Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

ORP1L, a new Rab7 effector and regulator of late endosome functions

Oxysterol binding protein (OSBP) homologues have been found in eukaryotic organisms ranging from yeast to humans. These evolutionary conserved proteins have in common the presence of an OSBP-related domain (ORD) which contains the fully conserved EQVSHHPP sequence motif. The ORD forms a barrel structure that binds sterols in its interior. Other domains and sequence elements found in OSBP-homologues include pleckstrin homology domains, ankyrin repeats and two phenylalanines in an acidic tract (FFAT) motifs, which target the proteins to distinct subcellular compartments. OSBP homologues have been implicated in a wide range of intracellular processes, including vesicle trafficking, lipid metabolism and cell signaling, but little is known about the functional mechanisms of these proteins. The human family of OSBP homologues consists of twelve OSBP-related proteins (ORP). This thesis work is focused on one of the family members, ORP1, of which two variants were found to be expressed tissue-specifically in humans. The shorter variant, ORP1S contains an ORD only. The N-terminally extended variant, ORP1L, comprises a pleckstrin homology domain and three ankyrin repeats in addition to the ORD. The two ORP1 variants differ in intracellular localization. ORP1S is cytosolic, while the ankyrin repeat region of ORP1L targets the protein to late endosomes/lysosomes. This part of ORP1L also has profound effects on late endosomal morphology, inducing perinuclear clustering of late endosomes. A central aim of this study was to identify molecular interactions of ORP1L on late endosomes. The morphological changes of late endosomes induced by overexpressed ORP1L implies involvement of small Rab GTPases, regulators of organelle motility, tethering, docking and/or fusion, in generation of the phenotype. A direct interaction was demonstrated between ORP1L and active Rab7. ORP1L prolongs the active state of Rab7 by stabilizing its GTP-bound form. The clustering of late endosomes/lysosomes was also shown to be linked to the minus end-directed microtubule-based dynein-dynactin motor complex through the ankyrin repeat region of ORP1L. ORP1L, Rab7 and the Rab7-interacting lysosomal protein (RILP) were found to be part of the same effector complex recruiting the dynein-dynactin complex to late endosomes, thereby promoting minus end-directed movement. The proteins were found to be physically close to each other on late endosomes and RILP was found to stabilize the ORP1L-Rab7 interaction. It is possible that ORP1L and RILP bind to each other through their C-terminal and N-terminal regions, respectively, when they are bridged by Rab7. With the results of this study we have been able to place a member of the uncharacterized OSBP-family, ORP1L, in the endocytic pathway, where it regulates motility and possibly fusion of late endosomes through interaction with the small GTPase Rab7.