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Research in our laboratory is increasingly focusing on three major areas: First, what molecular and biochemical mechanisms are responsible for triggering cell senescence? Second, how does the stroma of a tumor, such as a carcinoma, influence the biology of the tumor as a whole? Third, how do cancer cells within a primary tumor acquire the ability to invade and metastasize?
Molecular and Biochemical Mechanisms of Cell Senescence. Senescence is a cell phenotype that has been defined in the context of in vitro culture of cells. Thus, after a certain number of cell divisions, many types of cultured cells will halt proliferation and enter into a non-growing state that is often termed replicative senescence. The precise molecular mechanisms that are responsible for triggering entrance into this state are complex and confounding.
Much evidence implicates suboptimal conditions of culture as a key element that is responsible for triggering entrance into senescence. Thus, if cells are cultured at a level of ambient oxygen that more closely approximates that within living tissues, in vitro proliferation is extended. Moreover, certain types of epithelial cells can be coaxed to proliferate longer if they are provided with stromal support, i.e., support by mesenchymal cells such as fibroblasts and the extracellular matrix that the latter construct.
One key source of senescence is clearly the reactive oxygen species (ROS) that are generated as a consequence of normal metabolism and may conspire with ambient oxygen to induce oxidized DNA bases. The most commonly observed of these bases is 8-oxo-dG. Thus, the accumulation of these oxidized bases in the DNA may overwhelm the ability of the DNA repair apparatus to restore normal DNA structure, resulting in turn in the accumulation of these bases in the DNA. This in turn might trigger a halt to cell proliferation that is induced by proteins such as p53 and p16INK4A.
In fact, this oxidation may alter the guanosine before it is incorporated into the DNA. This oxidation is normally countered by an enzyme, 8-oxo-dGTPase, normally termed MTH1, which hydrolyzes this oxidized nucleoside triphosphate in order to avoid its inadvertent incorporation into the DNA. We have found that knockdown of MTH1 expression induces rapid senescence and the accumulation of significant damage in the genomic DNA of cells. This provides us with a powerful tool to measure the influence of DNA oxidation on the entrance into cell senescence.
It remains unclear precisely how a halt to cell proliferation is imposed, once a cell has sensed extensive physiologic stress and incurred damage. A prominent effector of this halt is p16INK4A, which blocks the advance of cells through the G1 phase of the cell cycle. We have recently been exploring the possibility that its mechanism of activation, which has been elusive until now, depends on the activation of stress-activate protein kinases of the p38 family and are currently exploring how inhibition of these enzymes will affect the expression of the important p16INK4A cyclin-dependent kinase inhibitor.
