Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Incidence of hyperoxia and related in-hospital mortality in critically ill patients: a retrospective data analysis.

BACKGROUND: Mechanical ventilation with oxygen is life-saving, however, may result in hyperoxia. The aim was to analyse the incidence and duration of hyperoxia burden and related in-hospital mortality in critically ill patients. METHODS: Patients of all ages admitted to intensive care units (ICUs) and with mechanical ventilation for at least seven consecutive days were included in this single centre retrospective medical record audit. The main outcome measure was time-weighted arterial partial pressure of oxygen (PaO2 ) over 7 days. Logistic regression for association with in-hospital mortality and propensity score matching was performed. RESULTS: In total, 20,889 arterial blood gases of 419 patients were analysed. Time-weighted mean PaO2 was 14.0 ± 2.4 kPa. Time-weighted mean FiO2 was 49.2 ± 12.1%. Seventy-six (18.1%) patients showed continuous hyperoxia exposure, defined as time-weighted mean PaO2 > 16 kPa. Duration of hyperoxia, hypoxia (PaO2 < 8 kPa) and normoxia (PaO2 8-16 kPa) were 37.9 ± 31.0 h (23.7%), 4.9 ± 9.5 h (3.1%), and 116.8 ± 29.6 h (73.2%). Hyperoxia occurred especially at low to moderate FiO2 in patients of first and second age quartiles (1-57 years) with smaller SAPS2 score. In-hospital mortality of patients with hyperoxia (32.9%) or normoxia did not differ (35.9%; P = 0.691). Conditional logistic regression showed no association between hyperoxia and in-hospital mortality (OR 1.46; 95%CI 0.72-2.96; P = 0.29). CONCLUSION: Substantial hyperoxia burden was observed in ICU patients. Young patients with less comorbidities showed hyperoxic episodes more often, especially with lower FiO2 . Hyperoxia during 7 days of mechanical ventilation did not correlate to increased in-hospital mortality.

Detection and differentiation of cerebral microemboli in patients undergoing major orthopaedic surgery using transcranial Doppler ultrasound.

Background: Cerebral microemboli (ME) are frequently generated during orthopaedic surgery and may impair cerebral integrity. However, the nature of cerebral ME, being either of solid or gaseous origin, is poorly investigated. Our primary aim was to determine both the frequency and nature of cerebral ME in generally anaesthetised patients undergoing major orthopaedic surgery. Methods: Fifty patients (hip/knee/shoulder prosthesis, spine surgery) were enrolled. Cerebral ME and cerebral blood flow velocity (CBFV) were determined in both middle cerebral arteries for 15 min preoperatively and postoperatively, using transcranial Doppler ultrasound. Cerebral tissue oxygen index, determined by near-infrared spectroscopy, was further examined. Statistical analysis was carried out using the Wilcoxon matched-pairs signed-ranks test (median (25 th ; 75 th percentile), P < 0.05). Results: Overall the frequency of postoperative cerebral ME rose to 600% of preoperative values. Primarily gaseous ME occurred preoperatively and postoperatively [19 (6; 63) vs 116 (24; 373), P < 0.001], while the number of solid ME was negligibly small [1 (0; 2) vs 2 (0; 6), P < 0.001]. CBFV and cerebral tissue oxygen index remained unaltered bilaterally before and after surgery. Conclusions: Our findings indicate that cerebral ME considerably increase after major orthopaedic surgery under general anaesthesia. The predominant accumulation of gaseous ME and their preoperative occurrence, suggest that the general anaesthesia and individual patient factors may contribute to the embolic load in addition to orthopaedic surgery. Clinical trial registration: . NCT02340416.

Cyclic and constant hyperoxia cause inflammation, apoptosis and cell death in human umbilical vein endothelial cells.

