Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis.

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here, we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small-hairpin RNA (shRNA) inhibition, proteomic and metabolomic technology, we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, HAMLET sensitivity was modified by the glycolytic state of tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen, hexokinase 1 (HK1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 and hypoxia-inducible factor 1α modified HAMLET sensitivity. HK1 was shown to bind HAMLET in a protein array containing ∼8000 targets, and HK activity decreased within 15 min of HAMLET treatment, before morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 min. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene addiction or the Warburg effect.

TLR- and CXCR1-dependent innate immunity: insights into the genetics of urinary tract infections.

The susceptibility to urinary tract infection (UTI) is controlled by the innate immune response and Toll like receptors (TLRs) are the sentinels of this response. If productive, TLR4 signalling may initiate the symptomatic disease process. In the absence of TLR4 signalling the infected host instead develops an asymptomatic carrier state. The activation of mucosal TLR4 is also influenced by the properties of the infecting strain, and pathogens use their virulence factors to trigger 'pathogen-specific' TLR4 responses in the urinary tract but do not respond to the asymptomatic carrier strains in patients with asymptomatic bacteriuria (ABU). The TLR4 dependence has been demonstrated in mice and the relevance of low TLR4 function for protection for human disease was recently confirmed in children with asymptomatic bacteriuria, who expressed less TLR4 than age matched controls. Functional chemokines and functional chemokine receptors are crucial for neutrophil recruitment, and for the neutrophil dependent bacterial clearance. Interleukin (IL)-8 receptor deficient mice develop acute septic infections and chronic tissue damage, due to aberrant neutrophil function. This mechanism is relevant for human UTI as pyelonephritis prone children express low levels of the human CXCL8 (Il-8) receptor, CXC chemokine receptor 1 (CXCR1) and often have heterozygous CXCR1 polymorphisms. This review illustrates how intimately the innate response and the susceptibility to UTI are linked and sophisticated recognition mechanisms that rely on microbial virulence and on host TLR4 and CXCR1 signalling.

Effects of epithelial and neutrophil CXCR2 on innate immunity and resistance to kidney infection.

The murine chemokine receptor 2 (mCXCR2) controls resistance to urinary tract infection. We have previously shown that mCXCR2 knockout mice develop severe acute pyelonephritis and renal tissue damage with sub-epithelial neutrophil entrapment. In this study we examined the relative importance of neutrophil- and epithelial-specific mCXCR2 expression for bacterial clearance in bone marrow chimeric mice infected with uropathogenic Escherichia coli. Mice expressing mCXCR2 on their neutrophils responded rapidly to experimental urinary tract infection, clearing the infection from the kidneys. Mice lacking epithelial mCXCR2, however, showed delayed exit of neutrophils from the tissues. Mice lacking neutrophil mCXCR2 and mice with no mCXCR2 had no neutrophil recruitment and bacterial clearance. Mice expressing mCXCR2 only in their epithelial cells had a transient epithelial chemokine response; however, neutrophil recruitment was inhibited and bacteria grew without constraint. Our study shows that the expression of mCXCR2 on hematopoietic cells was crucial for bacterial clearance, while its expression on non-bone marrow-derived cells influenced the neutrophil response. These results emphasize the importance of mCXCR2 for the innate defense against urinary tract infection.

Urinary tract infections revisited.

Urinary tract infections (UTIs) remain a significant clinical problem, despite antibiotic treatment and surgical correction of reflux and malformations. Here we propose that novel molecular tools may be applied to modernize and individualize the diagnosis and therapy of UTI. Determinants of bacterial virulence and host resistance are relatively well understood at the molecular level, and technology for their detection is within reach.

Fimbrial lectins influence the chemokine repertoire in the urinary tract mucosa.

