Natural Suppressor Cell Line Research Articles

Effects of apoptosis-inducing nucleosides released from CD57+HLA-DRbright natural suppressor cell line on human breast cancer cell death and growth

The CD57+HLA-DRbright-natural suppressor (57.DR-NS) cell line induced apoptosis in estrogen-non-responsive human breast carcinoma MDA-MB-435 cells by apoptosis-inducing nucleosides (AINs) released into the cultures. We obtained six active AINs isolated by high performance liquid chromatography (HPLC) from 57.DR-NS cell cultures. Each AIN isolated from 57.DR-NS cell cultures induced apoptosis in MDA-MB-435 cells. We found the occurrence of DNA strand breaks followed by the activation of caspase-3 during AIN-induced apoptosis in MDA-MB-435 cells. The data obtained here indicated that 57.DR-NS cells could induce apoptosis in MDA-MB-435 cells mediated by AINs through DNA strand breaks and activation of caspase-3. Furthermore, the administration of AINs into MDA-MB-435 tumor-bearing SCID mice culminated in strong suppression of tumor growth with no change of body weight of experimental mice suggesting no side effects of AINs.

Human prostate cancer cell death by novel anticancer compounds, apoptosis-inducing nucleosides from CD57+ HLA-DR(bright) natural suppressor cell line.

We validated the induction of apoptosis in human prostate cancer PC3 cells by apoptosis-inducing nucleosides (AINs) released from the CD57(+)HLA-DR(bright)-natural suppressor (57.DR-NS) cell line. We analyzed the molecular signaling pathway during AINs-induced apoptosis in PC3 cells. Direct and indirect co-cultures between 57.DR-NS and PC3 cells were performed. AINs were isolated by high-performance liquid chromatography (HPLC) from 57.DR-NS cell cultures. Apoptosis in PC3 cells was analyzed by DNA fragmentation, sub-G(1) DNA content with flow cytometry, and terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) method. The DNA strand breaks and activation of caspase-3 in PC3 cells were measured by DNA unwinding and flow cytometry assay. The 57.DR-NS cell line generated apoptosis in PC3 cells via AINs. AINs isolated from 57.DR-NS cell cultures induced apoptosis in PC3 cells. Furthermore, we found DNA strand breaks followed by activation of caspase-3 during AINs-induced apoptosis in PC3 cells. The data obtained here indicated that AINs could induce apoptosis in PC3 cells through DNA strand breaks and activation of caspase-3.

Human esophageal cancer cell death mediated by apoptosis-inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line.

We approached the induction of apoptosis in human esophageal cancer cells (T.Tn) by apoptosis-inducing nucleosides (AINs) from CD57+HLA-DRbright-natural suppressor (57.DR-NS) cell line originated in decidua. The 57.DR-NS cells generated apoptosis in T.Tn cells by AINs released into the cultures. We isolated a series of AINs by high performance liquid chromatography (HPLC) from 57.DR-NS cell culture fluids. Each AIN and its mixture induced apoptotic cell death in T.Tn cells, detected by DNA fragmentation in agarose gels, terminal deoxynucleotide transferase mediated dUTP-nick end labeling (TUNEL) method and accumulation of sub-G1 DNA content with flow cytometry. Furthermore, we found the occurrence of DNA strand breaks followed by the activation of caspase-3 during AINs-induced apoptosis in T.Tn cells. Thus, we validated that AINs could induce apoptosis in T.Tn cells mediated through DNA strand breaks and activation of caspase-3.

Human malignant cell death by apoptosis-inducing nucleosides from the decidua derived CD57 +HLA-DR bright natural suppressor cell line

The CD57 +HLA-DR bright natural suppressor (57.DR-NS) cell line derived from human decidual tissue mediated apoptosis of human leukemia Molt4 and carcinoma BeWo/GCIY cells but not human fibroblast WI-38 cells, and apoptosis-inducing nucleosides (AINs) appeared to be involved. Six AINs were released into 57.DR-NS cell culture media and were isolated by the combination of physicochemical procedures of C18 preparative column, thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). Subsequently, we demonstrated that AINs could induce apoptosis in the human malignant Molt4/BeWo/GCIY cell line but not human normal WI-38 fibroblasts. Apoptosis was characterized by DNA strand breaks and activation of the caspase cascade, especially caspase-3. The administration of AINs into GCIY tumor bearing SCID mice culminated in suppression of tumor growth due to apoptosis of tumor cells.