BACKGROUND: Perioperative high-dose oxygen (O2 ) exposure can cause hyperoxia. While the effect of constant hyperoxia on the vascular endothelium has been investigated to some extent, the impact of cyclic hyperoxia largely remains unknown. We hypothesized that cyclic hyperoxia would induce more injury than constant hyperoxia to human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were exposed to cyclic hyperoxia (5-95% O2 ) or constant hyperoxia (95% O2 ), normoxia (21% O2 ), and hypoxia (5% O2 ). Cell growth, viability (Annexin V/propidium iodide and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) lactate dehydrogenase (LDH), release, cytokine (interleukin, IL and macrophage migration inhibitory factor, MIF) release, total antioxidant capacity (TAC), and superoxide dismutase activity (SOD) of cell lysate were assessed at baseline and 8, 24, and 72 h. A signal transduction pathway finder array for gene expression analysis was performed after 8 h. RESULTS: Constant and cyclic hyperoxia-induced gradually detrimental effects on HUVECs. After 72 h, constant or cyclic hyperoxia exposure induced change in cytotoxic (LDH +12%, P = 0.026; apoptosis +121/61%, P < 0.01; alive cells -15%, P < 0.01; MTT -16/15%, P < 0.01), inflammatory (IL-6 +142/190%, P < 0.01; IL-8 +72/43%, P < 0.01; MIF +147/93%, P < 0.01), or redox-sensitive (SOD +278%, TAC-25% P < 0.01) markers. Gene expression analysis revealed that constant and cyclic hyperoxia exposure differently activates oxidative stress, nuclear factor kappa B, Notch, and peroxisome proliferator-activated receptor pathways. CONCLUSIONS: Extreme hyperoxia exposure induces inflammation, apoptosis and cell death in HUVECs. Although our findings cannot be transferred to clinical settings, results suggest that hyperoxia exposure may cause vascular injury that could play a role in determining perioperative outcome.

Induction of passive Heymann nephritis with antibodies specific for a synthetic peptide derived from the receptor-associated protein.

Passive Heymann nephritis (pHN) is an experimental rat model for human membranous glomerulopathy. In pHN, the formation of subepithelial immune deposits (ID) involves as antigenic targets the membrane glycoprotein gp330/megalin and the 44-kD receptor-associated protein (RAP). A single binding site for ID- inducing antibodies (Abs) was previously mapped to the 86 NH2-terminal amino acids of RAP (RAP1-86). To further narrow this epitope, Abs eluted from the glomeruli were immunoblotted on membranes that were loaded with overlapping synthetic peptides representing the amino acid sequence of RAP (SPOTs system). Two adjacent Ab-binding domains with the sequences PVRLAF, (amino acids 39-44) and HSD-LKIQE (amino acids 46-53), which were separated by a single L residue at amino acid 45, were detected. Rabbit Abs raised against synthetic peptides containing these domains individually (P31-44 and P46-53) failed to procedure glomerular IDs. By contrast, Abs raised against a larger composite peptide (P31-53) induced IDs within 3d that were firmly cross linked to the glomerular basement membrane. These data suggest that Ab binding in vivo depends on the conformation of the antigenic target sequence that is preserved in the synthetic peptide P31-53, which covers the entire Ab-binding domain of RAP but not in its subdomains, P31-44 and P46-53. Collectively, these results locate the sole ID-inducing epitope of RAP to amino acids 39-53.

Two Plasmodium knowlesi-specific antigens on the surface of schizont-infected Rhesus monkey erythrocytes induce antibody production in immune hosts.

Purified schizonts (6--10 nuclei) and membranes of schizont-infected erythrocytes from the Malaysian and Philippine strain of Plasmodium knowlesi are analyzed immunochemically using immunoglobulin of rhesus monkey hyperimmune sera against schizonts and of sera from naturally immune monkeys. The anti-schizont Ig identifies less than 20 immune components in Triton X-100-solubilized schizonts and membranes of infected cells. Of these antigens, 9 (component 1, 3, 4, 5, 6, 10, 11, 18, and 20) are common to parasites and membranes of infected erythrocytes, and 12 (2A,B, 6, 8, 9, 12, 13p, 14, 16A,B, 19 A,Bp, 21, 22p, and 23) are predominantly found in the parasite; 4 components (13i, 19A,Bi, 22A, B, and 24) are unique to the membrane of infected erythrocytes. Only three parasite-specific components (1, 13, and 19) are exposed on the surface of parasitized erythrocytes as revealed by both lactoperoxidase-catalyzed radioiodination and extensive absorption of anti-schizont Ig using intact infected erythrocytes. Two plasmodium-specific antigens (1 and 13) on the surface of infected erythrocytes are recognized by sera of rhesus monkeys rendered naturally immune against P. knowlesi infections and, therefore, represent antigens in vivo. Analyses of schizonts and membranes of parasitized erythrocytes of the two different strains of P. knowlesi yields only some minor quantitative, but no qualitative differences when analyzed with both types of antisera. Importantly, components 1 and 13 appear identical in both strains.

Protective Plasmodium knowlesi Mr 74,000 antigen in membranes of schizont-infected rhesus erythrocytes.

