Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.

Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.     

Mitotic activity of rat muscle satellite cells in response to serum stimulation: relation with cellular metabolism

Cellular and molecular adaptations of satellite cells isolated from rat hindlimb muscles (n = 10) were investigated in response to serum stimulation. Flow cytometry analysis of the amounts of DNA and RNA indicated that 97.7 +/- 0.7% of satellite cells were in G0 at the end of the isolation procedure, whereas 93.2 +/- 2.0% of cells were cycling after serum exposure. The length of cell division was 34.0 +/- 2.8 h. Myoblast proliferation was asynchronous, suggesting the existence of heterogeneous cell populations in skeletal muscle. Myoblast proliferation was also accompanied by a significant increase in c-met expression, and major adaptations of energetic and proteolytic metabolisms, including an increase in the relative contribution of glycolytic metabolism for energy production, an increase in proteasome and matrix metalloproteinases 2 and 9 activities, and a decrease in plasminogen activator activities. Our data suggest that, along with molecular adaptations leading to cell cycle activation itself, adaptations in energetic and proteolytic metabolisms are crucial events involved in satellite cell activation and myoblast proliferation.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

Genebet1 involved in vesicular transport is differentially transcribed in transformed cells of different metastatic potential

The method of differential display of cDNA produced on the RNA from highly and poorly metastasizing cell lines revealed a number of genes with different expression depending on the metastatic potential. An mRNA corresponding to one of these cDNA fragments (7HM) was found in increased amounts in high-metastasis but not in low-metastasis cell lines. The nucleotide sequence of the 7HM fragment showed high homology with that of genebet1. The Bet1 protein is involved in vesicular trasport from the endoplasmic reticulum to the Golgi complex. Enhanced expression ofbet1 in high-metastasis cell lines is probably induced by the oncogenev-src.     

Distribution of fibroblast growth factors in cultured tumor cells and their transplants

Summary The distributions of acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in extracts of various cultured mammalian cells were determined from their elution profiles on heparin-affinity chromatography, and assay of activity as ability to stimulate DNA synthesis in BALB/c3T3 cells. Only aFGF was found in extracts of mouse melanoma B 16 cell and rat Morris hepatoma cell (MH₁C₁) lines. Other tumor cell lines established from solid tumors and some normal cells contained bFGF as a main component, but blood tumor cell lines contained no aFGF or bFGF. The FGFs in extracts of solid tumor tissues derived by transplantations of these cultured tumor cells and various normal tissues of mice were also examined. Tumors formed by all cell lines, regardless of whether they produced aFGF, bFGF, or neither, contained bFGF that was probably derived from host cells including capillary endothelial cells, in addition to the tumor-derived aFGF or bFGF, if produced. The content of bFGF, possibly derived from the host, in these tumor tissues was comparable to those of various mouse organs other than thymus, lung, spleen, and testis, which have higher bFGF contents. Tumor tissues derived from cultured cells producing bFGF had relatively higher bFGF contents. Like bFGF, aFGF was distributed almost ubiquitously in normal mouse tissues.     

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells

Summary Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C₂, and H_5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C₂ and H_5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C₂ and H_5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.     

Role of proteins of the macroglobulin family in regulation of tumor growth

Proteins of the macroglobulin family are an ancient and evolutionarily conservative link of the immune system, which is actively involved in both inhibition of tumor growth cells and proliferation of tumor cells. Two basically different binding sites and a great conformational plasticity of all representatives of the macroglobulin family, as well as the presence of two to four representatives of the family in the blood of most species allow them to transport diverse substances and exert various regulatory influences on both the tumor and the entire organism. For example, the capacity of macroglobulins for binding hydrolases makes it possible to inhibit enzyme mediated tumor invasion. At the same time, an excess of macroglobulin/hydrolase complexes can activate apoptosis. The tumor is able of using macroglobulins, especially pregnancy-associated proteins, for its own protection. Specifically, pregnancy-associated α₂-glycoprotein, which is actively produced by human tumor cells, blocks the his to compatibility complex antigens. On the contrary, the capacity of binding zinc stimulates the thymulin-dependent activation of natural killer cells. Nevertheless, the actively growing tumor expresses many receptors to macroglobulins, which are the main carriers of some cytokines and growth factors essential for proliferation.     

High expression of Met/hepatocyte growth factor receptor suppresses tumorigenicity in NCI-H1264 lung carcinoma cells.

