A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

Cytotoxic interactions of bare and coated NaGdF4:Yb(3+):Er(3+) nanoparticles with macrophage and fibroblast cells.

The lanthanide nano-compounds are well suited to serve as fluorescent and magnetic contrast agents and luminescent labels. Although they are considered as promising materials for bio-imaging and bio-sensors in vivo or in vitro, the amount of data is still insufficient for deep understanding the toxicity of these nanomaterials. This knowledge is of great importance in the light of growing use of the biofunctionalized nanoparticles, which raises some questions about safety of these materials. Despite lanthanide-doped NaGdF4 nanocrystals are considered as non-toxic, here we present the data showing the fatal effect of newly synthetized NaGdF4:Yb(3+):Er(3+) on chosen types of cells. Our studies were performed on two cell lines NIH3T3 fibroblasts, and RAW264.7 macrophages. Cytotoxic properties of NaGdF4:Yb(3+):Er(3+) nanoparticles and their biological effects were studied by assessing cell culture viability (MTS), proliferation and apoptosis. Bare NaGdF4:Yb(3+):Er(3+) nanocrystals were cytotoxic and induced apoptosis of both NIH3T3 and RAW264.7 cells. Their cytotoxicity was reduced by PEGylation, at the expense of minimizing direct interactions between the compound and the cell. On the other hand, coating with silica reduced cell death induced by Yb(3+):Er(3+) codoped NaGdF4 nanocrystals (but proliferation was still inhibited). The NH2-modified silica coated nanoparticles were clearly less cytotoxic than pristine nanoparticles, which suggests that both, silica and PEG coatings are reasonable approaches to decrease cytotoxicity of the nanocrystal labels. The silica and PEG shell, should also enable and simplify further bio-functionalization of these luminescent labels. The authors acknowledge the financial support from: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (IITD PAN) grant no. 3/15, Polish Ministry of Science and Higher Education, Grant N N507 499538 and from the Wroclaw Research Centre EIT+ within the project "The Application of Nanotechnology in Advanced Materials" - NanoMat (POIG.01.01.02-02-002/08) financed by the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2).

Changes in glucocorticoid-induced apoptosis and in expression of Bcl-2 protein during long-term culture of thymic lymphoma.

Lymphomagenesis is a multistep process progressively freeing transformed thymocytes from external regulatory signals, i.e. thymic developmental program controlling growth, differentiation or apoptosis. Here we report that cells of thymic lymphoma overexpressing Ras/Raf proteins, initially resistant to TCR-dependent apoptosis but sensitive to dexamethasone- and etoposide-induced cell death, became insensitive to dexamethasone after long-time culture. That transition correlated with a strong increase in the expression of the anti-apoptotic Bcl-2 protein. Interestingly, lymphoma cells were still sensitive to p53-mediated apoptosis induced by etoposide. It suggests that the anti-apoptotic activity of Bcl-2 is correlated with a resistance to glucocorticoid-induced apoptosis but not to p53-mediated apoptosis. The sequence of mutations in the process of lymphomagenesis seems to be composed of at least 3 main hits which equip the cells with independence from external mitogenic signals (activation of Ras/Raf), resistance to inducers of apoptosis (activation of Bcl-2) and generation of cellular heterogeneity (deletion of p53) important in tumor progression.

Thymic lymphomas are resistant to Nur77-mediated apoptosis.

We reported previously that thymic lymphomas from mice expressing transgenic TCR autoreactive against male (HY) antigen were resistant to anti-CD3 antibody-mediated induction of apoptosis although they were responding to TCR triggering. To test whether thymic lymphomas were specifically resistant to TCR-dependent Ca(++)-mediated induction of apoptosis, we have measured apoptosis of cells treated with Ca(++)-dependent (ionomycin, A23187) and Ca(++)-independent (etoposide, dexamethasone) inducers of apoptosis. Here we show that, unlike thymocytes, all thymic lymphomas were resistant to Ca(++)-dependent but not to Ca(++)-independent induction of apoptosis. These results excluded a general defect of apoptosis in lymphoma cells and suggested a specific inhibition of the calcium-mediated (TCR-dependent) pathway of apoptosis in lymphomas. Interestingly however, nuclear expression of a specific mediator of TCR-dependent apoptosis Nur77 was induced in ionomycin-resistant lymphomas indicating that, unlike normal thymocytes, thymic lymphomas are resistant to Nur77-mediated apoptosis.