The immunogenic Plasmodium knowlesi (H strain) Mr 74,000 protein in membranes of schizont-infected rhesus erythrocytes was purified on a large scale, free of other polypeptides as monitored by dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. In a limited vaccination trial, four rhesus monkeys were immunized four consecutive times with the Mr 74,000 protein and Freund's complete and incomplete adjuvants. Two monkeys were injected with adjuvant only. Upon challenge with 10(4) viable P. knowlesi schizonts of the heterologous W strain, the control monkeys developed fatal parasitemias after 7 d. In contrast, the vaccinated monkeys exhibited a delayed onset of patent parasitemias and underwent self-cure on days 14 to 16 after peak parasitemias of between 7 and 11%. The protective immunity that was induced crossed different strains of P. knowlesi. Blood smears at the time of cure demonstrated limited reinfection, as indicated by the presence of normally appearing ring and trophozoite stages. The absence of schizont stages in the peripheral blood suggested a specific interruption of the erythrocytic schizogony at that stage. Immunochemical analyses of the rhesus sera revealed antibody only against the Mr 74,000 protein after the first two immunizations. Upon repeated antigen injection, antibodies reacted with components of Mr of approximately 102,000, 140,000, and 230,000 in addition to the Mr 74,000 protein. Besides immunological cross-reactivity, relatedness between all four immune-precipitated proteins was indicated by a greater than 50% tryptic peptide homology, suggesting that the Mr 230,000 component represents a precursor protein that is cleaved within the infected erythrocyte into proteins with Mr of approximately 140,000, 102,000, and 74,000.

Human-human hybridomas secreting monoclonal antibodies to the Mr 195,000 Plasmodium falciparum blood stage antigen.

Using the human lymphoblastoid cell line, GM 4672, and PBL of Gambian adults immune to Plasmodium falciparum (Pf) malaria, we have produced human-human hybridomas and selected those that produce mAb against Pf antigens. The fusion frequency, using PWM-stimulated donor lymphocytes was between 6.8 X 10(-5) and 1.5 X 10(-6). Using immune fluorescence, immune precipitation, and Pf in vitro growth inhibition, we cloned four hybridomas that reacted with the Pf Mr 195,000 schizont/merozoite protein. The differences in proteins immune precipitated and in growth inhibition indicate that, during development of protective immunity against Pf malaria, a spectrum of antibodies is produced reacting with different epitopes on the same antigen. Only a portion of these antibodies exhibits biological activity, suggesting that the recognition of certain epitopes is required for the development of a protective immune response.

Determinants on surface proteins of Plasmodium knowlesi merozoites common to Plasmodium falciparum schizonts.

In this report and (R. Schmidt-Ullrich, L. H. Miller, and D. F. H. Wallach. Manuscript in preparation.), we have demonstrated that malaria proteins on the surface of merozoites and infected erythrocytes cross-react between at least two primate malarias, Plasmodium knowlesi and P. falciparum. Sera from five Gambian adults who were highly immune to P. falciparum were used as a reagent to study the cross-reactivity between P. falciparum schizonts and surface proteins on P. knowlesi merozoites. Although the sera bound to the surface of viable, intact P. knowlesi merozoites, the sera did not block invasion of rhesus erythrocytes. 125I-lactoperoxidase-labeled surface proteins on merozoites formed complexes with the antibody. All major protein bands seen in the electrophoresis of the original Triton extract were bound by the immune sera. Because Gambians have never been exposed to P. knowlesi malaria, the antibodies that reacted with P. knowlesi merozoites must be directed against antigens of another parasite such as P. falciparum. We tested this hypothesis by competition for antibody in a Gambian serum between Triton-extracted antigens from P. falciparum schizont-infected erythrocytes and from surface-labeled P. knowlesi merozoites. P. falciparum inhibited the reaction, thus indicating cross-reaction between antigens in P. falciparum schizonts and P. knowlesi merozoites.

Comparative analysis of mononuclear cells isolated from mucosal lymphoid follicles of the human ileum and colon.

Studies of human mucosal lymphoid follicles are rare and have been limited to children's Peyer's patches, which are visible at endoscopy. We investigated lymphoid follicles in ileum biopsies of 87 patients and surgical colon specimens from 66 cancer patients, and examined phenotype and function of isolated follicular immune cells. Two (0-10) and 12 (0-117) follicles per patient were found in ileum and colon samples respectively (P < 0.001). The number of lymphoid follicles mononuclear cells (LFMC) that could be isolated per patient was higher from colon compared with ileum specimens [725 000 (0-23 Mio) versus 100 000 (0-1.3 Mio), P < 0.001]. T cells were predominant in both LFMC and lamina propria mononuclear cells (LPMC), but B cells were more and plasma cells less frequent in LFMC. T cells from mucosal follicles were more frequently CD4-positive and CD62L-positive, but less frequently CD8-positive, CD103-positive and CD69-positive than lamina propria T cells. LFMC from ileum compared with colon showed no differences in mononuclear cell composition. Anti-CD3/CD28 stimulation induced similar proliferation of LFMC and LPMC from ileum and colon, as well as secretion of high levels of interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-2, but lower levels of IL-4, IL-6 and IL-10. LFMC from colon secreted more IL-2 than those from ileum. Our study shows that mucosal lymphoid follicles can be identified clearly in adult human colon and yield viable immune cells sufficient for phenotypical and functional analysis. The cellular composition of LFMC from ileum and colon is similar, and both secrete predominantly T helper type 1 cytokines.

