The correlation of information obtained from in vitro investigations and in vivo experiments has frequently evaded researchers, especially in the area of tumor cell invasion and metastasis. In order to better understand and associate in vitro tumor cell invasion through basement membranes with in vivo tumor metastasis in syngeneic animal models, and the subsequent modulation of these processes, the following studies have been undertaken. Malignant murine melanoma cell lines designated B16F1 and B16F10, syngeneic to the C57BL6 mouse, a melanotic variant of the Cloudman S-91 melanoma cell line (denoted Mel-11a) with the syngeneic host being the DBA/2J mouse, and a malignant human melanoma line referenced as A375P (parental) and A375M (metastatic) were used for this dissertation project. Tumor cells were labeled with either ¹⁴C-thymidine or ¹²⁵I-deoxyuridine using previously established protocols. Radiolabeled tumor cells were introduced into the Membrane Invasion Culture System (MICS) in vitro, a system developed in our lab, and concomitantly into the lateral tail vein by injection or intracutaneously into the appropriate syngeneic host in the presence or absence of such biological response modifying agents as [Nle⁴, D-Phe⁷]-MSH, and α-MSH. The superpotent analogue of α-MSH ([Nle⁴, D-Phe⁷] -MSH) showed a proliferative and survival enhancing effect on tumor metastasis in vivo with no effect on in vitro tumor cell invasion, with similar effects demonstrated by α-MSH. The effects of these melanotropins on spontaneous metastasis formation appears to be negligible. The A375M and A375P human melanoma cells parallel their metastatic profile in vitro when assayed in MICS. In concert with these studies, the development of a control cell line, comprised of neural crest-derived melanocytes, and the study of their subsequent invasiveness in vitro were pursued. The neural crest-derived melanocytes were unable to invade the basement membranes (BM); although co-culturing neural crest cells with B16F10 melanoma cells produced an effect such that the neural crest cells did significantly invade the BMs. These studies demonstrate the ability of the MICS in vitro invasion assay to discriminate between tumor cells with differing metastatic propensities and could possibly be used in future studies to predict the effectiveness of biological response modifying agents in vivo.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Rap1a and Rap1b are two closely related members of the Ras family of small GTPases. Despite their high sequence similarity, the two proteins serve non-redundant functions in cells and organs. Rap1a plays critical roles during mouse development, and both Rap1a and Rap1b are required for angiogenesis. In glioblastoma cells, however, Rap1b plays a more unique role in tumor cell invasion. Loss of rap1a in mice resulted in 40% embryonic lethality, and caused cardiac defects in mouse embryos and cardiac hypertrophy in adult mice. These phenotypes, distinct from those of the rap1b knockout mice, suggest differential roles of the two GTPases during mouse development. Angiogenesis, the formation of new blood vessels by endothelial cells, is impaired by the loss of rap1. Blood vessel growth into FGF2-containing Matrigel plugs was absent from rap1a-/- mice and aortic rings derived from rap1a-/- mice failed to sprout primitive endothelial tubes in response to FGF2 when embedded in Matrigel. Knocking down of either rap1a or rap1b in human micro-vascular endothelial cells (HMVECs) confirmed that Rap1 plays key roles in endothelial cell function. The knockdown of rap1a or 1b resulted in decreased adhesion to extracellular matrices and impaired cell migration. Rap1 deficient endothelial cells failed to form 3-D tubular structures when plated on Matrigel in vitro. The activation of ERK, p38, and Rac, important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either Rap1 protein was depleted. In U373 human glioblastoma multiforme cells, depletion of rap1b, but not rap1a drastically reduced tumor cell invasion by decreasing the activity of secreted matrix metalloproteinase 2 (MMP2). The adhesion of cells to the extracellular matrices collagen or fibronectin, but not to vitronectin, was decreased upon rap1b depletion. However, a mild increase in proliferation associated with elevation in ERK1/2, p38, Akt and ribosomal S6 protein activation was observed in cells depleted of either rap1a or rap1b. When an MEK1/2 inhibitor U0126 was used, the phosphorylation of p38, Akt and S6 were decreased, however, to various levels, suggesting complex regulatory pathways mediate Rap1 action in glioblastoma cells.

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Research in Medicine

A study was undertaken to determine whether cytogenetic abnormalities can be identified in an invasive melanoma cell population that has been selected in vitro out of a larger cell population of low invasive potential. The selecting agent was a denuded human amniotic membrane situated within Mega-Membrane Invasion Culture System chambers. Invasive cells were collected, grown, and harvested for cytogenetic analysis. Metaphases of these cells were examined for chromosomal abnormalities and for evidence of gene amplification in the form of double minute chromosomes. Invasive cell lines evinced changes in their degree of aneuploidy which were not seen in parental control lines of the same passage number. Significant karyotypic abnormalities identified in invasive cell lines were an increased dosage of chromosome 7 and multiple 1q translocation marker chromosomes. Double minute chromosomes were found in up to 18% of invasive cell metaphases examined and in 3% of parental controls. The incidence of double minutes was found to decrease as a function of passage number.

Cheung Suet Ling. / Thesis (M.Ph.)--Chinese University of Hong Kong. / Bibliography: leaves 110-135.

Thesis (M.Res.(Med.))--University of Hong Kong, 2009. / Includes bibliographical references (p. 105-124).

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 171-225). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 171-225). Also available on the Internet.