Genetic analysis of factors regulating mesenchymal cell fates

Formation of cartilage and bone involves sequential processes in which undifferentiated mesenchyme aggregates into primordial condensations which subsequently grow and differentiate, resulting in morphogenesis of the adult skeleton. While much has been learned about the structural molecules which comprise cartilage and bone, little is known about the nuclear factors which regulate chondrogenesis and osteogenesis. MHox is a homeobox-containing gene which is expressed in the mesenchyme of facial, limb, and vertebral skeletal precursors during mouse embryogenesis. MHox expression has been shown to require epithelial-derived signals, suggesting that MHox may regulate the epithelial-mesenchymal interactions required for skeletal organogenesis. To determine the functions of MHox, we generated a loss-of-function mutation in the MHox gene. Mice homozygous for a mutant MHox allele exhibit defects of skeletogenesis, involving the loss or malformation of craniofacial, limb and vertebral skeletal structures. The affected skeletal elements are derived from the cranial neural crest, as well as somitic and lateral mesoderm. Analysis of the mutant phenotype during ontogeny demonstrated a defect in the formation or growth of chondrogenic and osteogenic precursors. These findings provide evidence that MHox regulates the formation of preskeletal condensations from undifferentiated mesenchyme. In addition, generation of mice doubly mutant for the MHox and S8 homeobox genes reveal that these two genes interact to control formation of the limb and craniofacial skeleton. Mice carrying mutant alleles for S8 and MHox exhibit an exaggeration of the craniofacial and limb phenotypes observed in the MHox mutant mouse. Thus, MHox and S8 are components of a combinatorial genetic code controlling generation of the skeleton of the skull and limbs. ^

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

TUMOR PROGRESSION: ANALYSIS OF THE INSTABILITY OF THE METASTATIC PHENOTYPE, SENSITIVITY TO RADIATION AND CHEMOTHERAPY (PHENOTYPIC DRIFT, BREAST CANCER)

The major complications for tumor therapy are (i) tumor spread (metastasis); (ii) the mixed nature of tumors (heterogeneity); and (iii) the capacity of tumors to evolve (progress). To study these tumor characteristics, the rat 13762NF mammary adenocarcinoma was cloned and studied for metastatic properties and sensitivities to therapy (chemotherapy, radiation and hyperthermia). The cell clones were heterogeneous and no correlation between metastatic potential and therapeutic sensitivities was observed. Further, these phenotypes were unstable during passage in vitro; yet, the changes were clone dependent and reproducible using different cryoprotected cell stocks. To understand the phenotypic instability, subclones were isolated from low and high passage cell clones. Each subclone possessed a unique composite phenotype. Again, no apparent correlation was seen between metastatic potential and sensitivity to therapy. The results demonstrated that (1) tumor cells are heterogeneous for multiple phenotypes; (2) tumor cells are unstable for multiple phenotypes; (3) the magnitude, direction and time of occurrence of phenotypic drift is clone dependent; (4) the sensitivity of cell clones to ionizing radiation (gamma or heat) and chemotherapy agents is independent of their metastatic potential; (5) shifts in metastatic potential and sensitivity to therapy may occur simultaneously but are not linked; and (6) tumor cells independently diverge to form several subpopulations with unique phenotypic profiles. ^

Loss of activator protein-2alpha results in overexpression of the thrombin receptor and correlates with the malignant phenotype of human melanoma

Increasing evidence demonstrates that the thrombin receptor (protease activated receptor-1, PAR-1) plays a major role in tumor invasion and contributes to the metastatic phenotype of human melanoma. We demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. The promoter of the PAR-1 gene contains multiple putative AP-2 and Sp1 consensus elements. We provide evidence that an inverse correlation exists between the expression of AP-2 and the expression of PAR-1 in human melanoma cells. Re-expression of AP-2 in WM266-4 melanoma cells (AP-2 negative) resulted in decreased mRNA and protein expression of PAR-1 and significantly reduced the tumor potential in nude mice. ChIP analysis of the PAR-1 promoter regions bp −365 to −329 (complex 1) and bp −206 to −180 (complex 2) demonstrates that in metastatic cells Sp1 is predominantly binding to the PAR-1 promoter, while in nonmetastatic cells AP-2 is bound. In vitro analysis of complex 1 demonstrates that AP-2 and Sp1 bind to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic cells had increased promoter activity compared to low and nonmetastatic melanoma cells. Our data shows that exogenous AP-2 expression decreased promoter activity, while transient expression of Sp1 further activated expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrates that the regulation of PAR-1 is mediated through interactions with AP-2 and Sp1. Moreover, loss of AP-2 in metastatic cells alters the AP-2 to Sp1 ratio and DNA-binding activity resulting in overexpression of PAR-1. In addition, we evaluated the expression of AP-2 and PAR-1 utilizing a tissue microarray of 93 melanocytic lesions spanning from benign nevi to melanoma metastasis. We report loss of AP-2 expression in malignant tumors compared to benign tissue while PAR-1 was expressed more often in metastatic melanoma cells than in benign melanocytes. We propose that loss of AP-2 results in increased expression of PAR-1, which in turn results in upregulation of gene products that contribute to the metastatic phenotype of melanoma. ^

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

Alterations in cell adhesion and migration are transcriptionally regulated by the tumor suppressor, TP53

