Induction of class I major histocompatibility complex antigens in human teratocarcinoma cells by interferon without induction of differentiation, growth inhibition, or resistance to viral infection

The behavior of human teratocarcinoma cells, and especially their stem cells (embryonal carcinoma cells), may provide insights into the properties of human early embryonic cells. We report here that human recombinant gamma-interferon (IFN-gamma) induced the expression of major histocompatibility complex Class I (HLA-A, B, C) antigens and beta 2-microglobulin in the two human embryonal carcinoma cell lines, 2102Ep cl.4D3 and NTERA-2 cl.D1, and in the yolk sac carcinoma cell line, 1411H; human recombinant IFN-alpha and IFN-beta were less effective inducers of these cell surface molecules. No induction was observed in the gestational choriocarcinoma cell line, JAR. Neither IFN-alpha, IFN-beta, nor IFN-gamma caused growth inhibition, expression of major histocompatibility complex Class II (HLA-DR) antigens, resistance to viral (vesicular stomatitis virus) infection, or expression of 2',5'-oligo(A)synthetase in any of the cells. Also, IFN-gamma neither induced differentiation of NTERA-2 cl.D1 cells, which are pluripotent human stem cells, nor influenced their differentiation induced by retinoic acid. However, developmental regulation of responsiveness to IFN was evident, since IFN-gamma induced higher levels of surface expression of HLA-A, B, C and beta 2-microglobulin in the retinoic acid-induced differentiated NTERA-2 cl.D1 cells than in the undifferentiated parental cells. Also, 2',5'-oligo(A)synthetase was inducible in the NTERA-2 cl.D1 differentiated cells by IFN-alpha and -beta, although not by IFN-gamma, and slight resistance to vesicular stomatitis virus infection was evident in aged cultures of differentiated cells exposed to IFN-alpha. The effect of recombinant mouse IFN-gamma on major histocompatibility complex expression by several murine teratocarcinoma cells was also examined: H-2 Class I (H-2Db), but not class II (I-Ab), antigens were induced in the parietal yolk sac carcinoma lines, PYS and F9Ac cl.9; in cultures of PCC3/A/1 containing both embryonal carcinoma (EC) and differentiated cells; and in cultures of the EC cells, PCC4azaR and PCC4AO, without evidence of differentiation. No induction was observed in the murine EC cell lines, F9 or FA (H-2Kk). Our results indicate that human EC cells, like murine EC cells, exhibit only a partial response to the interferons, and that the extent of this response is developmentally regulated.     

Differentiation of F9 cells is independent of c-myc expression.

The level of c-myc expression rapidly decreases during in vitro induced differentiation of mouse F9 embryonic teratocarcinoma to endoderm cells, raising the question of whether down regulation of c-myc expression is a part of the mechanism regulating differentiation. We have investigated the effect of enforced increases or decreases in c-myc RNA expression in F9 cells on growth and differentiation. The enforced expression of c-myc RNA in clones transfected with sense c-myc did not inhibit their terminal differentiation. Dramatically decreased c-myc RNA expression in antisense c-myc transfected clones also did not substantially alter the differentiation pathway, although the transformed cells withdraw from the cell cycle slightly earlier than control cells during the differentiation induction. These results suggest that the mechanism controlling differentiation operates independently of the level of c-myc RNA expression in F9 teratocarcinoma cells.     

Characterization of interleukin 2-dependent cytotoxic T-cell clones: specificity, cell surface phenotype, and susceptibility to blocking by Lyt antisera

Cytotoxic T-cells were derived from the peritoneal cavity of a C57BL/6 mouse immunized with BALB/c sarcoma Meth A and from the spleens of BALB/c x C57BL/6 F1 (hereafter called CB6F1) mice immunized with BALB/c leukemia RL male 1. The cells were cultured in interleukin 2 and cloned by limiting dilution, and their specificity was determined by direct tests and competitive inhibition assays. C57BL/6 anti-Meth A effector cells recognized H-2Dd determinants. CB6F1 anti-RL male 1 effector cells recognized a unique cell surface antigen of leukemia RL male 1. The specificity was maintained in long-term culture. The cell surface phenotype of the cloned effector cell lines as determined by absorption analysis was Thy-1.2+, Lyt-1.2+, 2.2+, and 3.2+. Cytotoxicity was blocked at the target cell level by antisera against H-2Dd, but not H-2Dk or H-2b, and at the effector cell level by antisera against Lyt-2.2 and 3.2, but not Lyt-1.2, Ly-5.1 or Thy-1.2.     

