The regulation of mTORC2 kinase activity and complex integrity

Growth factor signaling promotes anabolic processes via activation of the PI3K-Akt kinase cascade. Deregulation of the growth factor-dependent PI3K-Akt pathway was implicated in tumorigenesis. Akt is an essential serine/threonine protein kinase that controls multiple physiological functions such as cell growth, proliferation, and survival to maintain cellular homeostasis. Recently, the mammalian Target of Rapamycin Complex 2 (mTORC2) was identified as the main Akt Ser-473 kinase, and Ser-473 phosphorylation is required for Akt hyperactivation. However, the detailed mechanism of mTORC2 regulation in response to growth factor stimulation or cellular stresses is not well understood. In the first project, we studied the regulation of the mTORC2-Akt signaling under ER stress. We identified the inactivation of mTORC2 by glycogen synthase kinase-3β (GSK-3β). Under ER stress, the essential mTORC2 component, rictor, is phosphorylated by GSK-3β at Ser-1235. This phosphorylation event results in the inhibition of mTORC2 kinase activity by interrupting Akt binding to mTORC2. Blocking rictor Ser-1235 phosphorylation can attenuate the negative impacts of GSK-3β on mTORC2/Akt signaling and tumor growth. Thus, our work demonstrated that GSK-3β-mediated rictor Ser-1235 phosphorylation in response to ER stress interferes with Akt signaling by inhibiting mTORC2 kinase activity. In the second project, I investigated the regulation of the mTORC2 integrity. We found that basal mTOR kinase activity depends on ATP level, which is tightly regulated by cell metabolism. The ATP-sensitive mTOR kinase is required for SIN1 protein phosphorylation and stabilization. SIN1 is an indispensable subunit of mTORC2 and is required for the complex assembly and mTORC2 kinase activity. Our findings reveal that mTOR-mediated phosphorylation of SIN1 is critical for maintaining complex integrity by preventing SIN1 from lysosomal degradation. In sum, our findings verify two distinct mTORC2 regulatory mechanisms via its components rictor and SIN1. First, GSK-3β-mediated rictor Ser-1235 phosphorylation results in mTORC2 inactivation by interfering its substrate binding ability. Second, mTOR-mediated Ser-260 phosphorylation of SIN1 preserves its complex integrity. Thus, these two projects provide novel insights into the regulation of mTORC2.

Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention

Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated b y mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure shows that different amino acids substituted at the codon 12 position influences how KRas interacts with its effecters. In the absence of a direct inhibitor of mutant KRas several agents have recently entered clinical trials alone and in combination directly targeting two of the common downstream effecter pathways of KRas, namely the Mapk pathway and the Akt pathway. These inhibitors were evaluated for efficacy against different KRAS activating mutations. An isogenic panel of colorectal cells with wild type KRas replaced with KRas G12C, G12D, or G12V at the endogenous loci differed in sensitivity to Mek and Akt inhibition. In contrast, screening was performed in a broad panel of lung cell lines alone and no correlation was seen between types of activating KRAS mutation due to concurrent oncogenic lesions. To find a new method to inhibit KRAS driven tumors, siRNA screens were performed in isogenic lines with and without active KRas. The knockdown of CNKSR1 (CNK1) showed selective growth inhibition in cells with an oncogenic KRAS. The deletion of CNK1 reduces expression of mitotic cell cycle proteins and arrests cells with active KRas in the G1 phase of the cell cycle similar to the deletion of an activated KRas regardless of activating substitution. CNK1 has a PH domain responsible for localizing it to membrane lipids making KRas potentially amenable to inhibition with small molecules. The work has identified a series of small molecules capable of binding to this PH domain and inhibiting CNK1 facilitated KRas signaling.

Galectin-3 Enhances the Malignant Melanoma Phenotype by Regulating Autotaxin

In melanoma patient specimens and cell lines, the over expression of galectin-3 is associated with disease progression and metastatic potential. Herein, we have sought out to determine whether galectin-3 affects the malignant melanoma phenotype by regulating downstream target genes. To that end, galectin-3 was stably silenced by utilizing the lentivirus-incorporated small hairpin RNA in two metastatic melanoma cell lines, WM2664 and A375SM, and subjected to gene expression microarray analysis. We identified and validated the lysophospholipase D enzyme, autotaxin, a promoter of migration, invasion, and tumorigenesis, to be down regulated after silencing galectin-3. Silencing galectin-3 significantly reduced the promoter activity of autotaxin. Interestingly, we also found the transcription factor NFAT1 to have reduced protein expression after silencing galectin-3. Electrophoretic mobility shift assays from previous reports have shown that NFAT1 binds to the autotaxin promoter in two locations. ChIP analysis was performed, and we observed a complete loss of bound NFAT1 to the autotaxin promoter after silencing galectin-3 in melanoma cells. Mutation of the NFAT1 binding sites at either location reduces autotaxin promoter activity. Silencing NFAT1 reduces autotaxin expression while over expressing NFAT1 in NFAT1 negative SB-2 melanoma cells induces autotaxin expression. These data suggest that galectin-3 silencing reduces autotaxin transcription by reducing the amount of NFAT1 protein expression. Rescue of galectin-3 rescues both NFAT1 and autotaxin. We also show that the re-expression of autotaxin in galectin-3 shRNA melanoma cells rescues the angiogenic phenotype in vivo. Furthermore, we identify NFAT1 as a potent inducer of tumor growth and experimental lung metastasis. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

