Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

FANCM AND FAAP24 MAINTAIN GENOMIC STABILITY THROUGH COOPERATIVE AND UNIQUE FUNCTIONS

Fanconi anemia (FA) is a rare recessive genetic disease with an array of clinical manifestations including multiple congenital abnormalities, progressive bone marrow failure and profound cancer susceptibility. A hallmark of cells derived from FA patients is hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC) and cisplatin, suggesting that FA- and FA-associated proteins play important roles in protecting cells from DNA interstrand crosslink (ICL) damage. Two genes involved in the FA pathway, FANCM and FAAP24, are of particular interest because they contain DNA interacting domains. However, there are no definitive patient mutations for these two genes, and the resulting lack of human genetic model system renders their functional studies difficult. In this study, I established isogenic human FANCM- and FAAP24-null mutants through homologous replacement-mediated gene targeting in HCT-116 cells, and systematically investigated the functions of FANCM and FAAP24 inchromosome stability, FA pathway activation, DNA damage checkpoint signaling, and ICL repair. I found that the FANCM-/-/FAAP24-/- double mutant was much more sensitive to DNA crosslinking agents than FANCM-/- and FAAP24-/- single mutants, suggesting that FANCM and FAAP24 possess epistatic as well as unique functions in response to ICL damage. I demonstrated that FANCM and FAAP24 coordinately support the activation of FA pathway by promoting chromatin localization of FA core complex and FANCD2 monoubiqutination. They also cooperatively function to suppress sister chromatid exchange and radial chromosome formation, likely by limiting crossovers in recombination repair. In addition, I defined novel non-overlapping functions of FANCM and FAAP24 in response to ICL damage. FAAP24 plays a major role in activating ICL-induced ATR-dependent checkpoint, which is independent of its interaction with FANCM. On the other hand, FANCM promotes recombination-independent ICL repair independently of FAAP24. Mechanistically, FANCM facilitates recruitment of nucleotide excision repair machinery and lesion bypass factors to ICL damage sites through its translocase activity. Collectively, my studies provide mechanistic insights into how genome integrity is both coordinately and independently protected by FANCM and FAAP24.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

A novel treatment planning methodology for high dose 166Ho-DOTMP therapy in patients with multiple myeloma

Bone marrow ablation, i.e., the complete sterilization of the active bone marrow, followed by bone marrow transplantation (BMT) is a comment treatment of hematological malignancies. The use of targeted bone-seeking radiopharmaceuticals to selectively deliver radiation to the adjacent bone marrow cavities while sparing normal tissues is a promising technique. Current radiopharmaceutical treatment planning methods do not properly compensate for the patient-specific variable distribution of radioactive material within the skeleton. To improve the current method of internal dosimetry, novel methods for measuring the radiopharmaceutical distribution within the skeleton were developed. 99mTc-MDP was proven as an adequate surrogate for measuring 166Ho-DOTMP skeletal uptake and biodistribution, allowing these measures to be obtained faster, safer, and with higher spatial resolution. This translates directly into better measurements of the radiation dose distribution within the bone marrow. The resulting bone marrow dose-volume histograms allow prediction of the patient disease response where conventional organ scale dosimetry failed. They indicate that complete remission is only achieved when greater than 90% of the bone marrow receives at least 30 Gy. ^ Comprehensive treatment planning requires combining target and non-target organ dosimetry. Organs in the urinary tract were of special concern. The kidney dose is primarily dependent upon the mean transit time of 166 Ho-DOTMP through the kidney. Deconvolution analysis of renograms predicted a mean transit time of 2.6 minutes for 166Ho-DOTMP. The radiation dose to the urinary bladder wall is dependent upon numerous factors including patient hydration and void schedule. For beta-emitting isotopes such as 166Ho, reduction of the bladder wall dose is best accomplished through good patient hydration and ensuring a partially full bladder at the time of injection. Encouraging the patient to void frequently, or catheterizing the patient without irrigation, will not significantly reduce the bladder wall dose. ^ The results from this work will produce the most advanced treatment planning methodology for bone marrow ablation therapy using radioisotopes currently available. Treatments can be tailored specifically for each patient, including the addition of concomitant total body irradiation for patients with unfavorable dose distributions, to deliver a desired patient disease response, while minimizing the dose or toxicity to non-target organs. ^

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

Multiple gradient echo propeller (MGREP) MRI: Technical development and potential applications

