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Article No. Title
TCA1021 Soft agarose culture human tumour colony forming assay for drug sensitivity testing: {3+ H}- Thymidine incorporation vs colony counting
  C.A. JONES, T. TSUKAMOTO, P.C. O'BRIEN, C.B. UHL, M.C. ALLEY & M.M. LIEBER
PDF Icon In vitro drug sensitivity testing, both by optical colony counting and by a {3+H}-TdR incorporation assay, was performed on human tumour cells proliferation in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Tegression analysis comparing the results of the colony counting assay and the {3+H}-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The {3+H}-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.
 
TCA1022 Growth of human renal carcinoma in soft agar colony formation assays measured by computer-assisted volume analysis
  TAIJI TSUKAMOTO, MICHAEL C. ALLEY, KARL-HEINZ KURTH, AND MICHAIL M. LIEBER
PDF Icon Technical methods for assessing the growth and chemotherapy sensitivity of human tumor cells growing in soft-agar culture have veen less than ideal. Within the past year, there have been reports of studying the extent of growth of human tumor cells in these cultures by quantitating the change in cumulative volume analysis applied to soft-agar cultures of cells from 74 primary renal cell carcinomas, 14 primary transitional cell carcinomas of the renal pelvis, and four different human renal cell carcinoma xenografts. The extent of growth in vitro observed for cells from fleshly excised human renal tumors showed the expected and statistically significant relationship to tumor grade and stage. The renal cell carcinoma xenografts proliferated to a much greater extent in vitro than the cells from freshly excised human renal carcinomas. The fundamental growth limit of 10 +9 um.+3 cumulative growth unit volume per plate was confirmed by this series of experiments. Computer-assisted volume analysis appears to be a useful method to study the growth of freshly excised human renal carcinoma cells in vitro.
 
TCA1023 Activation and inactivation of cancer chemotherapeutic agents by rat hepatocytes cocultured with human tumor cell lines
  M.C. ALLEY, G. POWIS, P.L. APPEL, K.L. KOOISTRA, AND M.M. LIEBER
PDF Icon While colony formation assays provide sensitive indices of tumor cell proliferation and growth inhibition imposed by many chemotherapeutic agents, drugs which require metabolic activation lack activity in such assays. In the present study, we have utilized freshly isolated rat hepatocytes for the activation of drugs which are metabolized by hepatic microsomal as well as extramicrosomal enzymes. Hepatocytes in fluid medium are placed over soft-agarose matrix containing tumor-derived cells (e.g., A204, A549) within 35-mm culture dishes; drug and/or drug vehicle is added directly to the hepatocyte layer, and cultures are incubated for 24 hr prior to removal of the hepatocyte layer. Tumor cell colony formation is assessed following 7 to 10 days of incubation. Cyclophosphamide was used as a prototype agent to assess utility of the coculture methodology. In vivo treatment of rats with phenobarbital prior to hepatocyte isolation enhances cyclophosphamide toxicity in vitro, whereas pretreatment with carbon tetrachloride markedly reduced subsequent in vitro cyclophosphamide sytotoxicity.
 
TCA1024 Metabolic stability of experimental chemotherapeutic agents in hepatocyte:tumor cell co-cultures
  PEGGY L. APPEL, MICHAEL C. ALLEY, MICHAEL M. LIEBER, ROBERT SHOEMAKER, AND GARTH POWIS
PDF Icon A U.S. national Cancer Institute screening program for new anticancer drugs, based on the growth of primary human tumor cells in an in vitro soft agar colony formation assay, has resulted in the identification of a number of compounds that have cytotoxic activity against primary human tumor cells in vitro but are inactive in the conventional in vivo murine P388 leukemia animal model pre-screen. To investigate whether metabolic inactivation of the compounds might be a factor in the lack of in vivo cytotoxicity we have co-cultured rat hepatocytes with A204 rhabdomyosarcoma and murine P388 leukemia cell lines in soft afarose colony formation assay for 24 h during exposure to the compounds. Twenty compounds with a range of in vitro activities were studied. Thirteen compounds exhibited cytotoxicity against A204 cells in culture; nine of them were less active when co-cultured with hepatocytes, two were activated by hepatocyte co-culture, and two showed no effect of hepatocyte co-culture. P388 cells were more sensitive to the antiproliferative effects of the compounds than A204 cells. Two compounds that were not active against A204 cells exhibited cytotoxicity against P388 cells. One compound was inactivated by hepatocyte co-culture and one showed no effect. Five compounds showed no sytotoxicity toward either A204 cells or P388 cells. Thus, evidence for metabolic inactivation in hepatocyte co-culture is not always an indication for lack of in vivo antitumor activity. Hepatocyte co-culture methodology provides a somple and objective means, amenable to large-scale screening, of distinguishing metabolic activation or inactivation of a given compound from other pharmacokinetic and pharmacodynamic factors with a minimum of material.
 
