Does incorporation of gene for green fluorescent protein in BP6 fibrosarcoma tumor cells depress their intraperitoneal growth in rats? (In honour of Nobel Prize laureates 2008--Osamu Shimomura, Martin Chalfie, Roger Y. Tsien).

This manuscript was in honour of Nobel Prize in chemistry "for the discovery and development of the green fluorescent protein, GFP" to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien, simultaneously a brief information about experience with GFP in experimental tumorigenesis used this study is also presented. The experimental data have showed that BP6 cells incorporated with GFP gene have had smaller ability to induce both experimental intraperitoneal and subcutaneous tumor process. It was anticipated that incorporation of GFP gene might change physiological properties of cytoskeleton and worsen adhesive characteristics of tumor cells. It was also supposed that aftertime GFP will enable to monitor proliferation of cells not only within experimental work, but also in human medicine. GFP could help (supposedly) as reporter of proliferation, but also can serve as "target" for guide of tumorigenesis inhibiting substances. These ideas which are consequences of our experiments we append as congratulation to Nobel Prize in chemistry of the 2008 (Fig. 2, Ref. 44). Full Text (Free, PDF) www.bmj.sk.

Establishment, morphological, growth and cytoskeletal properties of 135-BCA carcinoma cell line derived from lung brain metastasis.

Many cell lines have been established from lung cancer but carcinoma cell lines derived from brain metastases occur rarely. The carcinoma cells growth relatively slowly in comparison with brain cells which often overgrow the tumor cells in early passages. The origin of these rapidly dividing brain cells in carcinoma cultures is discussed with respect to the previous studies on adult human brain tissue cultures. It was found that the majority of cells in adult human brain cultures derived from brain biopsies of patients with non-cancer diseases do not express glial markers. Based on the previous studies we suggest that they are glial precursor cells. The high proliferative capacity and non-glial phenotype of these brain cells may lead to the suggestion that they are of cancer origin. In this study the establishment and characterization of a new carcinoma cell line 135-BCA is described. The tissue cultures were derived from brain metastasis of lung large cell carcinoma. The cell line is specific by the epithelial cell morphology and evident cytokeratins expression during the whole subcultivation. All tumor cells were strongly immunoreactive for vimentin and negative stained for glial fibrillary acidic protein (GFAP). The new cell line may prove of value in biological and therapeutic studies of lung cancer. In addition, the further comparative analysis may reveal the environmental influence of brain tissue on carcinoma cells.

Heterogeneity of keratin intermediate filaments expression in human glioma cell lines.

Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin-positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines.

Characterization of two new permanent glioma cell lines 8-MG-BA and 42-MG-BA.

The establishment and characterization of two permanent glioma cell lines (8-MG-BA and 42-MG-BA) are described. Both cell lines were derived from the human glioblastoma multiforme. Analyzed cells were within the passage 200 to 220. The cells in both cultures showed similar morphology. In majority they consisted from flat polygonal cells. Growth kinetic studies demonstrated a population doubling time of 20 to 24 h in cell line 8-MG-BA and 48 to 54 h in cell line 42-MG-BA. The cell lines showed different hyperdiploid karyotypes. The immunofluorescence staining was performed for glial fibrillary acidic protein (GFAP) and vimentin. In the culture 8-MG-BA only a small amount of cells showed the GFAP-positive staining. At confluent 42-MG-BA culture the GFAP-positive cells reached 50 to 70% of all cells. Vimentin was found in all glioma cells in both cultures.

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.

Conformation-dependent accessibility of the linear epitopes located on the rabies virus glycoprotein.

Seven monoclonal antibodies (MAbs) were derived from mice immunized with the rabies virus glycoprotein of the Pitman-Moore (PM) strain. These antibodies recognized at least five partially overlapping sites located in one immunodominant region. A panel of MAbs was then used to characterize antigenic relationship between PM strain and SAD-Vnukovo strain of these rabies viruses. In immunoblot, all tested antibodies bound to the glycoprotein of both rabies strains, indicating shared antigenic determinants located on the corresponding immunodominant regions. The pattern of reactivity in immunoblot suggested the specificity of antibodies against linear epitopes. However, the supposed close antigenic relation between PM and SAD-Vnukovo strains (evidenced by immunoblot) was not fully confirmed by immunoenzymatic assay. Data provided by ELISA demonstrated two distinct patterns of MAbs reactivity with both antigens. Four antibodies showed specificity for PM strain glycoprotein only, while three MAbs bound with both PM and SAD-Vnukovo strain antigens. We supposed the strain-specific conformation of the native glycoprotein to be responsible for selective access of single MAbs to the respective common linear epitopes.

One-step method for establishing 8-azaguanine-resistant hybridomas suitable for the preparation of triomas.

A simple and rapid one-step method for establishing azaguanine resistant (Agr) hybridomas, which can be used as a fusion partner for the construction of triomas (hybridoma x splenocyte), has been developed. The method relies on cloning the hybridoma cells in soft agar supplemented with 20 micrograms/ml 8-azaguanine. The drug-resistant subclones were isolated after 3-5 days, in comparison with 4-5 weeks reported for the conventional adaptation method. The high frequency (about 10(-3) of Agr-mutants achieved by the cloning method was demonstrated with five different hybridoma clones. One of the derived Agr-hybridomas was fused with mouse immune spleen cells in order to demonstrate its suitability for the generation of triomas secreting bispecific monoclonal antibodies.

Distinct effect of pH 2 on a common antigenic structure found in human interferons-alpha 1 and -alpha 2 in the region 30-35.

The antigenic similarity between molecules of recombinant human interferon-alpha 1 (IFN-alpha 1) and recombinant human IFN-alpha 2 was demonstrated with neutralizing monoclonal antibody (mAb) 1-46. The common epitope for the mAb 1-46 was localized into amino-terminal region of IFN-alpha molecule around residues 30-35. Following pH 2 treatment, the biological activity of both IFN-alpha 1 and IFN-alpha 2 was retained but the antigenic relatedness between corresponding sequences 30-35 was diminished. The common structure on the IFN-alpha 1 molecule proved acid stable and the mAb 1-46 retained the ability to neutralize the pH 2 treated IFN-alpha 1. However, the neutralization of pH 2-treated IFN-alpha 2 by specific antibody was completely suppressed. These results complemented our earlier finding of the dramatic effect of acidic pH on the antigenic structure of region 132-137 of the IFN-alpha 2 molecule. We conclude that pH 2 may induce a conformational rearrangement of the IFN-alpha 2 molecule, resulting in an altered tertiary structure with deviating antigenic characteristics.

Mapping of two immunodominant structures on human interferon alpha 2c and their role in binding to cells.

Structure-function studies of human recombinant interferon (IFN) alpha 2c were performed using a panel of specific monoclonal antibodies in the binding and neutralizing assays. Two immunodominant structures, designated sites I and II, were detected and localized within two conserved hydrophilic regions of IFN-alpha molecule. Using the NK2 antibody as a marker, site I was mapped into a carboxy-terminal domain around residues 112-148. This site was shown to be, most probably, responsible for inducing the antiviral and antiproliferative activities of the receptor-bound IFN-alpha 2c in the cell. Site II that mapped into the amino-terminal domain of IFN-alpha 2c was, at least partially, formed by the amino acid residues 36-41. This region was shown to be most probably involved in the binding of IFN to its cellular receptor. These findings fit with Sternberg and Cohen's model (Int. J. Biol. Macromol. 4, 137-144, 1982) for the tertiary structure of human IFN-alpha.

Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.