Examples of low dose radiation studies

To determine whether radiation promotes aberrant ECM interactions, we examined integrin and E-cadherin localization in preneoplastic human cells surviving radiation. Integrins are a family of epithelial receptors for the ECM, while E-cadherin maintains normal cell-cell interactions and architecture. We used the HMT-3522 (S1) human breast cell line cultured within a reconstituted ECM. These cells are genomicaly unstable but phenotypically normal in that they recapitulate normal mammary architecture in the form of a multicellular, three-dimensional acinus. These clusters express integrins in a polarized fashion and develop an organized ECM over the course of 7-10 days in culture.

We examined the consequences of exposing these cells to ionizing radiation and a protein modifier known as TGF- as shown in Figure . Antibodies to E-cadherin, beta 1 integrin or alpha 6 integrin were detected using a green fluorescent label while nuclei were counterstained with a red fluorescent DNA dye. These were imaged using confocal fluorescence microscopy and were recorded using a 12-bit CCD camera. Cells that survived either 2 Gy or TGF-(400 pg/ml) exhibited decreased beta 1 or alpha 6 integrin localization, respectively. However when cells were exposed to both radiation and TGF-, additional perturbations were noted. The clusters were disorganized, did not polarize the integrins at the cell surface and failed to express E-cadherin, indicative of a lack of structural organization. Statistical analysis of treated and untreated samples indicates that TGF-beta and radiation treatment alters symmetric organization of acinus. Organization is quantified by representing the projection of each colony by an ellipse followed by measuring deviation of each nuclei from the ellipse. Processed images show the result of ellipsoidal and hyperquadratic representation of each nucleus in the image.

Experimental protocol for studying the 
effect of low dose radiation and TGF-beta
Treated sample Untreated sample
Hyper-rep Treated sample Hyper-rep Untreated sample

A number of experiments have been designed to understand the impact of cell-cell communications on the organization of cultured colonies. One experiment deals with a type of gap junction known as connexing-42. However, study of cell-cell communication is often three dimensional. The 2D segmentation technique has been extended to 3D for detecting nuclear formation. The nuclear formation is subsequently used as context to localize protein complexes associated with gaps junctions that modulate transfer of molecules between cells. Preliminary results indicate significant reduction of gaps junctions in treated colonies, which contributes to their abnormal growth state. The movie shows the structure of a 3D cultured colony and corresponding gap juctions in small green spheres.

Click here to see a movie of nuclear structure in colony and connexin expression.