High expression of endogenous bcl-2 and bcl-xL in thymic lymphomas do not diminish their sensitivity to etoposide-induced apoptosis.

It was previously reported that thymic lymphomas of anti HY-TCR transgenic mice were resistant to ionomycin but were sensitive to 50 microM of etoposide. We selected lymphoma clones with strong expression of anti-apoptotic proteins Bcl-2 or Bcl-xL and found that in contrast to proteins encoded by exogenous genes in other model systems, over-expression of endogenous Bcl-2 and Bcl-xL proteins failed to protect against etoposide-induced apoptosis. Susceptibility of lymphoma cells was not diminished even to suboptimal concentration of 0.5 microM of etoposide. Treatment with etoposide did not decrease the ratio of anti-apoptotic Bcl-2 and Bcl-xL proteins to pro-apoptotic Bax protein. Results presented here suggest that high expression of Bcl-2 and Bcl-xL may not diminish susceptibility of T-cell lymphomas to chemotherapy with etoposide.

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.

Overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins is linked with resistance to TCR-mediated apoptosis and tumorigenesis in thymic lymphomas from TCR transgenic mice.

Mice with transgenic TCR anti H-Y/Db develop spontaneous thymic tumors with a high frequency (up to 50%). Oncogenicity of TCR transgenes could depend on the deregulated expression of oncoproteins engaged in transduction pathways leading to proliferation or apoptosis. In agreement with this possibility we have found that cells of thymic lymphomas from TCR transgenic mice were largely resistant to TCR-dependent Ca++-mediated apoptosis but not to TCR-independent, p53-mediated (etoposide) apoptosis. Here we show raised expression of Bcl-2 protein in some but not in all thymic lymphoma cell lines. It suggests that the antiapoptotic function of Bcl-2 is not necessary for the process of tumorigenesis and the resistance of these lymphomas to Ca++-mediated apoptosis. On the other hand we show that all thymic lymphomas overexpressed Ras/Raf and L-myc proteins. Stimulation of the Ras/Raf pathway was reported to be required to maintain cell viability by preventing programmed cell death in thymic tumors derived from lck transgenic mice. Similarly, in TCR transgenic lymphomas overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins may be responsible for the resistance of these lymphomas to TCR-mediated apoptosis but not affect p53-mediated apoptosis.

[The role of vav protein in TCR-mediated signaling with MHC/peptide complexes leading to positive or negative selection of thymocytes]. / Rola bialka vav w zamianie informacji o oddzialywaniach TCR z kompleksem MHC/peptyd na sygnaly inicjujace pozytywna lub negatywna selekcje tymocytów.

On the basis of recent reports we discuss the role of Vav in TCR-dependent signaling pathways. The Vav protein is GDP/GTP exchange factor for Rac, which initiates transduction of signals in JNK pathway. Upon stimulation of TCR by antigenic peptides, Vav associates with Zap-70 in TCR/CD3 signaling complex and becomes phosphorylated on Tyr-174 by tyrosine kinase Lck. The function of Vav is modulated by substrates and products of PI3-kinase activated by interaction of CD28 on thymocytes with B7 on antigen presenting cells. The PI3-kinase substrates inhibit activation of Vav, while the products enhance phosphorylation and activation of Vav by Lck. It seems that Vav functions in key point of TCR-mediated signaling pathway, which is regulated by costimulatory molecule (CD28) necessary for negative selection. The Vav-mediated integration of signals results in positive or negative selection of thymocytes.

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.

[Signaling pathways and their role in maturation and function of T lymphocytes]. / Szlaki przekazywania sygnalu i ich rola w dojrzewaniu i funkcji limfocytów T.

Signals delivered through the beta/gp33 (pre-TCR) and T-cell receptor alpha beta control proliferation and differentiation of thymocytes at two distinct control points of T cell maturation. Interaction between T-cell receptor (TCR) and peptide/MHC complex induce signaling pathways leading to activation of T cell. Signal transduction involves CD3 zeta phosphorylation by Lck tyrosine kinase and activation of ZAP-70 which regulates signaling pathways through PKC, Ca++ and Ras/Raf kinase cascade. Appropriate response of cell is preceded by integration of different signals in the nucleus.

Role of thymic selection in the development of thymic lymphomas in TCR transgenic mice.

