A cloned metastatic variant of a murine tumor cell line, Lewis lung carcinoma (LLC(C3), was synchronized by double exposure to thymidine (5 x 10-4 M) at G1/S boundary. The effects of Prostaglandin (PG)E1, PGE2, PG synthetase inhibitors, and an analog of cAMP (dbut-cANP) on the life cycle of synchronized LLC(C3) cells were examined. When added to cells in G1 phase, PGE1 and dbut-cAMP enhanced 3H-thymidine uptake during S phase. Both of these agents suppressed S phase when they were added to cells entering S phase. The addition of PGE2 to LLC(C3) cells in G1 phase had no effect on S phase of the synchronized LLC(C3) cells. Prostaglandin E2 suppressed S phase when it was added at the beginning of S phase. A low concentration of PGE2 was more suppressive to S phase than was a high concentration. Indomethacin, an irreversible PG synthetase inhibitor, and piroxicam, a reversible PG synthetase inhibitor, suppressed S phase of synchronized LLC(C3) cells. These results suggest that prostaglandins can regulate the cell cycle of LLC(C3) cells through modulation of cAMP levels.

Cellular transformation is accomplished in vitro through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in pathways that normally operate to control proliferation. Activation of genes that code for growth factors, their receptors, and/or molecules essential in the transduction of signals from the cell surface to the nucleus are all potential mechanisms by which tumor cells could establish a selective growth advantage over normal cells. This dissertation addresses the question of what oncogenic mechanisms are important in the development and progression of human melanoma. These studies show that melanoma growth is regulated by endogenous substances produced by the melanoma cells themselves (autocrine stimulation), as well as by exogenous substances supplied by neighboring cells and platelets (paracrine stimulation). These factors work to drive the expression of the transformed phenotype for melanoma as evidenced by induction of serum-free soft agar growth. Human platelets were found to the the richest source of paracrine growth promoters. The factor from human platelets was characterized and partially purified. Melanoma cells respond to this 60,000 molecular weight, disulfide-bond-containing protein in colony formation assays. In addition, the protein has endothelial cell growth factor activity. Purified fractions which promoted optimal colony formation for human melanoma cells also maximally stimulated monolayer growth of bovine aortic endothelial cells, while melanocytes were nonresponsive. This implies that melanoma cells are expressing receptors for a protein which plays no known or apparent role in the normal growth of melanocytes. Melanoma cells are sensitive to growth regulatory molecules of autocrine and paracrine nature. This dissertation provides clues to the genetic lesions which have occurred in these melanoma cells to influence their proliferation. The aberrations appear to reside in those genes important in growth factor pathways at the level of endogenous production and misguided response to exogenous factors through receptor expression. We can not hope to fully inhibit the proliferation of tumor cells until we identify and understand those forces which drive their growth. These studies have increased our knowledge of those signals which stimulate melanoma cellular proliferation, and thus provide insight into important therapeutic targets.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Progress in understanding cancer progression has been hampered over the years by the different types of mutations present and irregular changes of gene regulation associated with any given cancer. In this work, molecular interactions between TALI, LMO1, and decorin were investigated. Numerous studies have shown that ectopic expression of decorin protein induced growth suppression in neoplastic cells of various histogenic origins. Furthermore, ectopic expression of TAL1 and LMOI oncoproteins has been shown to occur in approximately 50% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). It was of interest then to determine the preventive or interactive role decorin played with the oncogenic activity of TAL I and LMO1. In this investigation, decorin was introduced into a murine T-cell line (AKR-DP-603) through the use of the mammalian expression vector pcDNA3.1 (-). This particular cell line was previously engineered to express TALI and LMO1. Protein expression patterns in all cell populations were analyzed using the Western blot technique and a proteoglycan with a molecular weight of 100 kDa before chondroitinase ABC treatment and a core protein of55 kDa after treatment with chondroitinase ABC was seen. This finding is significant since it implies that the pcDNA3. 1(-) vector containing decorin cDNA was present, and the corresponding decorin peptides were expressed in both cytoplasmic and nuclear extracts. Furthermore, Northern blot analysis was performed on total RNA extracts to determine the transcriptional state of endogenous decorin rRNA, as well as exogenously introduced decorin. Northern blot analysis revealed no decorin-specific mRNA transcripts from the various cell populations. This result did not imply a lack of possible regulatory effect on protein and mRNA levels of TALL and LMOI by decorin. Finally, cell growth assays were performed on all cell populations and cell counts were used to assess the growth pattern of each population after serum withdrawal. The results show possible growth suppressive effects of decorin on TAL1 and LMOI expressing cells. Results obtained from this study shed further light on the molecular interactions influencing T-ALL and may also help in the design of potentially beneficial cancer treatments using decorin. / Department of Biology

The aim of this study was to examine the claim that a single, mutant oncogene can transform NIH 3T3 mouse fibroblasts into transformed, tumorigenic cells, acting in a genetically dominant fashion. A c.Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt, which encodes the enzyme XPRT (xanthine-guanine phosphoribosyl transferase). This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells which had incorporated the plasmid were selected by growth in mycophenolic acidcontaining medium, to which gpt confers resistance. A number of clonal lines were established and their tumorigenicity tested. Tumour cell lines derived from these transfectants were back-selected using 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate, to isolate clones which no longer contained functional pSV2gptEJ sequences. Six sub-clones which did not express detectable levels of the ras oncogene product, p21^H.ras , were obtained. All were judged to be less transformed than the transfected parent cells: they appeared morphologically normal, were more serum-sensitive, showed clear saturation densities and were more anchorage-dependent. Three of these sub-clones were found to be tumorigenic at all sites tested. Cytological examination of the NIH 3T3 transfectants revealed that significant perturbation of their chromosome complement accompanied transfection. The transfection process, in the absence of DNA or with pSV2gpt alone, was found to be capable of transforming NIH 3T3 cells. Finally, a brief investigation of the effect of a "functional EJ-ras gene upon the differentiated phenotypes of these cell lines was attempted by comparing their ability to produce an extracellular matrix.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy