The p53 tumor suppressor pathway in culture and {\it in vivo\/}

p53 functions as a tumor suppressor through its ability to initiate either growth arrest or apoptosis in cells which have sustained DNA damage. p53 elicits these cellular phenotypes through its biochemical function as a transcriptional activator. By inducing the expression of a battery of target genes, p53 is able to prevent the propagation of cells with damaged DNA. However, the genes transcriptionally induced by p53 which have been identified to date do not fully explain p53 function. p53 has been demonstrated to activate genes involved in cell cycle inhibition, apoptosis and cell proliferation. The reasons for simultaneous activation of p53 targets with disparate, opposing functions are not clear, but may be due to the use of transformed cell lines in previous experiments. In the studies presented in this thesis, the pathway of p53 tumor suppression has been studied in detail in two systems chosen for their relevance to the natural cell environment. One utilizes a normal, unaltered cultured cell system; the other the whole mouse. In order to better understand the role of the known p53 targets in effecting p53 function in normal cells, early rat embryo fibroblasts were irradiated with ultraviolet light to induce DNA damage. It was discovered that p53 protein levels increased in response to irradiation. The known targets of p53, namely, $p21\sp{WAF1/CIP1},\ mdm2,\ cyclin\ G,$ and bax, were shown for the first time to have a differential temporal induction. The growth suppressor $p21\sp{WAF1/CIP1}$ was induced first, followed by cyclin G then mdm2, which is involved in proliferation through its inactivation of p53, and finally, the apoptosis promoter, bax. These findings indicated that p53 activates its target genes in a manner to allow maximum effectiveness of target function. The rat embryo fibroblasts were shown to undergo apoptosis 24 h after irradiation. Additionally, investigation of these cells for cell cycle alterations demonstrated a brief arrest in G1. In the second study, thymocytes from mice with wild type p53 were shown to undergo apoptosis and activate p53 target genes upon ionizing radiation treatment, while thymocytes from mice deficient in p53 could not. The p53 target genes mdm2 and fas were tested in vivo for their ability to mediate p53-regulated apoptosis, and were found dispensible for that cellular function. Therefore, the p53 targets identified to date do not fully explain the ability of p53 to function as a tumor suppressor. Potentially, functional redundancy between the known targets would account for the data seen in these experiments. Additionally, identification of additional target genes should add further understanding of the p53 pathway of tumor suppression. ^

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^