GENETIC METHODS FOR DETECTING GAP JUNCTION COMMUNICATION


Year of Award:
2005
Award Type:
R21
Project Number:
CA111993
RFA Number:
RFA-CA-05-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
WYMAN, ROBERT J
Other PI or Project Leader:
N/A
Institution:
YALE UNIVERSITY
There is strong evidence that gap junctional communication (GJC) is a regulator of cell proliferation and that interruption of this is one of the steps in the malignant transformations of cancer. Gap junctions occur in all animal species and in most tissues from extremely early in development: the eight cell stage in mice, gastrulation in Drosophila and the two-cell stage in nematodes. Yet gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. Gap junctions are difficult to detect. The standard way to determine whether cells are GJ coupled is to inject dye into one cell and see if it spreads to neighboring cells. In vivo this requires microinjection, which limits the technique to large and unusually accessible cells. We propose to develop a molecular biological method for the in vivo detection of both enduring and transient GJC without the need for intracellular injection. In the simplest version of the technology, transgenic animals will be made with tissue-specific expression of b-galactosidase (b-gal). The intact animal will be injected with a b-gal substrate (e.g., X-gal) which is taken up by cells and is hydrolyzed to a small colored reporter molecule. b-Gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing b-gal can be detected with antibodies; any cell not expressing b-gal, but filled with the reporter color must have received its color via GJC. The technology will be validated for uniform cells of a single tissue type, different cell types in a complex tissue, in gap junctions made from a variety of GJ proteins, and in heterotypic junctions made from two different GJ proteins. Quantitative measures will be taken of in-animal and across-animal statistical reliability, extent of spread, and spatial and temporal resolution of the method. Aside from b-gal and X-gal as an enzyme-substrate pair, the method will be validated using other b-gal substrates. Another similar method will be tested using tissue specific expression of transporters to load the presynaptic cells and a detection method for trans-junctional passage. The method will be applied to tumors to assess GJ coupling between tumor cells, between tumor cells and normal cells before and after tumor induction. GJ proteins will be expressed in tumors and the method used to assess GJ coupling after expression and to determine whether tumor growth has been suppressed.