Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

Familial aggregation, candidate genes and genome scans: Analyzing the role of genetics in stroke

The genetic etiology of stroke likely reflects the influence of multiple loci with small effects, each modulating different pathophysiological processes. This research project utilized three analytical strategies to address the paucity of information related to the identification and characterization of genetic variation associated with stroke in the general population. ^ First, the general contribution of familial factors to stroke susceptibility was evaluated in a population-based sample of unrelated individuals. Increased risk of subclinical cerebral infarction was observed among individuals with a positive parental history of stroke. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. ^ The need to identify specific gene variation associated with stroke in the general population was addressed by evaluating seven candidate gene polymorphisms in a population-based sample of unrelated individuals. Three polymorphisms were significantly associated with increased subclinical cerebral infarction or incident clinical ischemic stroke risk. These relationships include the G-protein β3 subunit 825C/T polymorphism and clinical stroke in Whites, the lipoprotein lipase S/X447 polymorphism and subclinical and clinical stroke in men, and the angiotensin I-converting enzyme Ins/Del polymorphism and subclinical stroke in White men. These associations did not appear to be obfuscated by the stroke risk factors adjusted for in the analysis models specifically blood pressure levels or anti-hypertensive medication use. ^ The final research strategy considered, on a genome-wide scale, the idea that genetic variation may contribute to the occurrence of hypertension or stroke through a common etiologic pathway. Genomic regions were identified for which significant evidence of heterogeneity was observed among hypertensive sibpairs stratified by family history of stroke information. Regions identified on chromosome 15 in African Americans, and chromosome 13 in Whites and African Americans, suggest the presence of genes influencing hypertension and stroke susceptibility. ^ Insight into the role of genetics in stroke is useful for the potential early identification of individuals at increased risk for stroke and improved understanding of the etiology of the disease. The ultimate goal of these endeavors is to guide the development of therapeutic intervention and informed prevention to provide a lasting and positive impact on public health. ^

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

MCAM/MUC18 REGULATES MELANOMA PROGRESSION BY MODULATING THE EXPRESSION OF ID-1

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3. Interestingly, ATF-3 was upregulated by 6.9 fold in our cDNA microarray analysis following MUC18 silencing. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing, while mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18-silenced cells. These Data suggest that MUC18 silencing promotes inhibition of Id-1 expression by increasing ATF-3 expression and binding to the Id-1 promoter. Rescue of MUC18 reverted the expression of Id-1 and ATF-3, thus validating that they are not off-target effects of MUC18. To further assess the role of Id-1 in melanoma invasion and metastasis, we overexpressed Id-1 in MUC18-silenced cells. Overexpression of Id-1 in MUC18-silenced cells resulted in increased cell invasion, as well as increased expression and activity of MMP-2. Our data further reveal that Id-1 regulates MMP-2 at the transcriptional level through Sp1 and Ets-1. This is the first report to demonstrate that MUC18 does not act exclusively in cell adherence, but is also involved in cell signaling that regulates the expression of genes, such as Id-1 and ATF-3, thus contributing to the metastatic melanoma phenotype.

Distribution of genes encoding MSCRAMMs and Pili in clinical and natural populations of Enterococcus faecium.

Enterococcus faecium has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical E. faecium isolates studied; one of these, acm, has been studied in further detail. To determine the prevalence of the other 14 genes among various E. faecium populations, we have now assessed 433 E. faecium isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (P = 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of E. faecium to nosocomial environments and/or patients.

