Results presented in this dissertation support a requirement for an integrative examination of cell cycle progression. DNA synthesis measurements must be correlated with protein synthesis- and amino acid transport measurements. The temporal coupling between DNA synthesis and protein synthesis may differ from growth factor to growth factor.
Comparative biochemical studies of the effect of rapamycin and wortmannin against TGF-β1-stimulated cell cycle entry showed that rapamycin, but not wortmannin, was an effective inhibitor of TGF-β1 stimulated DNA synthesis, protein synthesis, amino acid transport, and cell volume increase. Neither rapamycin nor wortmannin, affected TGF-β1-stimulated increase of p70 S6 kinase phosphorylation at the linker domain, Threonine 389. However, rapamycin inhibited phosphorylation of p70 S6 kinase at the pseudosubstrate domain. Future experiments are warranted to determine whether a constitutively phosphorylated pseudosubstrate domain preclude rapamycin inhibition of TGF-β1 signaling.
Unexpectedly, p27kip1 was not up-regulated by rapamycin against TGF-β1. Rapamycin alone did accumulate a significant level of p27kip1, although the significance of this remains to be determined. If p27kip1 is induced in stress (such as contact inhibition, serum arrest, or heat shock), then rapamycin may be imposing a stress signal to the cell. Preliminary data also suggested a role for stress-induced eIF-2α kinase in mediating TGF-β1 effects, although more data is required in order to make conclusions regarding a possible role. EIF-2α induction often correlated with stress, such as heat shock, serum starvation, or viral infection. Rapamycin and wortmannin accelerated TGF-β1 accumulation of eIF-2α and may suggest imposition of a stress-like response by these cell cycle inhibitors. Whether the induction of eIF-2α contribute to the delayed response characteristic of TGF-β1 or to its modulation of other early acting growth factors remains to be investigated.
Dissection of growth factor- and inhibitor signaling requires examination of global "networks" rather than of one isolated protein factor or pathway. Integration of biochemical processes with the regulation of possible target components is important to uncover the pervasiveness of an extracellular signaling molecule. Inhibition of DNA synthesis may result from inhibition of amino acid transporters, which prevents accumulation of available substrates for protein synthesis. This deprivation in turn prevents achievement of critical mass and volume. A weak or ineffective agent against any given growth factor may not be affecting key macromolecular networks or may not be an irreversible inhibitor for these processes.
I sincerely thank my mentors Dr. Charles E. Wenner and Dr. Kirk J. Leister for their inspiration and unwavering support of my graduate research effort. Dr. Wenner is a wonderful teacher who values the information that everyone offers - regardless of whether you are a new student or a seasoned research scientist. Dr. Leister is an insightful scientist whose enthusiasm pulled me through times of discouragement when experiments didn't work out. As a student researching off campus, I appreciate the valiant effort that Drs. Wenner and Leister made to help me connect academia and industry - I will miss the discussions that we had through conference calling, faxes, and emails.
I am also grateful to Dr. Richard Dicioccio, Dr. Alan Kinniburgh, and Dr. Tom Kalman for generously giving up their time to provide me with valuable suggestions for my dissertation. I want to thank Dr. Robert Donaldson for helpful discussions and insight. Dr. Edward McNiff, Dr. Tedd Collins, and Dr. Stephen Gozo have been a source of encouragement and I appreciate the time and advice they offered. Sheryl Hood, thank you for your friendship. When I was buried deep in the barracks of graduate research, Sheryl always found the time to check how I was doing and to point out free food in the office when there were some!
Finally, I thank my husband, Cass, for teaching me that "Quitting is not an option."