Imaging biological activity of a glioblastoma treated with an individual patient-tailored, experimental therapy regimen.

PURPOSE: To monitor the metabolic effects of an individual patient-tailored, experimental glioma therapy regimen that included repetitive multiple neurosurgical resections, radiosurgical interventions, and an adjuvant maintenance therapy based on the tyrosine kinase inhibitor imatinib in combination with the chemotherapeutic agent hydroxyurea (HU). PROCEDURES: Therapeutic effects were monitored in a 26-year-old male patient with a glioblastoma multiforme by multimodal imaging using sequential L: -[methyl-(11)C]-methionine positron emission tomography (MET-PET) and MRI. The normalized MET uptake and volume of the metabolically active tumor were assessed sequentially. RESULTS: The individual patient-tailored, experimental glioma therapy caused a continuous decline of metabolically active tumor volume, associated with clinical remission over a period of more than two years. CONCLUSIONS: MET-PET seems to be useful for monitoring patient-tailored, experimental glioma therapy regimens, especially when patients are treated with a multi-step therapeutic regimen. Monitoring and guidance of those experimental therapy regimens by MET-PET in a larger patient group are needed to confirm its clinical value.

Pilot study of freshly excised breast tissue response in the 300-600 GHz range.

The failure to accurately define tumor margins during breast conserving surgery (BCS) results in a 20% re-excision rate. The present paper reports the investigation to evaluate the potential of terahertz imaging for breast tissue recognition within the under-explored 300 - 600 GHz range. Such a frequency window matches new BiCMOS technology capabilities and thus opens up the opportunity for near-field terahertz imaging using these devices. To assess the efficacy of this frequency band, data from 16 freshly excised breast tissue samples were collected and analyzed directly after excision. Complex refractive indices have been extracted over the as-mentioned frequency band, and amplitude frequency images show some contrast between tissue types. Principal component analysis (PCA) has also been applied to the data in an attempt to automate tissue classification. Our observations suggest that the dielectric response could potentially provide contrast for breast tissue recognition within the 300 - 600 GHz range. These results open the way for silicon-based terahertz subwavelength near field imager design, efficient up to 600 GHz to address ex vivo life-science applications.

Hypoxic pulmonary blood flow redistribution and arterial oxygenation in endotoxin-challenged NOS2-deficient mice.

Sepsis and endotoxemia impair hypoxic pulmonary vasoconstriction (HPV), thereby reducing arterial oxygenation and enhancing hypoxemia. Endotoxin induces nitric oxide (NO) production by NO synthase 2 (NOS2). To assess the role of NO and NOS2 in the impairment of HPV during endotoxemia, we measured in vivo the distribution of total pulmonary blood flow (QPA) between the right (QRPA) and left (QLPA) pulmonary arteries before and after left mainstem bronchus occlusion (LMBO) in mice with and without a congenital deficiency of NOS2. LMBO reduced QLPA/QPA equally in saline-treated wild-type and NOS2-deficient mice. However, prior challenge with Escherichia coli endotoxin markedly impaired the ability of LMBO to reduce QLPA/QPA in wild-type, but not in NOS2-deficient, mice. After endotoxin challenge and LMBO, systemic oxygenation was impaired to a greater extent in wild-type than in NOS2-deficient mice. When administered shortly after endotoxin treatment, the selective NOS2 inhibitor L-NIL preserved HPV in wild-type mice. High concentrations of inhaled NO attenuated HPV in NOS2-deficient mice challenged with endotoxin. These findings demonstrate that increased pulmonary NO levels (produced by NOS2 or inhaled at high levels from exogenous sources) are necessary during the septic process to impair HPV, ventilation/perfusion matching and arterial oxygenation in a murine sepsis model.

Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/gp330 in passive Heymann nephritis.

Megalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.

Macrophage colony stimulating factor expression in human cardiac cells is upregulated by tumor necrosis factor-alpha via an NF-kappaB dependent mechanism.