The defense against mucosal infections relies on chemokines that recruit inflammatory cells to the mucosa. This study examined if the chemokine response to uro-pathogenic Escherichia coli is influenced by fimbrial expression. The CXC (CXCL1, CXCL5, CXCL8, CXCL9, CXCL10) and CC chemokines (CCL2, CCL3, CCL5) were quantified after in vitro infection of uro-epithelial cells with a fimbriated E. coli pyelonephritis isolate, or with P or type 1 fimbriated transformants of an avirulent E. coli K-12 strain. The response profile was shown to vary with the fimbrial type. Type 1 fimbriated E. coli elicited mainly CXCL1 and CXCL8, whereas P fimbriated E. coli stimulated CCL2 and CCL5 and class II were more potent chemokine inducers than class III P fimbriae. Chemokines were also quantified in urine samples from 73 patients with febrile urinary tract infection, and analyzed as a function of disease severity and fimbrial expression by the strain infecting each patient. A complex CXC and CC chemokine response was detected in patient urine, with a significant influence of the fimbrial type. The results show that virulence factors like fimbriae may modify the mucosal chemokine response. This mechanism may allow the host to adjust the inflammatory cell infiltrate to fit the infecting strain.

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin.

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.

The host response to urinary tract infection.

The authors use the UTI model to identify basic mechanisms of disease pathogenesis, host response induction, and defense. Their studies hold the promise to provide a molecular and genetic explanation for susceptibility to UTI, and to offer more precise tools for diagnosis and therapy of these infections. There are few infections where the host response is understood in such detail and where pathologic host responses can be linked to distinct disease states. The susceptibility to UTI varies greatly in the population. The studies suggest that distinct molecular defects can cause the clinical entity of acute pyelonephritis with renal scarring, and suggest that the susceptibility to UTI in certain patient groups may have a genetic basis. In addition, the distinct signal transduction pathways explain the development of symptoms, and propose that defects in those signaling mechanisms may occur in patients with ABU. In the future, it may be useful to include these host response parameters in the diagnostic arsenal, to help in early detection of patients susceptible to recurrent UTI and renal scarring. These patients may then be offered therapies that strengthen their defense, and be offered close surveillance for recurrences and other complications.

[Effect of P fimbriae on pyuria and bacterial colonization of the human urinary tract]. / Der Einfluss von P-Fimbrien auf eine Leukozyturie und den Schwellenwert einer persistierenden Bakteriurie.

This study investigated the role of P fimbriae in colonization of Escherichia coli, host response, and bacterial persistence in humans. Human volunteers were inoculated intravesically with the nonadherent ABU isolate E. coli 83972 and with P fimbriated transformants of the same strain. During the following 24 h all urine samples, and thereafter daily samples, were collected for urine culture, analysis of neutrophil numbers, and cytokine concentrations (IL-6 and IL-8). The P fimbriated transformants showed enhanced bacterial colonization in comparison to E. coli 83972 and lowered the bacterial numbers needed for persistent bacteriuria. The P fimbriated transformants also lowered the bacterial numbers needed for a significant neutrophil and cytokine host response. We conclude that P fimbriae enhance bacterial colonization and trigger the host response in the human urinary tract.

Neutrophil recruitment, chemokine receptors, and resistance to mucosal infection.

Neutrophil migration to infected mucosal sites involves a series of complex interactions with molecules in the lamina propria and at the epithelial barrier. Much attention has focussed on the vascular compartment and endothelial cells, but less is known about the molecular determinants of neutrophil behavior in the periphery. We have studied urinary tract infections (UTIs) to determine the events that initiate neutrophil recruitment and interactions of the recruited neutrophils with the mucosal barrier. Bacteria activate a chemokine response in uroepithelial cells, and the chemokine repertoire depends on the bacterial virulence factors and on the specific signaling pathways that they activate. In addition, epithelial chemokine receptor expression is enhanced. Interleukin (IL)-8 and CXCR1 direct neutrophil migration across the epithelial barrier into the lumen. Indeed, mIL-8Rh knockout mice showed impaired transepithelial neutrophil migration, with tissue accumulation of neutrophils, and these mice developed renal scarring. They had a defective antibacterial defense and developed acute pyelonephritis with bacteremia. Low CXCR1 expression was also detected in children with acute pyelonephritis. These results demonstrate that chemokines and chemokine receptors are essential to orchestrate a functional antimicrobial defense of the urinary tract mucosa. Mutational inactivation of the IL-8R caused both acute disease and chronic tissue damage.

pap genotype and P fimbrial expression in Escherichia coli causing bacteremic and nonbacteremic febrile urinary tract infection.