Loss of T cell receptor diminished tumorigenicity of thymocyte-derived lymphoma cells in the T cell receptor transgenic mice.

Mice with transgenic T cell receptor (TCR) recognizing H-Y male antigen developed spontaneous lymphomas originated from immature thymocytes, with the surface expression of transgenic TCR and CD4/CD8 co-receptors. During in vitro long-term culture (3 months) some lymphoma cell lines lost the surface expression of TCR and co-receptors. Interestingly, the proteins of transgenic receptor were expressed intracellularly but TCR was not detectable on the surface of the in vitro selected subline in contrast to TCR-positive parental cells maintained in vivo. TCR-negative subline has been found to be slowly growing in vivo (i.p. injection) and less tumorigenic (s.c. injection) than parental TCR positive lymphoma. It seems that the in vivo interactions of lymphoma cells with microenvironment preserve their TCR expression and endow with growth advantage, while the selected in vitro TCR-negative cells lose the tumorigenic potential.

Unexpected reaction of anti-H-2d serum with thymic lymphoma cells derived from transgenic B6-H-2b TCR anti-HY/Db mice.

Transgenic mice with alpha beta TCR specific for male antigen (HY) in the context of H-2Db MHC class I were prepared by introduction of transgens into (C57BL/6J x DBA/2J)F1 hybrid mice (H-2b/d), where only H-2b is selective for maturating thymocytes. Founder transgenic mouse was backcrossed to H-2b MHC background. Serological typing of H-2 antigens revealed that transgenic mice do not express H-2d antigens. Unexpectedly, cells of thymic lymphomas, spontaneously developed in about 45% of old B6 (H-2b) transgenic mice and peripheral blood lymphocytes of about 40% of transgenic mice, reacted with anti-H-2d serum (strain 129 mice (H-2b) immunized with thymocytes and splenocytes of BALB/c mice) but not with monoclonal antibodies against H-2d MHC class I antigens. Anti-H-2d serum has been shown to react with thymocytes but not peripheral blood lymphocytes from non-transgenic H-2b mice. The lymphocytes of transgenic mice reacting with anti-H-2d serum could represent disseminating preneoplastic or neoplastic cells expressing antigen encoded outside MHC region and present on the cell surface of immature thymocytes but not lymphocytes in healthy mice.

Interleukin 3-dependent, asialo-GM1-positive cell line with natural cytotoxic but without natural killer cytotoxic activity.

IL-3-dependent cell line (designated as A-3) from adherent spleen cells was developed. The surface phenotype of A-3 cells was analysed by flow cytometry and determined as ASGM1+, FcR+, Ia+. There was no expression of T cell markers (Th1-CD3-CD4-CD8-), B cell marker (surface Ig) as well as macrophage marker Mac-1 antigen. Although A-3 cells expressed ASGM1, they are lacking NK cytotoxic activity in 4 h Cr release assay with YAC-1 targets. In contrast, A-3 cells are endowed with NC cytotoxic activity as shown in 18 h Cr release assay with WEHI-164 target cells.

Non-cytotoxic asialo-GM1-positive cells exert antimetastatic activity.

Metastasis can be inhibited by asialo-GM1-positive spleen cells, and in this paper we show that there are two such spleen cell populations. One population is adherent and non-cytotoxic to YAC cells, whereas the other population is non-adherent and cytotoxic to YAC cells. Both cell populations exert an antimetastatic activity in cyclophosphamide-treated mice that are inoculated with LL2 Lewis lung carcinoma cells. We conclude that the antimetastatic activity is not only exerted by cytotoxic asialo-GM1-positive cells (apparently natural killer cells), but also by adherent, non-cytotoxic asialo-GM1+, Thy1.2-, IgG- cells. This means that the latter exert their antimetastatic activity by a non-cytotoxic mechanism.

Potentiation of cyclophosphamide-induced enhancement of experimental metastasis by splenectomy.

Role of spleen in CY-induced enhancement of experimental lung metastases of Lewis Lung Carcinoma LL2 cells was studied. Reconstitution with spleen cells abolished the enhancing effect of CY. Conversely, removal of spleen in CY treated mice caused about two-fold increase in the number of metastases. In addition in splenectomized mice, the effect of CY was augmented.