To investigate the role of antigen receptor-mediated interactions in lymphomagenesis we have analyzed the influence of alpha beta TCR-mediated selection on the development of spontaneous thymic lymphomas, which appear with a high (up to 50%) frequency in mice expressing a transgenic TCR specific for the male antigen (HY) in the context of H-2Db molecules. To this end we compared the kinetics and the incidence of thymic lymphomas developing in females and males with selecting (H-2b) and non-selecting (H-2k) MHC molecules. The kinetics of development of thymic lymphomas was similar in positively selecting (H-2b females) and non-selecting (H-2k females and males) environments but significantly slower (P < 0.01) in the negatively selecting environment (H-2b male). Injection of lymphoma cells derived from a H-2b female into the thymus of a H-2b male resulted in strong, antigen-specific inhibition of growth, indicating that the slower kinetics of lymphomagenesis in H-2b males could be due, at least partially, to the sensitivity of oncogenically transformed thymocytes to TCR-mediated negative selection. Phenotypic and functional analysis of lymphoma cells indicated that they originated from the stage of pre-TCR-dependent transition of immature CD4-CD8- to CD4+ CD8+ thymocytes, which in H-2b females and males developed into tumors under different environmental pressures. These results failed to provide convincing evidence for the role of positive selection but provided a strong indication that self antigen-induced negative selection, in addition to its well established role in self tolerance, can occasionally act as a tumor surveillance mechanism by eliminating or suppressing growth of thymocytes undergoing oncogenic transformation.

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.

[Use of anti-H-Y/Db transgenic mice in studies on T-lymphocyte development and mechanisms of leukemogenesis]. / Wykorzystanie myszy transgenicznych TCR anty-H-Y/Db w badaniach nad rozwojem limfocytów T i mechanizmami leukemogenezy.

During the development of T cells in the thymus, the alpha beta T-cell receptor (TCR) mediated interactions of immature CD4+ CD8+ cells with thymic major histocompatibility complex (MHC) molecules in the absence of specific antigens are required for their survival and further maturation (positive selection) whereas interactions with MHC molecules presenting specific antigens result in their death by apoptosis (negative selection). Preliminary results suggest association of antigenic stimulation via TCR with leukemogenesis of TCR transgenic mice. TCR transgenic mice monitored over a period of 2 years revealed spontaneous leukemia development in mice of both sexes by the age of 8-12 months, with the surface expression of transgene and markers of T cell development pathway.

Mutation and altered expression of p53 genes in experimental rat bladder tumor cells.

p53 genes were analyzed for mutations and expression in a series of 24 tumors or hyperplastic lesions of the urinary bladder induced in F344 rats by carcinogen treatment. Of these, 18 were analyzed as short-term urothelial cultures. Polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing were used to detect alterations in p53 genes or cDNAs, and the relative amounts of p53 protein per cell were estimated by immunohistochemical staining. Missense substitutions were found in the exon 5-9 region of two of five cell cultures analyzed from lesions induced by the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. One of these was a papillary nodular hyperplasia, indicating that p53 mutations can be present in low- as well as high-stage/grade bladder lesions. p53 mutations were not found in the exon 5-9 region in cells of any of eight bladder lesions induced by N-[4-(5-nitro-2-furyl)-2- thiazoly]formamide (FANFT), including five transitional cell carcinomas (TCCs), or either of two TCCs induced by N-methylnitrosourea. Two of nine TCCs induced by the N-glucuronide of N-hydroxy-2-aminofluorene were found to have p53 mutations. One of these was evidently altered by three genetic events: a missense substitution in exon 8, a nonsense mutation in exon 6, and silencing of the "nonsense" allele (i.e., only the p53 missense mutation was detected). Immunohistochemical analysis with monoclonal antibody PAb240 (which preferentially binds to mutant p53 protein) detected p53 antigen only in those samples in which missense p53 mutations were found. With monoclonal antibody PAb421 (which detects mutant and wild-type p53), p53 antigen was also detected in cells from F542, a bladder tumor induced by FANFT in which no p53 mutations were found. Northern blot hybridization analysis showed that p53 transcripts were elevated twofold to threefold in several cases, including F542, suggesting that constitutive overexpression of wild-type p53 may occur in some bladder neoplasias. These data support the view that p53 may be involved in multiple rate-limiting steps in neoplastic transformation and may be a continuing target during bladder carcinogenesis. The data also contribute to evidence that certain chemical carcinogens may directly alter p53 genes during tumorigenesis.