Escherichia coli strains from patients with febrile urinary tract infections (n=73) were examined for pap genotype and P fimbrial expression in relation to bacteremia and patients' background variables. Most isolates were pap(+) by DNA hybridization (n=51), and 36 were papG(IA2)(+) and 18 prsG(J96)(+) by polymerase chain reaction. The pap and papG genotypes of the infecting strain were shown to vary with host compromise, sex, and age. Bacteremia in noncompromised patients was caused by papG(IA2)(+) strains, but compromised hosts carried a mixture of papG(IA2)(+), prsG(J96)(+), and pap(-) strains. Women of all ages were infected with papG(IA2)(+) strains. Infected men carried prsG(J96)(+) or pap(-) strains and were older, and most had compromising conditions. papG(IA2)(+) strains predominated among patients with medical illness, whereas prsG(J96)(+) strains predominated among patients with urinary tract abnormalities. These findings emphasize the strong influence of host factors on the selection of E. coli strains causing febrile urinary tract infections.

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation.

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.

P-fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract.

Uropathogenic Escherichia coli elicit a host response that determines the severity of urinary tract infection (UTI). Specific adherence mechanisms allow the bacteria to initiate this process by targeting epithelial cells in the urinary tract mucosa. Epidemiological studies show a strong association of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo. The present study examined the ability of P-fimbriae to induce inflammation in the human urinary tract. Patients were subjected to intravesical inoculation with a non-fimbriated E. coli strain or transformants of this strain expressing P-fimbriae. The inflammatory response was analysed as a function of P-fimbrial expression. The P-fimbriated transformants invariably caused higher interleukin (IL)-8, IL-6 and neutrophil responses in the urinary tract than the ABU strain. Furthermore, loss of P-fimbrial expression in vivo was accompanied by a return to background levels of neutrophils, IL-6 and IL-8 in individual patients. The results demonstrate that the pap sequences confer on a non-fimbriated, avirulent strain the ability to induce a host response in the human urinary tract. P-fimbriae thus fulfil the 'molecular Koch-Henle postulates' linking a single virulence factor to host response induction.

Escherichia coli-induced inducible nitric oxide synthase and cyclooxygenase expression in the mouse bladder and kidney.

BACKGROUND: The host response to urinary tract infection includes the production of different inflammatory mediators. We investigated the cellular localization and time course of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression in the mouse bladder and kidney after bacterial infection. METHODS: Experimental urinary tract infection in mice was established by intravesical inoculation of a clinical uropathogen Escherichia coli (E. coli) AD 110. Urine was collected at 6-, 12-, 24-, and 72-hours postinstillation, and the nitrite concentration was determined. The induction of iNOS and COX-2 was studied by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Nitrite levels in the urine had increased threefold at 6 and 12 hours postbacterial instillation. Bladders from mice instilled with AD 110, but not with phosphate-buffered saline, showed a large number of iNOS-- and COX-2--expressing inflammatory cells. The inflammatory cell activation peaked at 6 and 12 hours postinstillation and had vanished by 72 hours. iNOS expression was detected in some urothelial cells after 24 and 72 hours, but COX-2 expression was not detected. In the kidney, infection activated an iNOS and COX-2 response, as shown by immunoreactivity in inflammatory cells at all time points. A strong epithelial iNOS response was observed in the renal pelvis at 12, 24, and 72 hours postinstillation, but COX-2 was not detected. Enhanced tissue expression of iNOS and COX-2 after bacterial instillation was also demonstrated by RT-PCR. CONCLUSIONS: E. coli AD 110 induced expression of iNOS and COX-2 in the urinary tract. Inflammatory cells expressed both iNOS-and COX-2, but epithelial cells expressed only iNOS and with a later onset than in the inflammatory cells. This suggests that the epithelial iNOS response is not caused by direct bacterial activation, but more likely is by mediators involved in the inflammatory response.