Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

The mammary gland consists of an organized network of epithelial ducts and lobules. This histoarchitecture can be recapitulated in vitro by culturing mammary epithelial cells as 3D spheroids embedded in a reconstituted basement membrane. I first used this assay to characterize the role of cell-cell and cell-ECM adhesion in the formation and polarization of the apical junction complexes in normal mammary epithelial cells. Cell-cell adhesion alone was sufficient to initiate polarized junction assembly. However, the addition of exogenous ECM generated a spatial polarity signal dependent on laminin-1 and α6 and β1 integrins. This caused clusters of mammary epithelial cells to re-localize the junctional complexes to the center of the spheroid prior to lumen formation. In ductal breast carcinoma, a critical hallmark is the loss of normal polarized tissue architecture without the induction of an epithelial-to-mesenchymal transformation (EMT). Thus, misregulation of molecules that function as polarity determinants may contribute to ductal tumor progression. Podocalyxin is an anti-adhesive glycoprotein that may be involved, as it is important in epithelial morphogenesis, and its overexpression in clinical breast tumors is associated with poor outcome. Despite this, overexpression of podocalyxin in normal mammary epithelial cells did not disrupt 3D morphogenesis or apicobasal polarity. However, its overexpression in non-metastatic breast tumor cells did perturb the architecture and growth of tumor spheroids in vitro and it facilitated subcutaneous tumor growth in vivo without causing an EMT. Mechanistically, podocalyxin localized to and expanded non-adhesive membrane domains and induced microvillus formation that was dependent on its extracellular domain and Rho GTPase-regulated actin polymerization. Podocalyxin also recruited its intracellular binding partners NHERF-1 and ezrin via its cytoplasmic tail. Strikingly, the formation of this protein complex was not required for microvillus formation. Additionally, podocalyxin delayed cell-cell aggregation and decreased the initial adhesion, spreading and strength of attachment of tumor cells to fibronectin where it restricted β1 integrin localization to the basal/attached domain. These alterations in adhesion possibly contributed to podocalyxin's ability to increase growth factor-dependent tumor cell migration. Altogether, these data indicate that podocalyxin overexpression may facilitate a ductal tumor-like progression that involves EMT-independent alterations in tissue architecture. / Medicine, Faculty of / Graduate

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Call number: LD2668 .T4 1985 B37 / Master of Science

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We studied genetic polymorphism and phylogeny using nuclear random amplified polymorphic DNA markers (RAPDs) and mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) in the three California Closed-Cone Pines: Pinus attenuata Lemm., P. muricata D. Don, and P. radiata D. Don. A total of 343 to 384 trees derived from 13 populations were analyzed using 13 mitochondria' gene probes and two restriction enzymes, and more than 90 RAPD loci generated by 22 primers. Southern hybridization was used to test homology among comigrating RAPD markers. Segregation analysis and Southern hybridization were carried out to distinguish between RAPD fragments of nuclear and organellar origin. Estimates of genetic diversity and population differentiation, and phylogenetic analyses based on RAPD and RFLP markers, were compared with those based on allozymes from a similar study. Twenty-eight distinct mtDNA haplotypes were detected among the three species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (Hs) averaged 0.22, and population differentiation (GsT and 0) exceeded 0.78. Analysis of molecular variance (AMOVA) also revealed that more than 90% of the variation resided among populations. Species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Twenty-eight of 30 (93%) comigrating RAPD fragments tested were homologous by Southern hybridization. Hybridization with enriched mtDNA, and chloroplast DNA (cpDNA) clones, identified one fragment as being of mtDNA origin and two as being of cpDNA origin, among 142 RAPD fragments surveyed. RAPD markers revealed moderately higher intrapopulation gene diversity and significantly higher total genetic diversity and population differentiation than did allozyme markers for each species. Simulation analysis to study effects of dominance on RAPD diversity suggested that dominance substantially depressed values of diversity within populations and inflated values of differentiation among populations. By comparison to our empirical analyses, we inferred that the underlying diversity of RAPD markers is substantially greater than that of allozymes. Results of phylogenetic analysis of RAPD markers were largely consistent with those from allozyme analysis, though they had many minor differences. Joint phylogenetic analysis of both the RAPD and allozyme markers strongly supported a common ancestor for P. radiata and P. attenuata, and south to north migration histories for all three species. Dendrograms based on mtDNA analysis, however, strongly disagreed with those based on allozymes, RAPDs, chloroplast DNA and morphological traits, suggesting convergent genome evolution. / Graduation date: 1999

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.