The p53 transcription factor is a tumor suppressor and a master regulator of apoptosis and the cell cycle in response to cell stress. In some advanced tumors, such as prostate cancers, the loss of p53 correlates with an increase in the occurrence of metastases. In addition, several groups have suggested that p53 status correlates with changes in cell migration and cell morphology associated with a migratory phenotype. Others have identified several genes with roles in cell migration that are directly transcriptionally regulated by p53. Even so, modulation of cell migration is not widely recognized as a p53 stress response. ^ In an effort to identify novel p53 target genes and expand our knowledge of the p53 transcriptional response, we performed Affymetrix gene expression analysis in p53-null PC3 prostate cancer cells following infection with a control virus or adenoviral construct expressing wild-type p53. Over 300 genes that had not been previously recognized as p53 target genes were identified. Of these genes, 224 were upregulated and 111 were downregulated (p<0.05). Functional over-representation analysis identified cell migration as a significantly over-represented biological function of p53. Further analysis identified two genes that are critical for the control of cell migration as potential p53 targets. One, hyaluronan mediated motility receptor (HMMR), has recently been shown to be a p53 target important for regulation of the cell cycle. Here, we show that HMMR is downregulated by p53 in several cell lines, and HMMR's regulation is dependent on the presence of the cdk inhibitor, p21, and histone deactelyase activity. The other gene, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), itself a tumor suppressor, is shown here, for the first time, as a p53 direct target by ChIP analysis. We next determined the effect of p53 activation on cell migration and found that p53 significantly slows the rate of cell migration in Boyden chamber migration assays and digital videomicroscopy wound healing studies. Further, our studies established the specific roles of CEACAM1 and HMMR in cell migration and determine that loss of CEACAM1 and overexpression of HMMR independently contribute to increased cell migration. Taken together, these studies provide a direct mechanistic link between p53 to the regulatory control of specific target genes that mediate cell adhesion and migration. ^

Polycomb repressive complex 2 and induced basal-like breast cancer phenotype mediated by cyclin E/Cdk2

Despite of much success of breast cancer treatment, basal-like breast cancer subtype still presented as a clinical challenge to mammary oncologist for its lack of available targeted therapy owing to their negative expression of targeted molecules, such as PgR, ERα and Her2. These molecules are all critical regulators in mammary gland development. EZH2, a histone methyltransferase, by forming Polycomb Repressive Complex 2(PRC2) can directly suppress a large array of developmental regulators. Overexpression of cyclin E has also been correlated with basal-like (triple-negative) breast cancer and poor prognosis. We found an important functional link between these two molecules. Cyclin E/Cdk2 can enhance PRC2 function by phosphorylating a specific residue of EZH2, threonine 416 and increasing EZH2's ability to complex with SUZ12. This regulation would further recruit whole PRC2 complex to core promoter regions of these developmental regulators. The local enrichment of PRC2 complex would then trimethylate H3K27 around the core promoter regions and suppress the expression of targeted genes, which included PgR, ERα, erbB2 and BRCA1. This widespread gene suppressive effect imposed by highly active PRC2 complex would then transform the lumina) type cell to adopt a basal-like phenotype. This finding suggested deregulated Cdk2 activity owing to cyclin E overexpression may contribute to basal phenotype through enhancing epigenetic silencing effects by regulating PRC2 function. Inhibition of Cdk2 activity in basal-like cancer cells may help release the suppression, reexpress the silenced genes and become responsive to existing anti-hormone or anti-Her2 therapy. From this study, the mechanisms described here provided a rationale to target basal-like breast cancer by new combinational therapy of Cdk2 inhibitors together with Lapatinib, or Aromatin. ^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.

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.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Transformation studies of a nontumorigenic rat prostatic epithelial cell line with activated {\it myc\/} and {\it neu\/} oncogenes

A cloned nontumorigenic prostatic epithelial cell line, NbE-1.4, isolated from Noble (nbl/crx) rat ventral prostate, was used to examine the potential role of activated myc and neu oncogenes in prostate carcinogenesis. Transfection of SV40 promoter/enhancer driven constructs containing either v-myc, truncated c-myc, or neu-T (activated neu) oncogenes was accomplished using calcium phosphate-mediated DNA transfer. Cells were cotransfected, as necessary, with pSV2neo, allowing for selection of positive clones using the antibiotic geneticin (G418). G418 resistant colonies were pooled in some cases or limiting dilution exclusion cloned in others as described. Transfection of NbE-1.4 cells with activated myc oncogenes resulted only in the partial transformation. These cells display an altered morphology and decreased dependence on serum factors in vitro; however, saturation density, soft agar colony formation and growth assay in male athymic nude mice were all negative. Transfection and overexpression of NbE-1.4 cells with an activated neu oncogene alone resulted in tumorigenic conversion. Cell transformation was evident following an examination of the altered cellular morphology, an increased soft agar colony formation, and an acquisition of a tumorigenic potential when injected s.c. into male athymic nude mice. neu-transformed NbE-1.4 cells displayed elevated activity of the neu receptor tyrosine kinase. Furthermore, qualitative changes in tyrosine phosphorylated proteins were found in neu transformed cell clones. These changes were associated with elevated expression of mRNAs for laminin $\beta$1, $\beta$2, and procollagen type IV. The expression of fibronectin and E-cadherin, which are often lost during tumorigenesis, did not correlate with the tumorigenic phenotype. Therefore, it appears that neu oncogene overexpression has been found to be associated with the transformation of rat prostatic epithelial cells, presumably through alterations in gene expression that regulate extracellular matrix. The possible interrelationship and functional significance between neu oncogene expression and the elevated extracellular matrix gene expression is discussed. ^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^