Altered tumorigenic phenotype in mice with a hypomorphic p53 mutant allele

Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Analysis of interval -censored events in hypertension studies: Evaluation of treatment of coronary heart disease

Many statistical studies feature data with both exact-time and interval-censored events. While a number of methods currently exist to handle interval-censored events and multivariate exact-time events separately, few techniques exist to deal with their combination. This thesis develops a theoretical framework for analyzing a multivariate endpoint comprised of a single interval-censored event plus an arbitrary number of exact-time events. The approach fuses the exact-time events, modeled using the marginal method of Wei, Lin, and Weissfeld, with a piecewise-exponential interval-censored component. The resulting model incorporates more of the information in the data and also removes some of the biases associated with the exclusion of interval-censored events. A simulation study demonstrates that our approach produces reliable estimates for the model parameters and their variance-covariance matrix. As a real-world data example, we apply this technique to the Systolic Hypertension in the Elderly Program (SHEP) clinical trial, which features three correlated events: clinical non-fatal myocardial infarction, fatal myocardial infarction (two exact-time events), and silent myocardial infarction (one interval-censored event). ^

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies.

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene.

Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8 x 10(-5)), establishing a novel phenotype for this genetic variant.

Multiple independent genetic factors at NOS1AP modulate the QT interval in a multi-ethnic population.

Extremes of electrocardiographic QT interval are associated with increased risk for sudden cardiac death (SCD); thus, identification and characterization of genetic variants that modulate QT interval may elucidate the underlying etiology of SCD. Previous studies have revealed an association between a common genetic variant in NOS1AP and QT interval in populations of European ancestry, but this finding has not been extended to other ethnic populations. We sought to characterize the effects of NOS1AP genetic variants on QT interval in the multi-ethnic population-based Dallas Heart Study (DHS, n = 3,072). The SNP most strongly associated with QT interval in previous samples of European ancestry, rs16847548, was the most strongly associated in White (P = 0.005) and Black (P = 3.6 x 10(-5)) participants, with the same direction of effect in Hispanics (P = 0.17), and further showed a significant SNP x sex-interaction (P = 0.03). A second SNP, rs16856785, uncorrelated with rs16847548, was also associated with QT interval in Blacks (P = 0.01), with qualitatively similar results in Whites and Hispanics. In a previously genotyped cohort of 14,107 White individuals drawn from the combined Atherosclerotic Risk in Communities (ARIC) and Cardiovascular Health Study (CHS) cohorts, we validated both the second locus at rs16856785 (P = 7.63 x 10(-8)), as well as the sex-interaction with rs16847548 (P = 8.68 x 10(-6)). These data extend the association of genetic variants in NOS1AP with QT interval to a Black population, with similar trends, though not statistically significant at P<0.05, in Hispanics. In addition, we identify a strong sex-interaction and the presence of a second independent site within NOS1AP associated with the QT interval. These results highlight the consistent and complex role of NOS1AP genetic variants in modulating QT interval.

Detecting spatiotemporal clusters of accidental poisoning mortality among Texas counties, U.S., 1980 – 2001

Background Accidental poisoning is one of the leading causes of injury in the United States, second only to motor vehicle accidents. According to the Centers for Disease Control and Prevention, the rates of accidental poisoning mortality have been increasing in the past fourteen years nationally. In Texas, mortality rates from accidental poisoning have mirrored national trends, increasing linearly from 1981 to 2001. The purpose of this study was to determine if there are spatiotemporal clusters of accidental poisoning mortality among Texas counties, and if so, whether there are variations in clustering and risk according to gender and race/ethnicity. The Spatial Scan Statistic in combination with GIS software was used to identify potential clusters between 1980 and 2001 among Texas counties, and Poisson regression was used to evaluate risk differences. Results Several significant (p < 0.05) accidental poisoning mortality clusters were identified in different regions of Texas. The geographic and temporal persistence of clusters was found to vary by racial group, gender, and race/gender combinations, and most of the clusters persisted into the present decade. Poisson regression revealed significant differences in risk according to race and gender. The Black population was found to be at greatest risk of accidental poisoning mortality relative to other race/ethnic groups (Relative Risk (RR) = 1.25, 95% Confidence Interval (CI) = 1.24 – 1.27), and the male population was found to be at elevated risk (RR = 2.47, 95% CI = 2.45 – 2.50) when the female population was used as a reference. Conclusion The findings of the present study provide evidence for the existence of accidental poisoning mortality clusters in Texas, demonstrate the persistence of these clusters into the present decade, and show the spatiotemporal variations in risk and clustering of accidental poisoning deaths by gender and race/ethnicity. By quantifying disparities in accidental poisoning mortality by place, time and person, this study demonstrates the utility of the spatial scan statistic combined with GIS and regression methods in identifying priority areas for public health planning and resource allocation.

Extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks.

