1985 — 1988 |
Bertram, John S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Interactions During Malignant Transformation @ University of Hawaii At Manoa
In the C3H/10T1/2 CL8 cell line we have demonstrated that the expression of cells malignantly transformed by chemical carcinogens can be repressed by contact of malignant cells with nontransformed cells and that this inhibition can be modulated by serum concentration and by inhibitors of phosphodiesterase. It is the major aim of this research to examine in greater detail correlations between alterations in cyclic nucleotide levels and alterations in growth inhibition and to examine how this intracellular communication occurs in vitro. A good correlation has been achieved between elevations of intracellular cAMP levels and growth inhibition of malignant cells in mixed cultures, and these cells have been shown to be in metabolic communication. Using mixed culture of carcinogen-initiated cells and parental 10T1/2 cells, the colony size attained by these initiated cells was shown to directly determine the probability for transformation of these cells, indicating the central role played by cell interactions in expression of malignancy. Probing of junctional communication using dye-injection procedures has shown that cAMP elevations cause enhancement of communication between 10T1/2 cells and their transformed counterparts. The extent of this enhancement correlates well with the extent of growth inhibition. Transformed cells resistant to growth inhibition fail to show enhancement of junctional communication. The role of cAMP will also be probed using kinase-deficient cell mutants. The relevance of the observed in vitro effects to the in vivo situation will be investigated by studying the growth rate of transplantable tumors, of differing sensitivity to growth inhibition in vitro, in host animals treated with drugs which modify the in vitro response. We will also extend these studies to human fibroblasts in culture which we have shown to secrete an inhibitory factor into the medium. We will characterize this factor biologically and biochemically. These studies, in addition to providing basic information on growth control mechanisms, will aid in the development of more reliable and quantitative in vitro assays for chemically induced malignant transformation in mouse and human cell lines. (A)
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0.91 |
1985 — 1993 |
Bertram, John S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Inhibition of in Vitro Transformation by Retinoids @ University of Hawaii At Manoa
The retinoids, a group of natural and synthetic analogues of Vitamin A, have been shown to possess anticarcinogenic properties in vivo. We have recently demonstrated that natural retinoids will completely eliminate the expression of carcinogen induced morphological transformation in cultured C3H/10T1/2CL8 cells. This proposal seeks to obtain a better understanding of the mechanism whereby retinoids can inhibit transformation. This proposal is divided into 2 sections. In section 1 the effects of retinoids on transformation will be examined in detail, attempts will be made to isolate initiated cells, the relationship between tumor promoters and retinoids will be investigated, and the effects of the retinoid content of feral serum studied. In section 2, it is planned to investigate the biochemical aspects of retinoid activity. Using parental C3H/10T1/2 cells, initiated cells and transformed cells, we proposed to study the effects of retinoids on a. the biosynthesis of membrane glycolipids and glycoproteins using labeled membrane precursors, and b. the behavior of cell membranes using the fluorescence recovery after photobleaching technique to measure membrane mobility, and by a modification of the technique membrane fluidity. Finally, we propose to measure the levels of retinoic acid binding protein and of retinol binding protein in the cytosol of parental, initiated and transformed cell lines.
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0.91 |
1986 — 1987 |
Bertram, John S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Interactins During Malignant Transformation @ University of Hawaii At Manoa
It is proposed to investigate the means whereby growth-inhibited non-transformed fibroblasts are able cause growth inhibition of neoplastically transformed fibroblasts. The biological model used is the 10T1/2 cell culture system which closely reflects events occurring in vivo. In this system cAMP enhances cell interactions leading to growth control. The goal is to better understand the physiological control of cell division at the cell population level so that growth control process can be manipulated in normal and neoplastic cells. The aims of this proposal are to critically examine the role of gap-junctions in transferring growth regulatory information between cells, and to amass evidence for or against the cAMP as the chemical messenger of growth control signals. The first phase of the study will be to determine in neoplastic cells sensitive or resistant to growth inhibition by contacting normal cells, whether cAMP can be transferred to the neoplastic cell and whether the extent of this transfer correlates with the extent of growth inhibition. To detect transferred cAMP we shall utilize a sensitive micro-fluorometric assay for the detection of free catalytic units liberated by the action of cAMP on cAMP dependent protein kinase. We will next examine alterations in protein phosphorylation in general and of gap junctional components in particular by polyacrylamide gel electrophoresis, in homogeneous cultures of normal 10T1/2 cells in which drug induced cAMP elevation also enhances growth control. Correlations between enhanced growth control, protein phosphorylation and gap junctional permeance, measured by a dye-injection technique, will be examined, and the effects of agents blocking communication such as the tumor promoters evaluated. Using the information gained in studies of normal cells, the question of whether similar events occur in growth inhibited neoplastic cells will be examined. Using fluorescent activated cell sorting, neoplastic cells will be separated from normal cells, their position in the cell cycle determined and the phosphorylation state of marker proteins determined. A secondary question to be addressed is whether carcinogen-initiated cells when growth arrested by normal cells are in a biological and biochemical state corresponding to the initiated or neoplastic state. By these studies of routes and means of informational transfer between cells, it is hoped to help identify defects that result in noeplasia. This research should narrow the search for the function of oncogene products, and suggest ways to correct these defects.
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0.91 |
1998 — 2000 |
Bertram, John S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemoprevention by Carotenoid Derivatives @ University of Hawaii At Manoa
DESCRIPTION: There is persuasive epidemiological evidence that consumption of dietary carotenoids, particularly beta-carotene, reduces cancer risk at many sites. Laboratory studies also support a protective role for beta-carotene and many other dietary carotenoids. Yet clinical intervention trials with purified beta-carotene have failed to show protection. This proposal seeks to resolve this paradox and achieve a more complete understanding of the pharmacology and molecular biological actions of two major dietary carotenoids, lutein and lycopene. Dr. Bertram and colleagues have identified two carotenoid derivative formed after ingestion of the parent compounds that possess biological activity. This application will compare and contrast the activity of these derivatives with that of the parent compound for their ability to 1) inhibit neoplastic transformation in a model animal cell system, 2) alter the differentiation state of human keratinocytes in organotypic culture, 3) alter the expression of connexin43, a potential intermediate marker of response, in mouse and in human keratinocytes. It is also proposed to examine the molecular mechanism whereby these carotenoid derivatives are anti-proliferative by use of flow cytometry and antibodies to cell cycle regulatory proteins. Finally, the project will examine the molecular mode of action of these derivatives by examining 1) their ability to activate nuclear receptors by monitoring transcriptional activation of genes controlled by known responsive elements after transfection with reporter constructs containing these responsive elements together with the appropriate nuclear receptor, 2) by use of ligand-specific antagonists. Should these studies indicate that derivatives of dietary carotenoids, rather than the parent carotenoids, are responsible for many of the biological effects attributed to these compounds, new approaches to cancer prevention would be indicated and a route opened to develop new classes of biologically active compounds.
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0.91 |