Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^

Characterization of fecal contamination targeting 16S rRNA bacteroides molecular markers in the Santa Cruz River, Arizona

Understanding the origins, transport and fate of contamination is essential to effective management of water resources and public health. Individuals and organizations with management responsibilities need to understand the risks to ecosystems and to humans from contact with contamination. Managers also need to understand how key contaminants vary over time and space in order to design and prioritize mitigation strategies. Tumacacori National Historic Park (NHP) is responsible for management of its water resources for the benefit of the park and for the health of its visitors. The existence of microbial contaminants in the park poses risks that must be considered in park planning and operations. The water quality laboratory at the Maricopa Agricultural Center (in collaboration with stakeholder groups and individuals located in the ADEQ-targeted watersheds) identified biological changes in surface water quality in impaired reaches rivers to determine the sources of Escherichia coli (E. coli); bacteria utilizing innovative water quality microbial/bacterial source tracking methods. The end goal was to support targeted watershed groups and ADEQ towards E. coli reductions. In the field monitoring was conducted by the selected targeted watershed groups in conjunction with The University of Arizona Maricopa Agricultural Center Water Quality Laboratory. This consisted of collecting samples for Bacteroides testing from multiple locations on select impaired reaches, to determine contamination resulting from cattle, human recreation, and other contributions. Such testing was performed in conjunction with high flow and base flow conditions in order to accurately portray water quality conditions and variations. Microbial monitoring was conducted by The University of Arizona Water Quality Laboratory at the Maricopa Agricultural Center using genetic typing to differentiate among two categories of Bacteroides: human and all (total). Testing used microbial detection methodologies and molecular source tracking techniques.^

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

A Genomic Approach to Identify The Notch Pathway as a Putative Tumor Suppressor in Endometrial Cancer

Endometrial cancer is the most common gynecological malignancy and the fourth most frequently diagnosed cancer among women. The molecular changes that distinguish normal endometrium from endometrial carcinoma are not thoroughly understood. Identification of these changes could potentially aid in identifying at-risk women who are especially prone to develop endometrial cancer, such as obese women and women with Lynch Syndrome. A microarray analysis was performed using normal endometrium from thin and obese women and cancerous endometrium from obese women. We validated the differential expression of ten genes whose expression was significantly up-regulated or down-regulated using qRT-PCR. All of the genes had distinct expression levels depending on the endometrial carcinoma histotype. As a result, they could serve as molecular markers to distinguish between normal endometrium and endometrial cancer, as well as between low grade endometrial carcinomas and high grade endometrial carcinomas. Two of the ten genes validated, HEYL and HES1, are down-stream targets of the Notch signaling pathway. HEYL and HES1 were identified by microarray and qRT-PCR to have a significant decrease in expression in endometrial carcinomas compared to normal endometrium. We further analyzed the differential expression of other components of the Notch signaling pathway, Notch4 and Jagged1. They were also identified by qRT-PCR to be significantly down-regulated in endometrial carcinomas compared to normal endometrium. Therefore, we believe the Notch signaling pathway to act as a tumor suppressor in endometrial carcinomas.

Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^

Identification and characterization of a molecular marker in epithelial ovarian cancer

Carcinomas that arise from the ovarian surface epithelium represent a great challenge in gynecologic oncology. Although the prognosis of ovarian cancer is influenced by many factors capable of predicting clinical outcome, including tumor stage, pathological grade, and amount of residual disease following primary surgery, the biological aspects of ovarian cancer are not completely understood, thus implying that there may be other predictive indicators that could be used independently or in conjunction with these factors to provide a clearer clinical picture. The identification of additional markers with biological relevance is desirable. To identify disease-associated peptides, a phage display random peptide library was used to screen immunoglobulins derived from a patient with ovarian cancer. One peptide was markedly enriched following three rounds of affinity selection. The presence of autoantibodies against the peptide was examined in a panel of ovarian cancer patients. Stage IV patients exhibited a high percentage of positive reactivity (59%). This was in contrast to stage III patients, who only displayed 7% positive reactivity. Antibodies against the peptide were affinity purified, and heat-shock protein 90 (Hsp90) was identified as the corresponding autoantigen. The expression profile of the identified antigen was determined. Hsp90 was expressed in all sections examined regardless of degree of anaplasia. This thesis shows that utilizing the humoral response to ovarian cancer can be used to identify a tumor antigen in ovarian cancer. The data show that certain antigens may be expressed in ovarian tumors independent of the disease stage or grade, whereas circulating antibodies against such epitopes are only found in a subset of patients. ^

Identifying and Targeting Molecular Deregulations in Uterine Leiomyosarcoma: A Role for mTOR Inhibition-Based Combination Therapies

Uterine leiomyosarcoma (ULMS) is an aggressive malignancy characterized by marked chemoresistance, frequent relapses, and poor outcome. Despite efforts to improve survival over the past several decades, only minimal advances have been made. Hence, there is an urgent and unmet need for better understanding of the molecular deregulations that underlay ULMS and development of more effective therapeutic strategies. This work identified several common deregulations in a large (n=208) tissue microarray of ULMS compared to GI smooth muscle, myometrium, and leiomyoma controls. Our results suggest that significant loss of smooth muscle and gynecological differentiation markers is common in ULMS, a finding that could help render improved ULMS diagnosis, especially for advanced disease. Similarly to reports in other malignancies, we found that several cancer-related proteins were differentially expressed; these could be useful together as biomarkers for ULMS. Notably, we identified significant upregulation and overexpression of the mTOR pathway in ULMS, examined the possible contribution of tyrosine kinase receptor deregulation promoting mTOR activation, and unraveled a role for pS6RP and p4EBP1 as molecular disease prognosticators. The significance of mTOR activation in ULMS and its potential as a therapeutic target were further investigated. Rapamycin abrogated ULMS cell growth and cell cycle progression in vitro but induced only sight growth delay in vivo. Given that effective mTOR therapies likely require combination mTOR blockade with inhibition of other targets, coupled with recent observations suggesting that Aurora A kinase (Aurk A) deregulations commonly occur in ULMS, the preclinical impact of dually targeting both pathways was evaluated. Combined therapy with rapamycin (an mTORC1 inhibitor) and MLN8237 (an investigational Aurk A inhibitor) profoundly and synergistically abrogated ULMS growth in vitro. Interestingly, the superior effects were noted only when MLN8237 was pre-administered. This novel therapeutic combination and scheduling regimen resulted in marked tumor growth inhibition in vivo. Together, these data support further exploration of dual mTOR and Aurk A blockade for the treatment of human ULMS.

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.