Both in situ (i.e. cell-based) and in vitro (i.e. chemical-based) assays are used to determine: 1.) the efficacy of drugs on certain cell types, 2.) the effects of artificially induced or introduced wild-type and mutant genes on biological pathways that are the target of certain drugs, 3.) interactions between proteins to sort out biological signaling pathways of relevance. Collaboration between cell biology and genomics is essential for determining gene expression patterns.

Cell culture: treatment of cells with drug to determine if they go from high density (left image) to low density (right image) or vice versa. Ideally proliferative cancer cells should stop growing and die off with treatment.

Micrographs of NMuMG cells

In addition to treatment with drugs, transfection assays are done in which either a defective gene or a functional gene is added to cells to determine effects on everything from morphology (e.g. epithelial-to-mesenchymal transition) to drug resistance (e.g. Gefitinib resistance).

Western blot: proteins are first analyzed by separating them on the basis of size through a gel subjected to an electrical field, then by changing the direction of this field and immobilizing them on a membrane. Antibodies specific to the protein(s) of interest are chemically linked to enzymes that catalyze a reaction that causes the location on the membrane to which the protein(s) have bound to fluoresce. Exposing X-ray film to this fluorescence leaves an indelible blot that confirms the existence of the protein(s).

Sample Western blot depicting quantity of ACTB in a dose curve

ELISA (Enzyme-Linked Immunosorbent Assay): This assay is fundamentally similar to Western blotting except that the enzyme linked to the antibodies produce a color change rather than fluorescence. The intensity of a soluble protein¿s color change is proportional to the amount of protein(s) of interest present.

IHC (Immunohistochemistry): This accomplishes many of the same goals as Western blotting, except the procedure is coupled with microscopy so that the location of a gene transcript, protein, or organelle can be visualized under a microscope. The procedure is considerably more challenging in that a tissue section (such as from a patient biopsy) is chemically treated to keep the cells from decomposing, then is embedded in wax, sliced and fixed to a microscope slide, and contrast stained. The cells then have to be permeabilized to the antibodies and special microscopes are required.

IHC micrograph of normal prostate tissue stained for CDH1


IHC micrograph of prostate tumor stained for CDH1

The cells are stained brown owing to the interaction between antibodies specific for CDH1 & a chemical substrate, DAB, that turns brown in the presence of an enzyme linked to the CDH1 antibody.