INTRODUCTION: Macrophage colony stimulating factor (M-CSF) is a key factor for monocyte and macrophage survival and proliferation. M-CSF has been implicated in cardiac healing and repair after myocardial infarction. METHODS AND RESULTS: We show by immunohistochemistry and Western blotting analysis that M-CSF protein is present in human heart tissue. Cultured human adult cardiac myocytes (HACM) and human adult cardiac fibroblasts (HACF) isolated from human myocardial tissue constitutively express M-CSF. When HACM and HACF were treated with tumor necrosis factor-alpha (TNF-alpha) M-CSF protein production and M-CSF mRNA expression, determined by ELISA or by using RT-PCR, respectively, was significantly increased. To determine a possible role of nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) in M-CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IkappaB kinase 2 (IKK2) were used. Only the NF-kappaB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor-1 (JNK-I), were able to block the TNF-alpha-induced increase in M-CSF production in these cells, suggesting that the induction of M-CSF through TNF-alpha is mainly dependent on the activation of the NF-kappaB pathway. The monocyte activation marker CD11b was significantly increased after incubating U937 cells with conditioned medium from HACM or HACF as determined by FACS analysis. CONCLUSIONS: Our in vitro data taken together with our immunohistochemistry data suggest that human cardiac cells constitutively express M-CSF. This expression of M-CSF in the human heart and its upregulation by TNF-alpha might contribute to monocyte and macrophage survival and differentiation.

Capsule endoscopy in refractory celiac disease.

BACKGROUND AND STUDY AIMS: Patients with refractory celiac disease (RCD) are at risk of intestinal T-cell lymphoma, which is difficult to diagnose because it often develops in the small bowel. We therefore studied whether wireless capsule endoscopy was able to detect ulcerative jejunitis or intestinal T-cell lymphomas that were missed by standard endoscopic and imaging procedures in patients with RCD. PATIENTS AND METHODS: Detection of ulcerative jejunitis and overt T-cell lymphoma by capsule endoscopy or by upper and lower endoscopy, abdominal computed tomography (CT) or abdominal magnetic resonance tomography (MRT) was compared in 14 consecutive patients with RCD: in seven patients who showed loss of T-cell antigens on intraepithelial lymphocytes and/or clonality of the T-cell receptor gene (i. e. type II RCD) and in seven patients who did not have these features (i. e. type I RCD). RESULTS: Complete evaluation of the small bowel by capsule endoscopy was achieved in 9/14 patients. Signs of ulcerative jejunitis or intestinal T-cell lymphoma, affecting further clinical management, were found in two patients with type II RCD: in one patient these signs were found only by capsule endoscopy (ulcerations and stenosis) and in another patient the abnormalities were identified by CT/MRT (mesenteric lymph nodes harboring lymphoma). No clinically relevant abnormalities were found in patients with type I RCD by lower endoscopy or by small-bowel imaging (capsule endoscopy, CT, or MRT). CONCLUSIONS: In patients with type II RCD, capsule endoscopy can detect additional cases with ulcerative jejunitis and could be included in the diagnostic armamentarium, subject to confirmation by larger series. In patients with type I RCD, our study confirmed the low diagnostic yield of imaging procedures, including wireless capsule endoscopy.

Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death.

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells.

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis.

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

Attenuation of hypoxic pulmonary vasoconstriction by endotoxemia requires 5-lipoxygenase in mice.

Sepsis and endotoxemia impair hypoxic pulmonary vasoconstriction (HPV), thereby reducing systemic oxygenation. To assess the role of leukotrienes (LTs) in the attenuation of HPV during endotoxemia, the increase in left lung pulmonary vascular resistance (LPVR) before and during left mainstem bronchus occlusion (LMBO) was measured in mice with and without a deletion of the gene encoding 5-lipoxygenase (5-LO). LMBO increased the LPVR equally in saline-challenged wild-type and 5-LO-deficient mice (96+/-20% and 94+/-19%, respectively). Twenty-two hours after challenge with Escherichia coli endotoxin, the ability of LMBO to increase LPVR was markedly impaired in wild-type mice (27+/-7%; P<0.05) but not in 5-LO-deficient mice (72+/-9%) or in wild-type mice pretreated with MK886, an inhibitor of 5-LO activity (76+/-10%). Compared with wild-type mice, endotoxin-induced disruption of lung structures and inflammatory cell influx in the lung were markedly attenuated in 5-LO-deficient mice. Administration of MK571, a selective cysteinyl LT(1) receptor antagonist, 1 hour before endotoxin challenge preserved HPV and attenuated pulmonary injury in wild-type mice but did not prevent the endotoxin-induced increase in pulmonary myeloperoxidase activity. Taken together, these findings demonstrate that a 5-LO product, most likely a cysteinyl LT, contributes to the attenuation of HPV and to pulmonary injury after challenge with endotoxin.