Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.

Cancer stem cells (CSCs) play an important role in the development, invasion, and drug resistance of carcinoma, but the exact phenotype and characteristics of ovarian CSCs are still disputable. In this study, we identified cancer stem cell-like cells (CSC-LCs) and investigated their characteristics from the ovarian adenocarcinoma cell line 3AO. Our results showed that CSC-LCs were enriched in sphere-forming test and highly expressed CD44(+)CD24⁻. The spheres and CD24⁻ cells possessed strong tumorigenic ability by transplantation into nonobese diabetic/severe combined immunodeficient mice. CD44(+)CD24⁻ cells expressed stem cell markers and differentiated to CD44(+)CD24(+) cells by immunofluorescence assay and fluorescence-activated cell-sorting analysis. In vitro experiments verified that CD44(+)CD24⁻ cells were markedly resistant to carboplatin and paclitaxol. In conclusion, our study identifies the CD44(+)CD24⁻ phenotype, self-renewal, high tumorigenicity, differentiation potential, and drug resistance of ovarian CSC-LCs. Our findings may provide the evidence needed to explore a new strategy in the treatment of ovarian cancer.

A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.

A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (i.e., PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximately 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like "tubes" on Matrigel. However, 1G11 cells exhibited a "cobblestone" morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.

Renal expression of monocyte chemoattractant protein-1 in lupus autoimmune mice.

Mononuclear cell infiltration in glomeruli and renal interstitium is a prominent feature of some types of glomerulonephritis, including lupus nephritis. The mechanism(s) underlying monocyte influx into the kidney is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a chemotactic factor involved in the recruitment of monocytes/macrophages in the glomeruli of rats with mesangioproliferative as well as anti-glomerular basement membrane glomerulonephritis. In the study presented here, renal MCP-1 mRNA expression in New Zealand Black x New Zealand White (NZB/W) F1 mice, a model of genetically determined immune complex disease that mimics systemic lupus in humans, was investigated. Northern blot analysis revealed a single 0.7 kb MCP-1 transcript of very low intensity in kidneys from 2-month-old NZB/W mice that had not yet developed proteinuria nor renal damage. Message levels, which increased markedly with the progression of nephritis and in association with mononuclear cell infiltration, were 10- and 15- fold higher in 8-10-month-old mice than in 2-month-old mice. By in situ hybridization, increased expression of MCP-1 mRNA was demonstrated in glomeruli and, even more striking, in tubular epithelial cells. Western blot analysis demonstrated increased expression of MCP-1 protein in kidneys of 10-month-old NZB/W mice, consistent with MCP-1 mRNA data. When NZB/W mice were treated with cyclophosphamide up to 12 months of age, expression of MCP-1 in the renal tissue remained low, the influx of inflammatory cells did not appear, and glomerular and tubular structures remained well preserved. These data suggest that elevated MCP-1 might act as a signal for inflammatory cells to infiltrate the kidney in lupus nephritis.

Anti-tumor activity of cytokines against opportunistic vascular tumors in mice.

Polyoma middle T (PmT)-transformed endothelial cells may represent a unique murine model for human opportunistic vascular tumors. The present study was designed to evaluate the anti-tumor potential of a panel of 13 cytokines against murine PmT-transformed endothelial cells. Interferon gamma (IFNgamma) and transforming growth factor beta 1 (TGFbeta1) substantially decreased in a dose-dependent manner the proliferation of a panel of 6 PmT-transformed cell lines. IFNalpha and tumor necrosis factor alpha(TNFalpha) had marginal anti-proliferative activity, whereas other molecules (interleukins-1, -2, -4, -6 and -13, IFNbeta, leukemia inhibitory factor, oncostatin M, granulocyte-macrophage colony-stimulating factor) caused no growth inhibition. IFNgamma and TGFbeta1 were therefore selected for further analysis of their mechanism of action and in vivo relevance. IFNgamma and TGFbeta1 reduced the activity of phosphatidylinositol-3-kinase and the production of phosphatidylinositol 3,4-biphosphate, without modifying the tyrosine kinase(s) activity associated with PmT. IFNgamma and TGFbeta1 were also tested for their ability to modify the in vivo growth of the PmT-transformed endothelial cells H5V in syngeneic C57B1/6 mice. Treatment with IFNnu and TGFbeta1 significantly delayed tumor growth and increased survival time. In contrast, treatment with IFNalpha and TNFalpha failed to prolong survival. In nude mice, IFNgamma and TGFbeta1 had a transient effect on tumor growth but no effect on survival, suggesting a contribution of T cells to the in vivo anti-tumor activity of these cytokines.

Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase.

Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from nontransformed controls, whereas the affinity for arginine did not differ. The biochemical characterization of NOS and the study of mRNA transcripts indicate that tEnd.1 cells express both the inducible and the constitutive isoforms. NOS hyperactivity is not a simple consequence of cell transformation but needs a tissue-specific mTa expression. Since tEnd.1-conditioned medium induces NOS activity in normal endothelial cells, most likely NOS hyperactivity in endothelioma cells is attributable to the release of a soluble factor. This NOS-activating factor, which seems to be an anionic protein, could stimulate tEnd.1 cells to express NOS by an autocrine way. By the same mechanism, tEnd.1 cells could induce NOS in the neighboring endothelial cells, and NO release could play a role in the hemangioma development. Such hypothesis is confirmed by our in vivo experiments, showing that the administration of the NOS inhibitor L-canavanine to endothelioma-bearing mice significantly reduced both the volume and the relapse time of the tumor.

Cytokine regulation of monocyte recruitment.

Phagocytes infiltrating neoplastic tissues have peculiar membrane phenotype and functional properties. Tumor-associated macrophages (TAM) play a complex, ambiguous role in the regulation of primary tumor growth and metastasis (a "macrophage balance"). Yet these cells are strategically located at the very interface between tumor and host and represent a potential target for immunomodulation. A better understanding of the regulation and function of TAM may provide a less empirical basis of or rational design of therapeutic approaches, as vividly illustrated by the antitumor activity of i.p. in IFN ovarian cancer patients with minimal residual disease resistant to chemotherapy.

Isolation of a mitomycin-resistant human lung adenocarcinoma cell subline to investigate the modulation by sodium butyrate of cell growth and drug resistance.

We developed a mitomycin C (MMC)-resistant human lung adenocarcinoma cell subline, SPC-A1/DM4, from cloned SPC-A1/D13 parent cells by 1 h exposures to escalating concentrations of the drug over 17 months. A 5.9-fold resistance to MMC and a 3.8-fold cross-resistance to cisplatin were present in resistant cells compared with parent cells. This phenotype was stable in the absence of drug exposure for at least 6 months. Sodium butyrate (NaBu), a widely used differentiating agent, was shown to inhibit cell proliferation in a dose-dependent manner, with the cytostatic concentration of 2 mM. This NaBu-induced growth inhibition was reversible. However, SPC-A1/DM4 cells, after recovery from the cytostasis induced by 2 days treatment with 2 mM NaBu, became 2-fold more sensitive to MMC than the cells not exposed to the agent. Meanwhile, the cisplatin response of these treated cells reached a level comparable to the parent cells. This modulation by NaBu of drug resistance could be retained for at least 1 month. Treatment with 2 mM NaBu for 2 days caused inhibition of DNA synthesis and accumulation of cells in the G1 and G2/M-phases of the cell cycle. Correlated with these were a marked increase of protein content in these cell subpopulations and an enhanced RNA synthesis. In addition, NaBu-treated cells acquired development of endoplasmic reticulum and accumulated lipid droplets. These morphological alterations were accompanied by a significant decrease in the ratio of nuclear to cytoplasmic areas. These findings suggest that NaBu is potentially useful in the treatment of drug-resistant non-small cell lung cancer. Information about the NaBu-induced phenotypic alterations may offer a clue to the understanding of its long-term effect on drug resistance.

Successful separation of xiphoomphalopagus twins.

A pair of female xiphoomphalopagus twins were delivered by Caesarean section at 35th week of gestation on March 2, 1982, their combined weight being 4,800 g. Examinations revealed that they were conjoined from the xiphoid process down to the umbilicus. Infant A also had congenital heart defect (VSD). X-ray and echography showed that they had a fused liver and two independent biliary systems and alimentary tracts. After 6 weeks, the twins gained weight up to 7,000 g. The separation operation was performed at 1 1/2 months of age. During operation it was demonstrated that the xiphoid process and costal cartilages were fused together and the peritoneal cavities were of free communication above the umbilicus, and the livers merged into a single common liver. The large single liver was divided by electrocautery, resulting in a section surface of 8 X 7.5 cm. After separation, the closure of the abdominal wall in both infants presented some difficulties which were resolved by making relaxation incisions on either flanks. In the post operative period, the ventral wounds of both infants were disrupted for several centimetres and infant B had wound infection. The granulating area of skin defect on either relaxation incision of the flanks and ventral denuded wound were covered with full-thickness dead-foetus homografts. The wounds were well healed. Eventually both infants were discharged in good condition at 2 1/2 months after operation. Now they live well at 2 1/2 years of age.