The protein product of c-met proto-oncogene, Met, is a tyrosine kinase receptor for the hepatocyte growth factor (HGF). Met receptor is expressed in normal human bronchial epithelium. In comparison, its expression in squamous cell carcinoma (SQCC) of the lung is markedly decreased in a great majority of cases. To understand further the role of Met receptor overexpression in non-small-cell lung carcinoma, we forced-expressed the full-length met cDNA in the NCI-H1264 (H1264) lung carcinoma cell line with low constitutive expression of this receptor. In vitro studies demonstrated that increased Met expression in H1264 cells resulted in strong inhibition of their ability to form soft agar colonies and in marked suppression of tumorigenicity in the subcutaneous tissue of immune-deficient mice. This is despite inconsistent alteration in the proliferation rate on plastic surfaces. Tumor cells explanted from occasional xenograft tumors formed by the Met-overexpressing H1264 cells also demonstrated marked down-regulation of the receptor protein levels as compared to the transplanted cells. The results suggest that constitutive overexpression of Met receptor may negatively regulate the malignancy of certain human lung cancer cells.     

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5 non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (–623 to –471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (–341 to –306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C+D+E, B+C+D, B+C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B+C+E is active only in vascular meristematic cells. Domain C (–216 to –158) seems to have a double regulatory role as construct B+E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.     

Cyclooxygenase-2 increased the angiogenic and metastatic potential of tumor cells

It seems certain that COX-2 is related to tumor and some data suggested that COX-2 might have relation to tumor malignance and angiogenesis. In order to elucidate the relationship between COX-2 and tumor invasive and angiogenic ability, we transfected human transitional cell carcinoma (TCC) cell line, EJ, permanently with a COX-2 expression vector or the mock vector. The EJ-COX(2) cells, which overexpressed COX-2, acquired increased invasiveness and angiogenic ability by activation of VEGF, uPA, and MMP-2. Increased invasiveness and angiogenic ability were reversed by treatment with either selective COX-2 inhibitor, NS-398, or dual COX inhibitor, indomethacin. These results demonstrate that overexpression of COX-2 can lead to phenotypic changes that alter the metastatic and angiogenic potential of TCC cancer cells.     

Purification and identification of transferrin as a major pituitary-derived mitogen for MTW9/PL2 rat mammary tumor cells

Summary Transferrin was identified as a major tissue-derived growth factor for MTW9/PL2 rat mammary tumor cells. Mitogenic activity was assayed by the ability to stimulate the increase in number of MTW9/PL2 cells over 4 d in Dulbecco's modified Eagle's medium containing only 15 mM HEPES, 2 mM glutamine, and 50 μg/ml gentamicin. This growth-promoting activity was purified from ammonium sulfate precipitates of phosphate buffered saline extracts of porcine pituitaries using DEAE-Sepharose, chromatofocusing, molecular sieve chromatography and reverse phase high performance liquid chromatography. Pig pituitary mitogen (PPM) migrated as a single band at molecular weight 78 000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, eluted from chromatofocusing at multiple pH values near 6.3, exhibited an absorption maximum at 465 nm which was diminished by removal of iron, showed a characteristic salmon-pink color in aqueous solution, and was similar in amino acid composition to previously reported values for porcine transferrin. Purified PPM similar to commercially available human transferrin (ED₅₀=160 to 350 ng/ml). We have concluded that using serum-free assay conditions with MTW9/PL2 cells, transferrin was a major source of the mitogenic activity present in extracts of porcine pituitary. This work was supported by grants CA-38024 and CA-26617 from the National Cancer Institute, and American Cancer Society grant BC-255.     

Ketone bodies and two-compartment tumor metabolism: Stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.     

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

Summary Ehrlich ascites tumor cells contain a Na⁺ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC₅₀ of 25 m and by dimethylamiloride with an IC₅₀ of 0.6 m at 1mm external Na⁺. Decrease of external Na⁺ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na⁺/H⁺ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na⁺/H⁺ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na⁺ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na⁺. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na⁺ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na⁺/H⁺ antiporter to internal protons. The Na⁺/H⁺ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.     

Uteroglobin promoter-targeted c-MYC expression in transgenic mice cause hyperplasia of Clara cells and malignant transformation of T-lymphoblasts and tubular epithelial cells

To investigate the influence of the proto-oncogene c-MYC on tumor development in different epithelial tissues which secrete Clara Cell Secretory Protein (uteroglobin, UG), transgenic mouse lines were established expressing the human c-MYC proto-oncogene under the control of the rabbit UG-promoter. These mice expressed the c-MYC transgene in Clara cells and other UG expressing tissues like uterus and prostate. In the bronchioalveolar epithelium of the lung hyperplasias developed originating from Clara cells. Surprisingly, transgenics most frequently developed T-lymphoblastic lymphomas, a polycystic kidney phenotype and renal cell carcinoma derived from tubular epithelial cells, which are both tissues that had so far not been known to express UG. Immunohistological studies in UG/MYC transgenics and in a transgenic line (UG/eGFP) expressing Green Fluorescent Protein confirmed that the uteroglobin promoter is not only active in Clara cells, but also in tubular epithelial cells of the kidney and in lymphatic tissue. The UG/MYC transgenics will be useful to investigate the biochemical mechanisms underlying the development of carcinomas and the oncogenic properties of c-MYC in epithelial cells of various tissues.