binding protein, is an essential regulator of human neutrophil migration and chemotaxis. Since human subjects and mice deficient in Rac2 display deficiencies in neutrophil functions similar to newborn infants, we postulated that newborn neutrophils may be deficient in Rac2. OBJECTIVES: The aim of the study was to measure Rac1 and Rac2 concentrations in neutrophils from umbilical cord blood. METHODS: Neutrophils from cord and adult blood were isolated, total cell lysates extracted, and Rac protein concentrations determined using Western blot analysis. RESULTS: Rac2 concentrations were significantly lower in the neutrophil protein lysates isolated from cord blood compared to adult blood despite similar levels of Rac1. CONCLUSIONS: Diminished Rac2 expression in cord blood neutrophils may contribute to the defects observed in cord blood neutrophil function.

Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo. Role of the C-terminal polybasic domain.

The Rho family GTPase Rac acts as a molecular switch for signal transduction to regulate various cellular functions. Mice deficient in the hematopoietic-specific Rac2 isoform exhibit agonist-specific defects in neutrophil chemotaxis and superoxide production, despite expression of the highly homologous Rac1 isoform. To examine whether functional defects in rac2(-/-) neutrophils reflect effects of an overall decrease in total cellular Rac or an isoform-specific role for Rac2, retroviral vectors were used to express exogenous Rac1 or Rac2 at levels similar to endogenous. In rac2(-/-) neutrophils differentiated from transduced myeloid progenitors in vitro, increasing cellular Rac levels by expression of either exogenous Rac1 or Rac2 increased formylmethionylleucylphenylalanine- or phorbol ester-stimulated NADPH oxidase activity. Of note, placement of an epitope tag on the N terminus of Rac1 or Rac2 blunted reconstitution of responses in rac2(-/-) neutrophils. In rac2(-/-) neutrophils isolated from mice transplanted with Rac-transduced bone marrow cells, superoxide production and chemotaxis were fully reconstituted by expression of exogenous Rac2, but not Rac1. A chimeric Rac1 protein in which the Rac1 C-terminal polybasic domain, which contains six lysines or arginines, was replaced with that of the human Rac2 polybasic domain containing only three basic residues, also reconstituted superoxide production and chemotaxis, whereas expression of a Rac2 derivative in which the polybasic domain was replaced with that of Rac1 did not and resulted in disoriented cell motility. Thus, the composition of the polybasic domain is sufficient for determining Rac isoform specificity in the production of superoxide and chemotaxis in murine neutrophils in vivo.

Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles.

The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific Rac2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of Rac2-null murine macrophages were characterized to examine whether Rac2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and Rac2 are present in similar amounts, Rac1 was approximately 4-fold more abundant than Rac2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of Rac2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in rac2(-/-) mice. FcgammaR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in Rac2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcgammaR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in rac2(-/-) macrophages. Macrophage morphology was also similar in wild-type and Rac2-null cells, as was actin polymerization induced by FcgammaR-mediated phagocytosis or M-CSF. Hence, Rac2-null macrophages have selective defects paralleling many of the observed functional defects in Rac2-null neutrophils. These results provide genetic evidence that although Rac2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.

The hemopoietic Rho/Rac guanine nucleotide exchange factor Vav1 regulates N-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions.

Vav1 is a hemopoietic-specific Rho/Rac guanine nucleotide exchange factor that plays a prominent role in responses to multisubunit immune recognition receptors in lymphoid cells, but its contribution to regulation of neutrophil functions is unknown. Activated Rho family GTPases are critical participants in neutrophil signaling cascades initiated by binding of FMLP and other chemoattractants to their cognate G protein-coupled receptors. Therefore, we investigated whether Vav1 regulates chemoattractant-induced responses in neutrophils. We found that superoxide production elicited by FMLP in Vav1(-/-) murine neutrophils isolated from either bone marrow or from peritoneal exudates was substantially reduced compared with that of wild type. Filamentous actin generation in FMLP-stimulated Vav1(-/-) neutrophils was also markedly reduced, whereas it was normal in response to IL-8 or leukotriene B(4). FMLP induced tyrosine phosphorylation of Vav1, whereas IL-8 or leukotriene B(4) did not, correlating with the requirement for Vav1 in chemoattractant-stimulated filamentous actin generation. Neutrophil motility in vitro and neutrophil mobilization into peripheral blood in vivo elicited by FMLP were both decreased in Vav1(-/-) mice. Hence, this study defines a new role for Vav1 in regulating granulocytic leukocytes as well as linking Vav1 to specific cellular responses downstream of a seven transmembrane domain receptor.

Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.

Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To elucidate the specific role of Rac1 in neutrophils, we generated mice with a conditional Rac1 deficiency restricted to cells of the granulocyte/monocyte lineage. As observed in Rac2-deficient neutrophils, Rac1-deficient neutrophils demonstrated profound defects in inflammatory recruitment in vivo, migration to chemotactic stimuli, and chemoattractant-mediated actin assembly. In contrast, superoxide production is normal in Rac1-deficient neutrophils but markedly diminished in Rac2 null cells. These data demonstrate that although Rac1 and Rac2 are both required for actin-mediated functions, Rac2 is specifically required for activation of the neutrophil NADPH oxidase.

Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect on neutrophil functions.

The hemopoietic-specific Rho family GTPase Rac2 shares 92% amino acid identity with ubiquitously expressed Rac1. Neutrophils from rac2(-/-) mice have multiple defects, including chemoattractant-stimulated NADPH oxidase activity and chemotaxis, which may result from an overall reduction in cellular Rac or mechanisms that discriminate Rac1 and Rac2. We show that murine neutrophils have similar amounts of Rac1 and Rac2, unlike human neutrophils, which express predominantly Rac2. An affinity precipitation assay for Rac-GTP showed that although FMLP-induced activation of both isoforms in wild-type neutrophils, approximately 4-fold more Rac2-GTP was detected than Rac1-GTP. Wild-type and Rac2-deficient neutrophils have similar levels of total Rac1. FMLP-induced Rac1-GTP in rac2(-/-) neutrophils was approximately 3-fold greater than in wild-type cells, which have similar levels of total Rac1, yet FMLP-stimulated F-actin, chemotaxis, and superoxide production are markedly impaired in rac2(-/-) neutrophils. Heterozygous rac2(+/-) neutrophils, which had intermediate levels of total and FMLP-induced activated Rac2, exhibited intermediate functional responses to FMLP, suggesting that Rac2 was rate limiting for these functions. Thus, phenotypic defects in FMLP-stimulated Rac2-deficient neutrophils appear to reflect distinct activation and signaling profiles of Rac1 and Rac2, rather than a reduction in the total cellular level of Rac.