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.

[Diversity of signal transduction pathways which induce apoptosis of thymocytes. Role of bcl-2, pim-1, c-myc and p53 genes in selection processes of thymocytes]. / Róznorodnosc szlaków przekazywania sygnalu do apoptozy tymocytów. Rola genów bcl-2, pim-1, c-myc i p53 w procesach selekcji tymocytów.

There are independent signaling pathways which transmit apoptotic signals in thymocytes. c-Myc and p53 proteins participate in the apoptosis induction, whereas Bcl-2 and Pim-1 proteins inhibit complex apoptotic machinery.

Antitumor activity evaluation of bromine-substituted analogues of ifosfamide. I. Stereodifferentiation of biological effects and selection of the most potent compounds.

The series of 9 compounds, including 3 racemates and 6 enantiomers of bromine-substituted analogues of ifosfamide (bromo-, chlorobromo- and dibromofosfamides) have been evaluated for antitumor activity against L1210 leukemia, Lewis lung carcinoma and B16 melanoma in mice. Effective and curative doses of tested compounds were estimated on the basis of computer-assisted elaboration of the dose-effect curves obtained from experimental data. Two oxazaphosphorine drugs, ifosfamide and its congener cyclophosphamide, were used as referentials. Elementary toxicity studies were conducted in parallel in healthy animals and lethal doses were determined. Selection of the most potent compounds was based on the comparison of their therapeutic indices, calculated from the ratio of lethal to effective doses. In effect four compounds which have been shown therapeutically more effective than both referential drugs, were selected for further evaluation in mice bearing advanced tumours. Stereodifferentiation of evaluated biologic effects favouring S(-) isomers was observed in all three groups of compounds. Preliminary observation was also made indicating significant lethality reduction after per os administration of selected agents, which was not paralleled by diminution of their antitumor effectivity.

Tumorigenicity and metastatic ability of MmB16 mouse melanoma cell line and its two Aleuria aurantia agglutinin resistant variants.

The availability of neoplastic cell lines with well defined growth characteristics has greatly facilitated study of the tumor phenotype, tumor progression and metastatic process. MmB16 cell line has been established in vitro from the B16 mouse melanoma serially passaged in C57BL/6 mice. From MmB16 cells two lectin-resistant (LecR) variants were selected with the use of Aleuria aurantia agglutinin (AAA). The correlation between the lectin resistance and their in vivo growth parameters, especially tumorigenicity and metastatic ability, were evaluated. The local tumor growth and the average survival time of mice after subcutaneous (s.c.) inoculation of AAAR variant cells did not differ significantly from those of the parent MmB16 cells. However, the AAAR variants revealed significantly higher experimental lung colonizing ability after intravenous (i.v.) administration and slightly increased spontaneous metastatic ability after s.c. inoculation, as compared to parent MmB16 cells.

Biological evaluation of mitoxantrone dihydrochloride synthesized by a new method. I. Acute toxicity and antitumor activity in mouse transplantable tumor systems.

A high antitumor activity of mitoxantrone dihydrochloride, synthesized according to a new method, was demonstrated in mice with i.p. growing tumors: P388 leukemia in CD2F1 and B16 melanoma in B6D2F1 hybrid strains. The preparation was ineffective when administered to mice with subcutaneously implanted solid tumors: Lewis Lung carcinoma, 16/C mammary adenocarcinoma or B16 melanoma. This finding is consistent with data reported by others for mitoxantrone produced by American Cyanamid Co. Acute toxicity of the tested compound was evaluated after single i.p. or i.v. administration to male and female CD2F1 and B6D2F1 mice.

Biological evaluation of mitoxantrone dihydrochloride synthesized by a new method. II. Comparison of Now 85/34 and Novantrone (Lederle) acute toxicity and antitumor activity.

A basic biological similarity of the newly synthesized mitoxantrone dihydrochloride preparation Now 85 with the Lederle's Novantrone was tested in simultaneously carried out comparative evaluations of acute toxicity and antitumor activity.

Comparative evaluation of acute toxicity and antitumor activity of IF-XXV preparation and holoxan.

The alkylating agent ifosfamide synthesized according to own method at the Institute of Pharmaceutical Industry, Warsaw, was compared in biological evaluations with Holoxan (Asta-Werke's ifosfamide). No significant differences between tested compounds were found in respect of acute toxicity and antitumor activity in experimental systems in mice.