Active roles of caspase-3 in human gastric carcinoma cell death by apoptosis inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line.

The six apoptosis inducing nucleosides (AINs), which were isolated by high performance liquid chromatography from CD57+HLA-DRbright natural suppressor (57.DR-NS) cell cultures, induced apoptosis in human gastric carcinoma (GCIY) cells demonstrating the accumulation of sub-G1 DNA content and morphological changes. By means of DNA unwinding assay, it has been revealed that DNA strand breaks were initially involved in the apoptotic cell death of GCIY cells treated with AINs followed by activation of caspase cascades, especially caspase-3. Actually, the cleavage of fluorogenic substrate for caspase-3 was identified in the reaction. Whereas, the addition of caspase-3 inhibitor into the reaction prevented the cleavage of fluorogenic substrate for caspase-3 and resulted in the blockage of the sub-G1 DNA accumulation and DNA fragmentation as apoptotic signals. Thus, it was definitely elucidated that the activation of caspase-3 displayed a key feature during AIN-induced apoptosis in GCIY cells.

Human T cell leukemia cell death by apoptosis-inducing nucleosides from CD57(+) HLA-DR(bright) natural suppressor cell line.

Apoptosis‐inducing nucleosides (AINs) released from CD57+HLA‐DRbright natural suppressor (57.DR‐NS) cell line, derived from human decidual tissue, were isolated from 57.DR‐NS cell culture supernatant by the combination of thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). Apoptotic cell death was strongly induced in human T cell leukemia Molt4 cells treated with AINs, absolutely depending on DNA strand breaks, with activation of the caspase cascade, especially caspase‐3. The administration of AINs to Molt4 tumor‐bearing severe combined immunodeficiency (SCID) mice resulted in drastic suppression of tumor growth, with a decrease of tumor size and the appearance of apoptotic signals in tumor tissue. Thus, AINs are candidates for development as anticancer agents.

Activation of Caspase-3 in Molt4 Cells by Apoptosis-Inducing Nucleosides from CD57+HLA-DRbright Natural Suppressor Cell Line

Apoptosis-inducing nucleosides (AINs), which were released and isolated from CD57+HLA-DRbright natural suppressor (57.DR-NS) cell line derived from human decidual tissue, induced apoptosis in Molt4 cells. The addition of caspase-3 inhibitor into the reaction blocked the cleavage of caspase-3 and apoptosis in Molt4 cells treated with AINs, detected by flow cytometrical or spectrofluorometrical analysis and DNA fragmentation assay. Furthermore, by means of immunoblotting, the processing of caspase-3 was shown with the appearance of their catalytically active subunits of 20 and 11 kDa during the generation of apoptosis in Molt4 cells treated with AINs. This processing of caspase-3 into active subunits was also blocked by the addition of caspase-3 inhibitor. Thus, it was definitely revealed that the activation of caspase-3 was a key feature in the caspase cascade of AINs-induced apoptosis in Molt4 cells.

Apoptotic cell death of human gastric tumor induced by nucleosides from CD57+HLA-DRbright natural suppressor cell line.

CD57+HLA-DRbright natural suppressor (57.DR-NS) cell line derived from human decidual tissue generated apoptosis in a human gastric carcinoma cell line (GCIY) but not in a human diploid normal cell line (WI-38). The factors released from 57.DR-NS cells were purified by thin-layer-chromatography (TLC) and separated by high performance liquid chromatography (HPLC) into six components. The physicochemical structures of the six components were determined as a series of nucleosides and their modified forms collectively termed <apoptosis inducing nucleosides (AINs)> in a previous study. They could generate apoptotic cell death of GCIY malignant target cells, but not disturb the viability of WI-38 normal target cells. The administration of AINs into GCIY tumor bearing SCID mice resulted in drastic suppression of tumor growth followed by the decrease in tumor size due to the occurrence of apoptosis in tumor tissues.