TCA1025 Breast cancer growth inhibition by delivery of the MDGI-derived peptide P108
  HUEY-LING WANG, AND ANDREAS KURTZ
PDF Icon Mammary derived growth inhibitor (MDGI) is a member of the family of cytoplasmic fatty acid binding proteins (FABPs), which bind hydrophobic ligands such as fatty acids, retinoids, eicosanoids and prostaglandines. MDGI and an 11 amino acid MDGI-derived conserved C-terminal peptide (P108) inhibits growth of normal mammary epithelial cells in tissue and organ culture, but fails to inhibit proliferation of many breast cancer cell lines in vitro. Here, the e€ects of peptide P108 on tumor growth of MCF-7, MDA-MB468 and MDA MB231 human breast cancer cell lines in nude mice were tested. To deliver P108 into tumors, a novel peptide production system was applied for expression and secretion of small bioactive peptides in mammalian cells. Functional di€erentiation was observed in MCF-7 and MDA-MB468 cells upon P108 expression. In addition, EGF-dependent colony formation in soft agar by MDA-MB468 cells was inhibited by secreted P108. Tumor growth in athymic nude mice was suppressed in all three cell lines tested. Furthermore, P108 expressed by MCF-7/P108 cells caused paracrine tumor growth inhibition of MDA-MB231 cells. These results indicate that breast cancer inhibition by P108 is independent of binding to hydrophobic ligands and is perhaps mediated by interference with EGF-dependent signaling pathways.
 
TCA1026 CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential
  RAMON G MANZANO, LUIS M MONTUENGA, MARK DAYTON, PAUL DENT, ICHIRO KINOSHITA, SILVESTRE VICENT, GINGER J GARDNER, PHUONGMAI NGUYEN, YUNG-HYUN CHOI, JANE TREPEL, NELLY AUERSPERG4 AND MICHAEL J BIRRER
PDF Icon Although early stage ovarian cancer can be e€ectively treated with surgery and chemotherapy, the majority of cases present with advanced disease, which remains essentially incurable. Unfortunately, little is known about the genes important for the development and progression of this disease. In this study, the expression of 68 phosphatases was determined in immortalized ovarian epithelial cells (IOSE) and compared to ovarian cancer cell lines. CL100, a dual speci®city phosphatase, displayed 10 ± 25-fold higher expression in normal compared to malignant ovarian cell lines. Immunohistochemical staining of normal ovaries and 68 ovarian cancer specimens confirmed this differential expression. Re-expression of CL100 in ovarian cancer cells decreased adherent and non-adherent cell growth and induced phenotypic changes including loss of ®lopodia and lamellipodia with an associated decrease in cell motility. Induced expression of CL100 in ovarian cancer cells suppressed intraperitoneal tumor growth in nude mice. These results show for the first time that CL100 expression is altered in human ovarian cancer, that CL100 expression changes cell morphology and motility, and that it suppresses intraperitoneal growth of human ovarian epithelial cancer. These data suggest that down-regulation of CL100 may play a role in the progression of human ovarian cancer.
 