High-molecular-weight glycoproteins of human teratocarcinoma defined by monoclonal antibodies to carbohydrate determinants

Three mouse monoclonal antibodies to distinct cell surface antigens were derived from immunizations with cells of Tera-1, a human teratocarcinoma cell line, and a membrane preparation of placental tissue. The distribution of the antigens on 165 cultured lines of various human tumors and normal cells was determined by mixed hemadsorption assays and on fresh tissues by immunofluorescence staining. K4 antigen is expressed on cell lines derived from teratocarcinomas but not on any other cultured cell tested. Normal adult colonic epithelium, some fetal tissues, and specimens of testicular teratocarcinoma were also K4 positive. K21 antigen was detected on teratocarcinoma cell lines and, at more than 100-fold lower levels, on cultures of normal and malignant kidney epithelium but not on other cultured cells. K21 expression in normal tissues is restricted to the epithelium of fetal intestine and bronchus. Other fetal tissues and all adult normal tissues tested lacked K21. A subset of teratocarcinoma specimens (5 of 8) was reactive with antibody K21. P12 antigen is represented on a wide range of cell lines and tissues, including a subset of teratocarcinomas. AbK4, AbK21, and AbP12 react with carbohydrate sequences present on high-molecular-weight glycoproteins. AbK21 and AbP12 recognize the lacto-N-tetraose and lacto-N-fucopentaose III (X-hapten) structures, respectively, whereas AbK4 reacts with a neuraminidase-sensitive determinant.     

Suppression of tumorigenicity in temperature-sensitive mutants of embryonal carcinoma cells by induction of cell differentiation at non-permissive temperature

The temperature-sensitive (ts) mutants of cell differentiation derived from the mouse teratocarcinoma cell line F9, when exposed to non-permissive temperature, undergo stem-cell differentiation concomitant with a transient retardation of cell-cycle progression. By incubating these mutant strains at non-permissive temperature, we were able to study the relationship between cell differentiation and tumorigenicity. Upon exposure to non-permissive temperature, the mutant cells undergo extensive differentiation and lose their ability to initiate tumors in vivo, but retain their in vitro proliferative potential. Our data suggest that the loss of tumorigenicity is not caused by altered proliferative potential, but rather by cell differentiation. We therefore suggest that the loss of proliferative potential and the onset of cell differentiation in teratocarcinoma F9 cells are 2 independent events which can be genetically dissected, and that there is (a) crucial step(s) in the differentiation pathway at which these ts mutant cells lose their tumorigenicity.     

Control of radiosensitivity of F9 mouse teratocarcinoma cells by regulation of histone H2AX gene expression using a tetracycline turn-off system

The mouse histone H2AX has unique COOH-terminal serine residues that are phosphorylated in response to double-strand DNA breaks introduced by ionizing radiation. This suggests that H2AX acts to maintain genomic stability. We constructed a tetracycline (tet)-directed turn-off vector and integrated it into F9 mouse teratocarcinoma cells by homologous recombination. In homozygously recombined cells, expression of the histone H2AX gene was repressed to 0.02% of the expression observed in wild-type cells by the addition of doxycycline, an analog of tet. Sensitivity of cells with repressed H2AX expression to X-irradiation was increased 1.95x, indicating that DNA repair was impaired by repression of H2AX. When we s.c. injected tet-regulated F9 cells into the flanks of mice, tumor growth was slightly suppressed by X-irradiation in H2AX-repressed tumors, whereas without X-irradiation, tumor growth did not differ by H2AX status. Thus, H2AX might be a potential molecular target for sensitizing cancer cells to radiotherapy to minimize required irradiation doses.     