It is system dynamics that determines the function of cells, tissues and organisms. To develop mathematical models and estimate their parameters are an essential issue for studying dynamic behaviors of biological systems which include metabolic networks, genetic regulatory networks and signal transduction pathways, under perturbation of external stimuli. In general, biological dynamic systems are partially observed. Therefore, a natural way to model dynamic biological systems is to employ nonlinear state-space equations. Although statistical methods for parameter estimation of linear models in biological dynamic systems have been developed intensively in the recent years, the estimation of both states and parameters of nonlinear dynamic systems remains a challenging task. In this report, we apply extended Kalman Filter (EKF) to the estimation of both states and parameters of nonlinear state-space models. To evaluate the performance of the EKF for parameter estimation, we apply the EKF to a simulation dataset and two real datasets: JAK-STAT signal transduction pathway and Ras/Raf/MEK/ERK signaling transduction pathways datasets. The preliminary results show that EKF can accurately estimate the parameters and predict states in nonlinear state-space equations for modeling dynamic biochemical networks.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

A pharmacological investigation of noradrenergic receptor mechanisms in neophobia

The dorsal noradrenergic bundle (DB) is a major ascending pathway which originates in the locus coeruleus of the brainstem and projects to the forebrain. The behavioral role of the DB remains unclear, despite a great deal of effort. Selective attention and anxiety are two areas which have been the focus of recent research. Some studies of the DB utilize the neurotoxin 6-hydroxydopamine (6-OHDA), since 6-OHDA injection into this pathway results in greater than 90 percent depletion of cortical and hippocampal norepinephrine (NE). Neophobia, the fear of novelty, has been reported to be either increased or decreased by 6-OHDA lesions of the DB, depending on conditions. The selective attention hypothesis would be supported by increased neophobia after 6-OHDA lesions, while the anxiety hypothesis would be supported by decreased neophobia. We have examined the effects of 6-OHDA DB lesions on neophobia under conditions in which the test environment and/or the test food were novel. We found that the lesion attenuates neophobia, defined as an increased preference for novel food, when both the environment and food were novel. The lesion had no effect on neophobia when only the environment or food was novel.^ We examined the effects of chronic intraventricular NE infusions on behavior in our neophobia test, in sham and 6-OHDA DB lesioned animals. We found that chronic NE infusions into lesioned animals significantly reversed the lesion-induced attenuation of neophobia. Sham/NE infused animals demonstrated a 40 percent greater preference for familiar food compared to sham/saline infused animals. These data suggest that infusions of NE have an effect opposite to lesion-induced attenuation of neophobia. Chronic infusions of the alpha adrenoceptor agonists had no consistent effects on neophobia. The beta adrenoceptor agonist, isoproterenol reversed the lesion-induced attenuation of neophobia but not to a statistically significant degree. Isoproterenol increased neophobia in sham animals. Forskolin, an adenylate cyclase activator, mimicked the effects of NE infusion by significantly reversing the lesion-induced attenuation of neophobia, while increasing neophobia in sham animals. These results suggest that increased release of NE during stress increases neophobia in part by stimulating beta adrenoceptors which activate adenylate cyclase. ^

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Quantitation of C4a and C4b in systemic lupus erythematosus patients

The fourth component of human complement (C4) exists in blood as two major forms or isotypes which differ in their biochemical and functional properties. Because C4A preferentially transacylates onto amino groups, it has been postulated that this isotype is more important in the clearance of immune complexes. Patients having systemic lupus erythematosus (SLE), an autoimmune disease, have an increased incidence of C4A null genes and presumably decreased levels of C4A. Currently accepted methods for the detection of C4, however, cannot accurately quantitate C4A and C4B. Thus, their role in disease susceptibility and activity has not been studied. A novel immunoassay, which utilized heat-aggregated IgG to activate and capture C4, was developed for accurate quantitation of total C4, C4A and C4B by monoclonal antibody conjugates. Higher mean total C4 values were found in a healthy Black control population when compared to White controls. This appeared to be due to an increase in C4B. In SLE patients, mean total C4 levels were significantly lower than controls regardless of disease activity. Serial patient studies showed that the ratio of C4A:C4B remained relatively constant. When the patient group was compared to controls based on C4 null gene status, the mean levels of C4A were identical while C4B was decreased in the patients. This suggests that the common HLA-B8, Dr3 C4A*Q0 gene deletion found in SLE patients may also adversely affect genetic control of the C4B genes. Furthermore, low levels of C4A cannot fully account for disease development in SLE patients having C4A null genes. ^

Patient compliance during drug therapy for hypertension

A retrospective study has been conducted examining the relationship between patient compliance and race among diagnosed hypertensives in NHANES II 1976-1980. The study includes the review/analysis of 403 blacks and 2,011 nonblacks. Patient compliance was measured using the frequency that patients took their hypertensive medication.^ A statistically significant trend of increasing compliance as age increased was found (p =.000) in blacks, nonblacks, and the study group. The number of times a person spoke with a doctor about high blood pressure was found to be statistically significant (p ==.000) in nonblacks and the study group. ^

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^