Establishment of a natural suppressor cell line producing soluble suppressor factor other than transforming growth factor-beta.

Natural suppressor (NS) cell line (Clone 59) was established from the bone marrow of adult C3H/Hej mice in the presence of WEHI-3 conditioned media. Clone 59 cells suppressed the generation of cytotoxic T lymphocytes from normal mouse spleen. This suppression was seen at a responder-to-suppressor cell ratio of 1000:1 and lacked antigen specificity or MHC restriction. Clone 59 cells expressed the 'null' surface phenotype (Thy1.2-, CD3-, Lyt-2-, L3T4-, surface Ig-, MAC-1-) by immunofluorescent staining. Clone 59 cells exhibited no cytolytic activity against NK cell-sensitive YAC-1 and natural cytotoxic L929 target cell lines. Non-specific suppression, with a cell-free supernatant from the Clone 59-NS cells, also was observed. The supernatant did not inhibit [3H]-thymidine uptake by CTLL-2 cells which were proliferating in response to IL-2. Anti-transforming growth factor-beta (TGF-beta) monoclonal antibody had no effect on suppression, suggesting that the non-specific suppression is mediated by some soluble factors other than TGF-beta. Clone 59 cells may be useful in identifying non-specific suppressor cells in adult bone marrow and studying their functional role in the regulation of tolerance and self-reactivity.

Studies of Mammalian Implantation In Vitro. (I): Production of Laminin from Human Decidual Cell Line and Its Effects on Murine Trophoblastic Outgrowth.

TTK-1 cell line from human first trimester decidual tissue carried the surface markers such as HNK-1 (Leu 7) and HLA-DR. This cell line was strongly suspected of being human natural suppressor cell line of bone marrow or lymphoid tissue origin. In addition, the cells were found to express laminin on its cell surface, as examined by indirect immunofluorescence (IIF) test, and also secrete it into culture supernates as examined by enzyme linked immunoassay (EIA) and immunostaining. The addition of the supernates from this cell line into the murine embryo culture induced the trophoblastic outgrowth of embryos in vitro in the same manner as murine EHS sarcoma derived laminin did. This phenomenon was considered to be due to laminin in the UK-1 supernates because the effect was completely blocked by the addition of anti-laminin antibodies into the embryo culture.

Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor.

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.

Cloned natural suppressor cell lines derived from the spleens of neonatal mice.

The establishment and characterization of cloned natural suppressor (NS) cell lines derived from the spleen of neonatal BALB/c mice are described. Cloned NS cells suppress the mixed leukocyte reaction (MLR) between normal adult responder and stimulator spleen cells with a 50-fold greater efficiency than fresh neonatal cells. Suppressive activity of both cells did not depend on the haplotype of the responder or stimulator cells, and was radioresistant. Cloned NS cells did not inhibit the uptake of [3H]thymidine by HT-2 cells proliferating in response to interleukin 2 (IL-2), nor the in vitro secretion of IL-1 by macrophages in response to lipopolysaccharide. Several experiments indicated that absorption of IL-2 could not explain the suppression of the MLR by the NS cells in the range of cell numbers tested. The results suggest that NS cells may suppress the MLR by interfering with early stages of T cell activation. The cell surface of a cloned NS cell line was examined using immunofluorescence staining, and was strongly positive for the Thy-1.2, Ly-5, and asialo-GM1 antigens. However, Lyt-1, Lyt-2, surface Ig, IE, MAC-1, and Fc and C3 receptor markers were not detected. In addition, NS cells showed no cytolytic activity against the YAC-1 target cell line. On the basis of these findings, cloned NS cells do not appear to be mature T cells, B cells, macrophages, or NK cells. The development of cloned NS cells may be useful in determining the identity and mechanism of action of nonspecific suppressor cells in the neonatal spleen, and their role in neonatal tolerance and maternal-fetal relationships.