Cessation of autonomous proliferation of mouse lymphoma EL4 by fusion with a T cell line

Benzanthracene-induced C57BL/6 (H-2b) mouse T-cell lymphoma EL4 (a thymidine kinase-deficient cell line) was fused by using polyethylene glycol with an Mlsa (Mls for minor lymphocyte stimulatory) antigen-dependent T cell line, which was designated G4 and had been derived from a C3H/He mouse (H-2k), and the fused cells were cultured in HAT medium. Although no growing cells appeared in most of these fusions, we consistently obtained growth-arrested H-2Kb-positive cells from the fused cell populations by the panning method. The cells were tetraploid and were able to proliferate in response to Mlsa antigen. Three H-2Kb-positive clones, isolated by limiting dilution from three different fusions, were shown to be EL4 x G4 hybrids, because (1) they had both H-2k and H-2b antigens; (2) each of the clones had one submetacentric chromosome which was a marker chromosome of EL4, and they were tetraploid with modal chromosome numbers of 74, 78, and 79, respectively; (3) they had 4 isozymes of both parental cells. These results indicate that EL4 lymphoma cells cease to proliferate when fused with T cell line G4. The malignant phenotype of lymphoma EL4 is thus suppressed at the level of cell transformation by the introduction of the G4 cell genome.     

Induction of antigen specific cellular immunity by vaccination with peptides from MN/CA IX in renal cell carcinoma

We investigated the induction of the specific immunity for renal cell carcinomas (RCC) using MN/CA IX, a tumor-associated antigen frequently expressed in RCC. We have generated 9-mer peptide derived from MN/CA IX and examined the antigenicity as a vaccine to induce specific immunity for RCC. To use mouse syngeneic system, we transfected human MN/CA9 cDNA into RenCa and BALB-3T3 cells originally from BALB/c mouse, and established MN/CA IX expressing mouse cell lines, i.e., MN-RenCa and MN-3T3. The immunization of BALB/c mouse with MN-RenCa cells resulted in the induction of cytotoxic T lymphocytes (CTL) against MN/CA IX expressing cells and the CTL clone was established from bulked CTL. This CTL clone specifically lyzed MN-3T3 cells, but not parental cells. To identify the targeted epitope binding to H-2Kd antigen, three 9-mer peptides (A, B, C-peptide) of human MN/CA IX compatible with the H-2Kd as well as HLA-A24 binding motif was synthesized. The cloned CTL targeted the B-peptide pulsed BALB-3T3 cells as well as MN-3T3 cells. Furthermore, spleen cells from BALB/c mouse immunized with B-peptide reacted against MN-RenCa cells. These results suggest that the peptides derived from MN/CA IX containing HLA-A24 binding motif may be useful as a potent tumor vaccine for the treatment of human RCC, and in mouse models.     

Studies on the humoral regulation of granulopoiesis in leukaemic RFM mice.

Intraperitoneal diffusion chambers have been used to investigate changes in humoral factors during the development of myeloid leukaemia in mice. Normal mouse bone marrow cells form colonies of granulocytes and macrophages when cultured in semi-solid agar medium within intraperitoneal diffusion chambers. The use of mice bearing transplanted myeloid leukaemia as Agar Diffusion Chamber (ADC) hosts enhances colony formation from normal marrow. The humoral basis for this stimulation has been shown by the colony stimulating activity of the fluid entering the diffusion chambers when assayed against normal mouse bone marrow cells in agar culture in vitro. The stimulus to colony growth in ADCs and the in vitro colony stimulating activity depend on the phase in the development of the leukaemia investigated, and the stimulation was abolished by splenectomy. There was no apparent relationship between the growth of the leukaemic cell population in vivo and the level of the stimulating factor detected in leukaemic mice.     

Comparison of primary hepatocytes and S9 metabolic activation systems for the C3H-10T 1/2 cell transformation assay

Three metabolic activation systems, primary rat hepatocytes,primary mouse hepatocytes and Aroclor 1254-induced rat liverS9 fraction were examined as exogenous activation systems forthe C3H-10T 1/2 cell transformation assay. Under the conditionsof the assay the primary mouse hepatocytes were more effectivethan the rat S9 fraction in mediating the transformation ofC3H-10T 1/2 cells by the antineoplastic drug cyclophosphamide.However, the S9 fraction was more consistent than the mousehepatocytes in the activation of dimethylnitrosamine. The primaryrat hepatocytes were ineffective for activating either cyclophosphamideor dimethylnitrosamine in the transformation of C3H-10T 1/2cells. The presence of mouse hepatocytes, but not the S9 fraction,inhibited transformation of C3H-10T 1/2 cells by 3-methylcholanthrene.These results demonstrate that the three systems were differentiallyeffective in the activation of procarcinogens.     

