In the previous period of support for this project we characterized an intracellular TGFalpha autocrine loop. Cells with an intracellular TGFalpha express high amounts of intracellular precursor TGFalpha which has an abnormally slow rate of processing relative to other cells. Consequently, they continue to express high intracellular levels of TGFalpha in non-dividing, non-cycling states such as the quiescent state generated by nutrient and growth factor deprivation. The intracellular location of the autocrine activity results in the inaccessibility of TGFalpha autocrine activity in these cells to TGFalpha neutralizing antibodies and EGFtau blocking antibodies. However, constitutive TGFalpha anti-sense vector expression results in the acquisition of EGF dependency. Anti-sense transfected cells do not express high levels of intracellular TGFalpha and show decreased TGFalpha expression and transcription in non- dividing states. They are similar to well-differentiated, growth factor dependent colon carcinoma cells in this regard as these cells also require exogenous EGF for clonal growth and release from quiescence. They also show low levels of intracellular TGFalpha and reduced expression of TGFalpha in non-dividing states. These results have led us to hypothesize that TGFalpha acts as an autocrine factor to aid in the initiation of clonal growth or the escape from quiescence in colon carcinoma cells and that its internal location in some cells provides a growth regulatory advantage due to inappropriately high expression in non-cycling growth states such as quiescence. It is further hypothesized that the factor does not have a stimulatory function for exponentially growing cells. Moreover, it has recently been found that other EGF related peptides, amphiregulin (AR), and cripto (CR), are also autocrine regulatory factors in colon cancer. We hypothesize that these factors will interrelate with TGFalpha in terms of control of expression and temporal control of function. These hypothesizes will be tested by: 1. Determining the autocrine function of TGFalpha in exponential and non- dividing growth regulatory states. 2. Determine interrelationship of control of expression among TGFalpha, AR and CR. 3. Determine the autocrine function of AR. 4. Determine the autocrine function of CR.
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