Regulation of gene expression and cell growth in vivo by tetracycline using the hollow fiber assay.

The hollow fiber assay presents a potentially unique tool to study the effects of regulated gene expression in cell lines that do not form tumors in vivo. The hollow fibers allow small molecules to pass freely through while keeping the cells within the fibers and segregated from host cells. OSp16.1 cells, derived from the U24 clone of the U2-OS osteogenic sarcoma tumor line, express the p16INK4a tumor suppressor under the regulation of tetracycline (tet) (Mitra J et al. Mol Cell Bio 19:3916, 1999). The in vitro induction of p16 in the OSp16.1 cell line is regulated by tet. The hollow fiber assay was used to determine whether the regulation of the p16 gene could be achieved in vivo, since these cells did not grow in the xenograft model. There were no differences in the in vivo growth pattern of U24 cells loaded into the hollow fibers with and without tet: 807% and 839% net growth, respectively. OSp16.1 cells in fibers in mice receiving 3.33 mg/kg/day tet had a 644% net growth after 21 days. There was a 194% net growth without tet. Immunoblotting of extracts prepared from the hollow fibers confirmed that p16 was induced in the absence of tet. These data demonstrate this assay is a useful tool for studying the effects of regulated gene expression in vivo.

Alpha(v)beta(3) integrin in angiogenesis and restenosis.

Various integrins are thought to be intimately involved in several pathological processes, including cancers (solid tumors and metastasis), cardiovascular diseases (stroke and heart failure), inflammatory diseases (rheumatoid arthritis) and ocular pathologies. The mechanism of the involvement of integrins in these acute and chronic disease states is slowly being elucidated. Recently, various therapeutic candidates, including antibodies, cyclic peptides and peptidomimetics, have been clinically evaluated and have been shown to successfully modulate certain disease processes. This review focuses on the key role of the alpha(v) integrin (alpha(v)beta(3)) in the angiogenic processes in diseases such as cancer, restenosis following percutaneous transluminal coronary angioplasty, stroke, ocular disease and rheumatoid arthritis.

Small molecule alpha(v) integrin antagonists: novel anticancer agents.

The members of the integrin family are targets that potentially provide both therapeutic and diagnostic opportunities. Advances in the understanding of the signalling pathways, transcriptional regulation and the structure/function relationships of the adhesion molecules to extracellular matrix proteins have all contributed to these opportunities. The role of the integrins in pathological processes in both acute and chronic diseases, include ocular, cancer (solid tumours and metastasis), cardiovascular (stroke and heart failure) and inflammatory (rheumatoid arthritis) conditions. Various therapeutic candidates, including antibodies, cyclic peptides and peptidomimetics, have been identified. This review will focus on the key role of the alpha(v) integrin (alpha(v)beta(3) and alpha(v)beta(5)) in angiogenic processes in tumours, including its potential use in cancer diagnostics and therapy.

Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

The hollow fiber assay: continued characterization with novel approaches.

The hollow fiber assay, a unique in vivo model, permits the simultaneous evaluation of compound efficacy against multiple cell lines in two physiological compartments. This assay has been used to characterize in vivo activity of cytotoxic compounds. The purpose of the present study was to characterize and optimize this assay for compounds with a defined mechanism of action, specifically cell cycle inhibition. Two human tumor cell lines and one normal human cell line were loaded into polyvinylidene fluoride hollow fibers at two or more cell concentrations and grown in mice for 3-10 days. The data demonstrate the importance of characterizing the initial loading density of various cell lines in the evaluation of compounds. All studies were performed with cells in the linear part of the cell growth curves. Initial loading densities of 1-2 x 10(4) cells/fiber gave the greatest opportunity for growth in the three human cell lines tested (HCT116 colon carcinoma, NCI-H460 non-small cell carcinoma, and AG 1523 normal fibroblast). Utilizing the MTT assay, standard curves were constructed to correlate the final number of cells with optical density (OD) readings at 540 nm in order to calculate cell numbers in the fibers. Insights into the mechanism of action of cisplatin have been gained using Western blot analysis of the cell cycle markers PCNA (a protein present throughout the cell cycle) and Rb (a protein that acts as a tumor suppressor gene product) from the hollow fiber cells. In cisplatin-treated NCI-H460 cells both PCNA and Rb phosphorylation decreased, suggesting the arrest of the cells prior to the S phase. Standard therapeutic agents, cisplatin, racemic flavopiridol, cyclophosphamide and mitomycin C, were evaluated independently in the hollow fiber assay and the xenograft model. The data demonstrate that compounds active in the hollow fiber assay are also active in the xenograft.

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Mechanism of action of DuP 721: inhibition of an early event during initiation of protein synthesis.

The mode of action of DuP 721 was investigated. This compound was active primarily against gram-positive bacteria, including multiply resistant strains of staphylococci. Although inactive against wild-type Escherichia coli, DuP 721 did inhibit E. coli when the outer membrane was perturbed by genetic or chemical means. Pulse-labeling studies with E. coli PLB-3252, a membrane-defective strain, showed that DuP 721 inhibited amino acid incorporation into proteins. The 50% inhibitory concentration of DuP 721 for protein synthesis was 3.8 micrograms/ml, but it was greater than 64 micrograms/ml for RNA and DNA syntheses. The direct addition of DuP 721 to cell-free systems did not inhibit any of the reactions of protein synthesis from chain initiation through chain elongation with either synthetic or natural mRNA as template. However, cell extracts prepared from DuP 721 growth-arrested cells were defective in initiation-dependent polypeptide synthesis directed by MS2 bacteriophage RNA. These cell-free extracts were not defective in polypeptide elongation or in fMet-tRNA(fMet)-dependent polypeptide synthesis stimulated by poly(G.U). We conclude, therefore, that DuP 721 exerts its primary action at a step preceding the interaction of fMet-tRNA(fMet) and 30S ribosomal subunits with the initiator codon.

Predominant microflora associated with human dental periapical abscesses.

The microflora of periapical abscesses of teeth free of periodontal disease was studied. Permucosal aspiration of abscess contents and anaerobic cultural, microscopic, and biochemical techniques indicated a mixed but somewhat specific and relatively limited facultative and obligate anaerobic flora with Fusobacterium nucleatum and Streptococcus mitis as a frequent pair.