Persistence of the immunoprotective effects of leukemia X fibroblast hybrid cells toward leukemia in histocompatible mice

Hybrids of ASL-1 murine leukemia cells and LM(TK-) cells, a cultured line of mouse fibroblast origin, stimulate partial immunity toward ASL-1 cells in (A/J X C3H/HeJ)F1 mice (F1 mice). Such mice ordinarily exhibit no resistance to the malignant proliferation of ASL-1 cells. Unprotected animals invariably die within 14-18 days after receiving as few as 200 ASL-1 cells. The hybrid cells, the mice used in the experimental studies and the leukemia cells used for challenge all share the same histocompatibility antigens. ASL-1 cells are H-2a; LM(TK-) cells are H-2k, both ASL-1 X LM(TK-) hybrid cells and A/J X C3H/HeJ)F1 mice are H-2a/k. The long-term persistence of the immunoprotective properties of the hybrid cells toward murine leukemia was investigated by using cells that had been in continuous culture for approx. 36 months. (A/J X C3H/HeJ)F1 mice injected previously with hybrid cells in continuous culture and then challenged with up to 10(7) ASL-1 cells survived longer (p less than 0.001) than mice who had not received hybrid cells previously. Some mice challenged with lesser number of ASL-1 cells survived indefinitely (greater than 200 days). The median survival time of F1 mice injected simultaneously with 10(7) hybrid cells and 200 or 2000 ASL-1 cells was significantly (p less than 0.001) prolonged as well, although the differences between experimental and control groups are less pronounced than if the hybrid cells were injected before challenge with ASL-1 cells. The hybrid cells like those freshly prepared continue to be rejected by histocompatible precipients. In no instance has there been evidence of a progressively growing tumor of hybrid cells in immunocompetent F1 mice. Hybrid cells like those investigated previously do form rapidly growing metastasizing tumors in immunodeficient nu/nu (BALB/c) or X-irradiated (550 R) F1 mice. The cells possess approx. 70 chromosomes (reduced from 85) including chromosomes identified as having originated in each parental source. Like (A/J X C3H/HeJ)F1 animals, they continue to express both H-2a and H-2k antigenic determinants.     

Mouse cellular retinoic acid binding protein: cloning, complementary DNA sequence, and messenger RNA expression during the retinoic acid-induced differentiation of F9 wild type and RA-3-10 mutant teratocarcinoma cells

Retinoic acid, a natural derivative of vitamin A (retinol), induces mouse F9 teratocarcinoma stem cells to differentiate into nontumorigenic parietal endoderm cells. The mouse cellular retinoic acid binding protein (CRABP) has been implicated in the mechanism of action of retinoic acid (RA), since a mutant F9 cell line, RA-3-10, which possesses less than 5% of the wild type level of [3H]RA:CRABP binding activity, fails to differentiate in response to RA. In order to study the CRABP in this RA-induced differentiation process, we have cloned and sequenced the full-length mouse CRABP complementary DNA and have characterized its expression in wild type F9 and mutant cells. The mouse CRABP mRNA is a single, low abundant mRNA approximately 800 bases in length. The steady state level of the CRABP mRNA was measured in untreated stem cells and after the addition of RA alone, dibutyryl cyclic AMP plus theophylline (CT), or retinoic acid, dibutyryl cyclic AMP and theophylline (RACT) to F9 wild type and the mutant RA-3-10 cells. The CRABP mRNA was present in wild type F9 stem cells, and the level of its expression was changed by RA. When RA was added to F9 wild type cells, the steady state level of CRABP mRNA decreased 2- to 3-fold. When RACT was added to wild type cells, the level of CRABP mRNA increased and then decreased, resulting in a peak of CRABP mRNA expression between 24 and 48 h. In contrast, untreated mutant RA-3-10 cells had a lower level of CRABP mRNA than wild type stem cells, and the mutant cells responded quite differently to the addition of RA and RACT. The addition of RA caused an impressive 60-fold increase in the steady state level of CRABP mRNA in RA-3-10 cells by 120 h. One interpretation of this result is that there is negative regulation of CRABP mRNA expression, mediated directly or indirectly by the wild type functional CRABP protein, and that this regulation is aberrant in the RA-3-10 cells.     