RESPONSES OF INDICATOR AND PATHOGENIC BACTERIA TO THE PRESENCE OF CARBARYL IN WATER

Biodegradability is a desirable, if not a necessary characteristic of pesticides. Carbaryl, as Sevin, is one of the more widely used insecticides for the control of agricultural pests and has been reported to be readily degraded by microorganisms. Because of its broad application, the concentration of Sevin in surface waters has been reported to reach nearly four parts per million (PPM) in surface waters, where it has been reported to affect the growth and metabolic rates of aquatic bacterial populations. Following these reports, it is of public health importance to determine the effects of this insecticide on the growth and metabolic rates of bacteria used to indicate water pollution, and on pathogenic organisms which are found in polluted water.^ This study was conducted to determine the effect of carbaryl on the growth and metabolic rates of indicator and pathogenic organisms. Escherichia coli and Streptococcus faecalis were used as indicators, while Staphylococcus aureus and Salmonella typhimurium were the pathogens studied. Pure and mixed cultures of these organisms were exposed to two concentrations of carbaryl (Sevin).^ The study demonstrated that the fecal pollution indicator organisms, E. coli and S. faecalis respond differently to the presence of small concentrations of carbaryl in water as do the two pathogens tested, (S. typhimurium and S. aureus). The growth of all test organisms as measured by spread plate counts, was reduced by the presence of either one mg/l or five mg/l carbaryl within a period of eight days. Survival of the organisms in the presence of five mg/l carbaryl varied dependent upon whether the organism was in pure or mixed culture. In the presence of five mg/l carbaryl, both pure and mixed culture of E. coli showed longer survival. S. faecalis survived for more than eight days in pure culture, neither S. typhimurium nor S. aureus survived for eight days in pure culture.^ The metabolic rate of S. faecalis and S. aureus was reduced by both five mg/l and one mg/l Sevin concentrations, contrary to E. coli and S. typhimurium which had reduced metabolic rate with the introduction of five mg/l Sevin but showed an increase in the metabolic rate with one mg/l Sevin. There was no difference between the test and control when mixed populations were exposed to five mg/l Sevin and the metabolic rate tested. A mixture of E. coli and S. typhimurium populations showed a respiration increase over the control when exposed to one mg/l Sevin concentration. If similar effects occur in polluted surface waters, misleading results from bacteriological water quality testing may occur. ^

Gene Therapy for Pediatric Cancer: State of the Art and Future Perspectives.

While modern treatments have led to a dramatic improvement in survival for pediatric malignancy, toxicities are high and a significant proportion of patients remain resistant. Gene transfer offers the prospect of highly specific therapies for childhood cancer. "Corrective" genes may be transferred to overcome the genetic abnormalities present in the precancerous cell. Alternatively, genes can be introduced to render the malignant cell sensitive to therapeutic drugs. The tumor can also be attacked by decreasing its blood supply with genes that inhibit vascular growth. Another possible approach is to modify normal tissues with genes that make them more resistant to conventional drugs and/or radiation, thereby increasing the therapeutic index. Finally, it may be possible to attack the tumor indirectly by using genes that modify the behavior of the immune system, either by making the tumor more immunogenic, or by rendering host effector cells more efficient. Several gene therapy applications have already been reported for pediatric cancer patients in preliminary Phase 1 studies. Although no major clinical success has yet been achieved, improvements in gene delivery technologies and a better understanding of mechanisms of tumor progression and immune escape have opened new perspectives for the cure of pediatric cancer by combining gene therapy with standard therapeutic available treatments.

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Identification of antigen-encoding genes of Enterococcus faecalis during human infections and characterization of a gene cluster for the biosynthesis of an antigenic polysaccharide

Genomic libraries of two Enterococcus faecalis strains, OG1RF and TX52 (an isolate from an endocarditis patient), were constructed in Escherichia coli and were screened with serum from a rabbit immunized with surface proteins of an E. faecalis endocarditis isolate and sera from four patients with enterococcal endocarditis. Thirty-eight immunopositive cosmid clones reacted with at least two of the patient sera and contained distinct inserts based on their DNA restriction patterns. These were chosen for further subcloning in a pBluescript SK ($-$) vector. Each sublibrary was screened with one of the five sera. Analysis of sequences from the immunopositive subclones revealed similarities to a range of proteins, including bacterial virulence factors, transporters, two-component regulators, metabolic enzymes, and membrane or cell surface proteins. Fourteen subclones did not show significant similarity to any sequence in the databases and may contain novel genes. Thirteen of the immunopositive cosmid clones did not yield immunopositive subclones and one such cosmid clone, TX5159, produced an antigenic polysaccharide in Escherichia coli. The insert of TX5159 was found to contain a multicistronic gene cluster containing genes similar to those involved in the biosynthesis and export of polysaccharides from both Gram-positive and Gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of TX5159. RT-PCR of genes within the cluster with total RNA from OG1RF showed that these genes are transcribed. The polysaccharide was detected in two recently reported E. faecalis mucoid strains using specific antibody, but not in the other strains tested. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide. ^