Senescent cells and terminally differentiated cells are similar in that neither are able to respond to growth stimuli. Aged cells and differentiated cells share a reduced capacity to express enzymes involved in DNA synthesis. A mechanism must exist for suppressing these enzyme activities in non-replicating cells. To investigate this mechanism I have been studying the regulation of the replicative enzyme thymidine kinase (TK). We have established that TK enzyme levels and TK mRNA levels decline as euploid cells become senescent. A similar decline in TK mRNA occurs during terminal muscle differentiation. Isolation of a TK- mouse myoblast line has facilitated analysis of the regulatory mechanism. Myoblasts transformed with cloned cellular TK genes regulate TK activity normally during differentiation. Myoblasts transformed with cloned viral TK genes do not. These results imply that the cloned cellular TK gene contains sufficient information to allow for its appropriate regulation. Subsequent experiments, in which the cellular TK gene was systematically modified in vitro, established that the regulatory information is located between the translation start and stop sites. Experiments described in this proposal are designed to precisely locate the cis-acting regulatory information within this intragenic region. In addition, the level at which this regulatory information is acted upon will be established by determining the rate of TK gene transcription in nuclei isolated from proliferating and differentiated cells. TK transcription rates will also be determined in senescent and early passage euploid cells to establish the relevance of our findings in the muscle cell system to cellular aging. A thorough understanding of the mechanism linking mitogenic stimulation and induction of replicative enzymes will elucidate the molecular basis for the limited replicative potential of eucaryotic cells.