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. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

HETEROGENEITY IN VIRUS EXPRESSION, TUMORIGENICITY, AND IN VITRO GROWTH PROPERTIES OF DRUG-RESISTANT CLONES OF AN RIII MOUSE MAMMARY TUMOR CELL LINE

Four 8-azaguanine (AG)-resistant and 5-bromodeoxyuridine (BUdR)-resistant clones of a mouse mammary adenocarcinoma cell line, RIII 7387, were developed and analyzed for their tumorigenic properties, in vitro characteristics, and virus expression. These characteristics were analyzed for relationships of any of the cellular parameters and the ability of these lines to produce tumors in syngeneic animals.^ The results of this study demonstrated that the parental line consists of a heterogeneous population of cells. Doubling times, saturation densities, and 2-deoxy-D-glucose uptake varied between sublines. In addition, while all sublines were found to express both B-type and C-type viral antigenic markers, levels of the major B-type and C-type viral proteins varied in the subclones. The sublines also differed markedly in their response to the presence of dexamethasone, glutathione, and insulin in the tissue culture medium.^ Variations in retrovirus expression were convirmed by electron microscopy. Budding and extracellular virus particles were seen in the majority of the cell lines. Virus particles in one of the BUdR-resistant lines, BUD9, were found however, only in inclusions and vacuoles. The AG-resistant subline AGE11 was observed to be rich in intracytoplasmic A particles. The examination of these cell lines for the presence of retroviral RNA-dependent DNA polymerase (RT) activity revealed that some B-type RT activity could be found in the culture fluid of most of the cell lines but that little C-type RT activity could be found suggesting that the C-type virus particles expressed by these RIII clones contain a defective RT.^ Tumor clones also varied in their ability to form tumors in syngeneic RIII mice. Tumor incidence ranged from 50% to 100%. The majority of the tumors regressed within 30 days post infection.^ Statistical analysis indicated that while these clones varied in their characteristics, there was no correlation between the ability of these cell lines to form tumors in syngeneic mice and any of the other characteristics examined.^ These studies have confirmed and extended the growing evidence that tumors, regardless of their natural origin, consist of heterogeneous subpopulations of cells which may vary widely in their in vitro growth behavior, their antigenic expression, and their malignant properties. ^

NEW TARGET GENES FOR TUMOR SUPPRESSORS p53 AND p73 IN REGENERATING LIVER

The p53-family of proteins regulates expression of target genes during tissue development and differentiation. Within the p53-family, p53 and p73 have hepatic-specific functions in development and tumor suppression. Despite a growing list of p53/p73 target genes, very few of these have been studied in vivo, and the knowledge regarding functions of p53 and p73 in normal tissues remains limited. p53+/-p73+/- mice develop hepatocellular carcinoma (HCC), whereas overexpression of p53 in human HCC leads to tumor regression. However, the mechanism of p53/p73 function in liver remains poorly characterized. Here, the model of mouse liver regeneration is used to identify new target genes for p53/p73 in normal quiescent vs. proliferating cells. In response to surgical removal of ~2/3 of liver mass (partial hepatectomy, PH), the remaining hepatocytes exit G0 of cell cycle and undergo proliferation to reestablish liver mass. The hypothesis tested in this work is that p53/p73 functions in cell cycle arrest, apoptosis and senescence are repressed during liver regeneration, and reactivated at the end of the regenerative response. Chromatin immunoprecipitation (ChIP), with a p73-antibody, was used to probe arrayed genomic sequences (ChIP-chip) and uncover 158 potential targets of p73-regulation in normal liver. Global microarray analysis of mRNA levels, at T=0-48h following PH, revealed sets of genes that change expression during regeneration. Eighteen p73-bound genes changed expression after PH. Four of these genes, Foxo3, Jak1, Pea15, and Tuba1 have p53 response elements (p53REs), identified in silico within the upstream regulatory region. Forkhead transcription factor Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative, cellular proliferation. p53 and p73 bind a Foxo3 p53RE and maintain active expression in quiescent liver. During liver regeneration, binding of p53 and p73, recruitment of acetyltransferase p300, and an active chromatin structure of Foxo3 are disrupted, alongside loss of Foxo3 expression. These parameters of Foxo3 regulation are reestablished at completion of liver growth and regeneration, supporting a temporary suspension of p53 and p73 regulatory functions in normal cells during tissue regeneration.