A resource for cell line authentication, annotation and quality control.

Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data.

A comprehensive transcriptional portrait of human cancer cell lines.

Tumor-derived cell lines have served as vital models to advance our understanding of oncogene function and therapeutic responses. Although substantial effort has been made to define the genomic constitution of cancer cell line panels, the transcriptome remains understudied. Here we describe RNA sequencing and single-nucleotide polymorphism (SNP) array analysis of 675 human cancer cell lines. We report comprehensive analyses of transcriptome features including gene expression, mutations, gene fusions and expression of non-human sequences. Of the 2,200 gene fusions catalogued, 1,435 consist of genes not previously found in fusions, providing many leads for further investigation. We combine multiple genome and transcriptome features in a pathway-based approach to enhance prediction of response to targeted therapeutics. Our results provide a valuable resource for studies that use cancer cell lines.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Outer membrane protein A of Escherichia coli K1 selectively enhances the expression of intercellular adhesion molecule-1 in brain microvascular endothelial cells.

Escherichia coli K1 meningitis is a serious central nervous system disease with unchanged mortality and morbidity rates for last few decades. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli at the vascular endothelium; however, the effect of E. coli invasion of endothelial cells on the expression of ICAM-1 is not known. We demonstrate here that E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) selectively up-regulates the expression of ICAM-1, which occurs only in HBMEC invaded by the bacteria. The interaction of outer membrane protein A (OmpA) of E. coli with its receptor, Ecgp, on HBMEC was critical for the up-regulation of ICAM-1 and was depend on PKC-alpha and PI3-kinase signaling. Of note, the E. coli-induced up-regulation of ICAM-1 was not due to the cytokines secreted by HBMEC upon bacterial infection. Activation of NF-kappaB was required for E. coli mediated expression of ICAM-1, which was significantly inhibited by over-expressing the dominant negative forms of PKC-alpha and p85 subunit of PI3-kinase. The increased expression of ICAM-1 also enhanced the binding of THP-1 cells to HBMEC. Taken together, these data suggest that localized increase in ICAM-1 expression in HBMEC invaded by E. coli requires a novel interaction between OmpA and its receptor, Ecgp.

Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.

Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kappaB activation.

Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-alpha, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1beta, IL-1beta, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA- E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of kappaB, thereby blocking the translocation of nuclear factor (NF)-kappaB to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-kappaB to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-kappaB and the production of cytokines and chemokines induced by OmpA- E. coli, indicating a role for these kinases in the NF-kappaB/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA- E. coli, suggesting that the modulation of upstream events common for NF-kappaB and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages.

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.

Amyloid peptide-induced cytokine and chemokine expression in THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger RNA.

In Alzheimer's disease (AD) one finds increased deposition of A beta and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (A beta(1-40) and A beta(1-42))-induced expression of inflammatory cytokines and chemokines. We observed that A beta peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-alpha, and IL-1 beta) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1 beta (MIP-1 beta)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that A beta causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, A beta mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-kappa B and CREB. Moreover, A beta-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that A beta-induced expression of TNF-alpha, IL-1 beta, MCP-1, IL-8, and MIP-1 beta was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that A beta-induced expression of cytokines (TNF-alpha and IL-1 beta) and chemokines (MCP-1, IL-8, and MIP-1 beta) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.

Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor.

Monocytes from patients with sickle cell disease (SCD) are in an activated state. However, the mechanism of activation of monocytes in SCD is not known. Our studies showed that placenta growth factor (PlGF) activated monocytes and increased mRNA levels of cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) and chemokines (monocyte chemotactic protein-1 [MCP-1], IL-8, and macrophage inflammatory protein-1beta [MIP-1beta]) in both normal monocytes and in the THP-1 monocytic cell line. This increase in mRNA expression of cytochemokines was also reflected in monocytes derived from subjects with SCD. We studied the PlGF-mediated downstream cellular signaling events that caused increased transcription of inflammatory cytochemokines and chemotaxis of THP-1 monocytes. PlGF-mediated cytochemokine mRNA and protein expression was inhibited by PD98059 and wortmannin, inhibitors of mitogen-activated protein kinase kinase (MAPK/MEK) kinase and phosphatidylinositol-3 (PI3) kinase, respectively, but not by SB203580, a p38 kinase inhibitor. PlGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), which was completely inhibited by wortmannin, indicating that activation of PI3 kinase preceded MEK activation. PlGF also induced transient phosphorylation of AKT. MEK and PI3 kinase inhibitors and antibody to Flt-1 abrogated PlGF-induced chemotaxis of THP-1 monocytes. Overexpression of a dominant-negative AKT or a dominant-negative PI3 kinase p85 subunit in THP-1 monocytes attenuated the PlGF-mediated phosphorylation of ERK-1/2, cytochemokine secretion, and chemotaxis. Taken together, these data show that activation of monocytes by PlGF occurs via activation of Flt-1, which results in activation of PI3 kinase/AKT and ERK-1/2 pathways. Therefore, we propose that increased levels of PlGF in circulation play an important role in the inflammation observed in SCD via its effects on monocytes.

Placenta growth factor activates monocytes and correlates with sickle cell disease severity.

Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 +/- 1.2 pg/mL (n = 9) compared with 15.5 +/- 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 +/- 0.7 pg/mL (n = 9) in healthy controls (P <.05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1beta, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P <.05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P <.05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.