The PROPELLER (Periodically Rotated Overlapping Parallel Lines with Enhanced Reconstruction) magnetic resonance imaging (MRI) technique has inherent advantages over other fast imaging methods, including robust motion correction, reduced image distortion, and resistance to off-resonance effects. These features make PROPELLER highly desirable for T2*-sensitive imaging, high-resolution diffusion imaging, and many other applications. However, PROPELLER has been predominantly implemented as a fast spin-echo (FSE) technique, which is insensitive to T2* contrast, and requires time-inefficient signal averaging to achieve adequate signal-to-noise ratio (SNR) for many applications. These issues presently constrain the potential clinical utility of FSE-based PROPELLER. ^ In this research, our aim was to extend and enhance the potential applications of PROPELLER MRI by developing a novel multiple gradient echo PROPELLER (MGREP) technique that can overcome the aforementioned limitations. The MGREP pulse sequence was designed to acquire multiple gradient-echo images simultaneously, without any increase in total scan time or RF energy deposition relative to FSE-based PROPELLER. A new parameter was also introduced for direct user-control over gradient echo spacing, to allow variable sensitivity to T2* contrast. In parallel to pulse sequence development, an improved algorithm for motion correction was also developed and evaluated against the established method through extensive simulations. The potential advantages of MGREP over FSE-based PROPELLER were illustrated via three specific applications: (1) quantitative T2* measurement, (2) time-efficient signal averaging, and (3) high-resolution diffusion imaging. Relative to the FSE-PROPELLER method, the MGREP sequence was found to yield quantitative T2* values, increase SNR by ∼40% without any increase in acquisition time or RF energy deposition, and noticeably improve image quality in high-resolution diffusion maps. In addition, the new motion algorithm was found to improve the performance considerably in motion-artifact reduction. ^ Overall, this work demonstrated a number of enhancements and extensions to existing PROPELLER techniques. The new technical capabilities of PROPELLER imaging, developed in this thesis research, are expected to serve as the foundation for further expanding the scope of PROPELLER applications. ^

Comparison of multiple comparison methods for identifying differential gene expression in simulated and real papillary thyroid cancer microarray data

The difficulty of detecting differential gene expression in microarray data has existed for many years. Several correction procedures try to avoid the family-wise error rate in multiple comparison process, including the Bonferroni and Sidak single-step p-value adjustments, Holm's step-down correction method, and Benjamini and Hochberg's false discovery rate (FDR) correction procedure. Each multiple comparison technique has its advantages and weaknesses. We studied each multiple comparison method through numerical studies (simulations) and applied the methods to the real exploratory DNA microarray data, which detect of molecular signatures in papillary thyroid cancer (PTC) patients. According to our results of simulation studies, Benjamini and Hochberg step-up FDR controlling procedure is the best process among these multiple comparison methods and we discovered 1277 potential biomarkers among 54675 probe sets after applying the Benjamini and Hochberg's method to PTC microarray data.^

Statistical Methods for Differential Expressions of Genes Detected in Multiple-Condition Experiment of Microarray

Most studies of differential gene-expressions have been conducted between two given conditions. The two-condition experimental (TCE) approach is simple in that all genes detected display a common differential expression pattern responsive to a common two-condition difference. Therefore, the genes that are differentially expressed under the other conditions other than the given two conditions are undetectable with the TCE approach. In order to address the problem, we propose a new approach called multiple-condition experiment (MCE) without replication and develop corresponding statistical methods including inference of pairs of conditions for genes, new t-statistics, and a generalized multiple-testing method for any multiple-testing procedure via a control parameter C. We applied these statistical methods to analyze our real MCE data from breast cancer cell lines and found that 85 percent of gene-expression variations were caused by genotypic effects and genotype-ANAX1 overexpression interactions, which agrees well with our expected results. We also applied our methods to the adenoma dataset of Notterman et al. and identified 93 differentially expressed genes that could not be found in TCE. The MCE approach is a conceptual breakthrough in many aspects: (a) many conditions of interests can be conducted simultaneously; (b) study of association between differential expressions of genes and conditions becomes easy; (c) it can provide more precise information for molecular classification and diagnosis of tumors; (d) it can save lot of experimental resources and time for investigators.^

Motivational interviewing and feedback for college drinkers: Handling missing values with complete case analysis and multiple imputation

Objective: In this secondary data analysis, three statistical methodologies were implemented to handle cases with missing data in a motivational interviewing and feedback study. The aim was to evaluate the impact that these methodologies have on the data analysis. ^ Methods: We first evaluated whether the assumption of missing completely at random held for this study. We then proceeded to conduct a secondary data analysis using a mixed linear model to handle missing data with three methodologies (a) complete case analysis, (b) multiple imputation with explicit model containing outcome variables, time, and the interaction of time and treatment, and (c) multiple imputation with explicit model containing outcome variables, time, the interaction of time and treatment, and additional covariates (e.g., age, gender, smoke, years in school, marital status, housing, race/ethnicity, and if participants play on athletic team). Several comparisons were conducted including the following ones: 1) the motivation interviewing with feedback group (MIF) vs. the assessment only group (AO), the motivation interviewing group (MIO) vs. AO, and the intervention of the feedback only group (FBO) vs. AO, 2) MIF vs. FBO, and 3) MIF vs. MIO.^ Results: We first evaluated the patterns of missingness in this study, which indicated that about 13% of participants showed monotone missing patterns, and about 3.5% showed non-monotone missing patterns. Then we evaluated the assumption of missing completely at random by Little's missing completely at random (MCAR) test, in which the Chi-Square test statistic was 167.8 with 125 degrees of freedom, and its associated p-value was p=0.006, which indicated that the data could not be assumed to be missing completely at random. After that, we compared if the three different strategies reached the same results. For the comparison between MIF and AO as well as the comparison between MIF and FBO, only the multiple imputation with additional covariates by uncongenial and congenial models reached different results. For the comparison between MIF and MIO, all the methodologies for handling missing values obtained different results. ^ Discussions: The study indicated that, first, missingness was crucial in this study. Second, to understand the assumptions of the model was important since we could not identify if the data were missing at random or missing not at random. Therefore, future researches should focus on exploring more sensitivity analyses under missing not at random assumption.^

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^