TCA1027 The angiogenic factor midkine is aberrantly expressed in NF1-de®cient Schwann cells and is a mitogen for neuro®broma-derived cells
  GEORGE A MASHOUR, NANCY RATNER, GALAM A KHAN, HUEY-LING WANG, ROBERT L MARTUZA, AND ANDREAS KURTZ
PDF Icon Loss of the tumor suppressor gene NF1 in neurofibromatosis type 1 (NF1) contributes to the development of a variety of tumors, including malignant peripheral nerve sheath tumors (MPNST) and benign neurofibromas. Of the different cell types found in neurofibromas, Schwann cells usually provide between 40 and 80%, and are thought to be critical for tumor growth. Here we describe the identification of growth factors that are upregulated in NF17/7 mouse Schwann cells and are potential regulators of angiogenesis and cell growth. Basic fibroblast growth factor (FGF-2), platelet-derived growth factor (PDGF) and midkine (MK) were found to be induced by loss of neurofibromin and MK was further characterized. MK was induced in human neurofibromas, schwannomas, and various nervous system tumors associated with NF1 or NF2; midkine showed an expression pattern overlapping but distinct from its homolog pleiotrophin (PTN). Immunohistochemistry revealed expression of MK in S-100 positive Schwann cells of dermal and plexiform neurofibromas, and in endothelial cells of tumor blood vessels, but not in normal blood vessels. Furthermore, MK demonstrated potent mitogenic activity for human systemic and brain endothelial cells in vitro and stimulated proliferation and soft agar colony formation of human MPNST derived S100 positive cells and fibroblastoid cells derived from an NF1 neurofibroma. The data support a possible central role for MK as a mediator of angiogenesis and neuro®broma growth in NF1.
 
TCA1028 ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
  REBECCA S. MURAOKA, ANNE E. G. LENFERINK, BRIAN LAW, ELIZABETH HAMILTON, DANA M. BRANTLEY, L. RENEE ROEBUCK, AND CARLOS L. ARTEAGA
PDF Icon ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27+/- primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27+/+ cells. In contrast, ErbB2- or cyclin D1-overexpressing p27-/- cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27-/- cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27+/+, p27+/-, and p27-/- cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27+/- mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27+/+ glands. However, MMTV-neu/p27-/- glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27+/+ glands. These results suggest that p27+/- mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.
 
TCA1029 Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTVyNeu 1 MMTVyTGF-a bigenic mice
  ANNE E. G. LENFERINK, JEAN F. SIMPSON, LAURA K. SHAWVER, ROBERT J. COFFEY, JAMES T. FORBES, AND CARLOS L. ARTEAGA
PDF Icon ErbB2/Neu destabOverexpression of ErbB-2yNeu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)yNeu 1 MMTVytransforming growth factor a bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogenactivated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27Kip1. In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1y2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 andMAPKprecipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478- treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.
 
TCA1030 Epidermal Growth Factor Receptor (EGFR) Antibody Down-regulates Mutant Receptors and Inhibits Tumors Expressing EGFR Mutations
  MARIANELA PEREZ-TORRES, MARTA GUIX, ADRIANA GONZALEZ, AND CARLOS L. ARTEAGA
PDF Icon Activating mutations in the kinase domain of the EGF receptor have been reported in non-small cell lung cancer. The majority of tumors expressing these mutants are sensitive to ATP mimetics that inhibit the EGFR tyrosine kinase. The effect of antibodies that bind to the ectodomain of the receptor is less clear. We report herein the effects and mechanisms of action of the antibody cetuximab in lung cancer cells that naturally express receptor mutations and in ErbB-null 32D hematopoietic cells transfected with mutant EGFR. Treatment with cetuximab down-regulated EGFR levels and inhibited cell growth both in vitro and in vivo. This was associated with inhibition of ligand-independent EGFR signaling. These effects were seen in 32D cells arguing the growth inhibitory action was not because of the blockade of autocrine ligand action. Both antibody-induced EGFRdown-regulation and inhibition of growth required receptor dimerization as monovalent Fab fragments only eliminated receptor levels or reduced cell proliferation in the presence of antihuman IgG. Finally, cetuximab inhibited growth of H1975 lung cancer cells and xenografts, which expressed L858R/T790M EGFR and were resistant to EGFR tyrosine kinase inhibitors. These data suggest that cetuximab is an effective therapy against mutant EGFR-expressing cancer cells and thus can be considered in combination with other anti-EGFR molecules.

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