Characterization of a human ovarian teratocarcinoma-derived cell line

A cell line (PA I), derived from human ovarian teratocarcinoma cells, was obtained by culturing ascitic fluid cells from a patient with recurrence of malignant ovarian teratoma. During early passages the cultured cells showed a variable morphology, a long doubling time, and a low plating efficiency (2 %). After about 50 passages in vitro, a cell population which was more homogenous and resembled embryonal carcinoma cells were obtained. These cells had a shorter doubling time (26 h), and increased plating efficiency (77 %). The early-passage cells were aneuploid (P 24) whereas the late-passage cells had a normal diploid karyotype with one balanced translocation between chromosomes No. 15 and No. 20 (P 224). Details of the karyotype suggest that the cells are heterozygous, i.e. derived from a stage before the first meiotic division. One of the two X chromosomes was inactive, and the cells expressed HLA antigens (A28 and B12), and β₂-microglobulin. Expression of F9 antigen, characteristic of two-cell and later preimplantation embryos, was absent, while expression of PCC4 antigen, expressed also by blastocysts, was present. This finding suggests that the line might express some embryonic characteristics. The PA I cell line maintained in monolayer cultures showed several characteristics of malignant cells. The proportion of malignant cells increased with successive passages in vitro. The late-passage cells represented a fairly homogenous population of malignant cells similar to embryonal carcinoma cells. Late-passage PA I cells, when seeded under conditions that prevented attachment of cells to the substratum, formed embryoid bodies consisting of an inner core of cells similar to embryonal carcinoma cells, surrounded by a rind of endoderm-like cells. These two cell layers were separated by a basement membrane-like structure containing fibronectin. The core embryonal carcinoma cells expressed high alkaline phosphatase activity whereas the endoderm-like cells had low alkaline phosphatase activity. Embryoid bodies seeded on an adhesive substratum formed polycystic structures divided by layers of epithe-lial-like cells and containing extracellular fibrils similar to collagen type I or III. In these cultures, further limited differentiation into endoderm-like, epithelial-like cells and pigmented cells was observed. Morphological differentiation of undifferentiated PA I cells into endoderm-like cells in monolayer cultures could be obtained by treatment with BrdUrd or by plating in low serum concentration and at low density. Cells with characteristic fibrillar distribution of fibronectin and actin microfilament bundles were then observed, indicating formation of cells lacking properties of malignant cells. As indicated by these results, the PA I cell line, in spite of a limited capacity to differentiate in vitro, shares some of the properties of mouse teratocarcinoma cell lines and might therefore serve as a useful model for studies on some developmental mechanisms in human cells.     

The sensitivities of human and murine hemopoietic cells exposed to cytotoxic drugs in an in vivo culture system.

An agar diffusion chamber technique has been used to measure the sensitivities of human and murine hemopoietic colony-forming cells to cytotoxic drugs. The cells were held in i.p. diffusion chambers and exposed to the cytotoxic drugs by i.v. injection of the host mice. This method allows some account to be taken of the continuous changes in activity during the metabolic degradation of the drug. To determine how far this system provides a valid measure of the sensitivity of the cells in hemopoietic tissue, the responses of mouse bone marrow exposed to the drugs in situ in the donor mouse were compared with those of mouse cells exposed in diffusion chambers. The dose-response curves for cyclophosphamide and 5-fluorouracil were exponential in all cases. Exponential survival curves were also seen when human and mouse colony-forming cells were exposed to vinblastine or methotrexate in diffusion chambers. The plateaus seen when mouse cells were exposed to these drugs in situ could, however, be regained by omitting agar from the chambers during the exposure period. The results indicate that there are differences between the sensitivities of human and mouse marrow cells to cytotoxic drugs and that any extrapolation from mouse to humans must be viewed with caution.     

Antigenic phenotype of NIH 3T3 cell line

The antigenic phenotype of the NIH 3T3 cell line was examined by use of a panel of monoclonal antibodies and alloantisera specific for H-2 and several non-H-2 antigens. The binding of antibodies to cell surface antigens was examined by a complement-dependent microcytotoxicity test and indirect immunofluorescence quantitated by flow cytometry. The phenotype of the tested NIH 3T3 cell line was H-2q, Qa-2, Ly-6.2, Thy-1.2, Ly-23.2, and 9F 3+. The expression of H-2 antigens (Kq, Dq/Lq) was lower than that in the T-lymphocytes of the B10.Q (H-2q) strain, and the expression of Qa-2 was very low. From the Ly-6 complex, the antigen Ly-m6.2A was strongly expressed, while Ly-m.6B, Ly-m.6C, and antigens ThB and H9/25 were not detected. A monoclonal antibody specific for the nonpolymorphic antigen 9F 3 brightly stained 100% of NIH 3T3 cells. Inasmuch as the NIH 3T3 immortalized cell line was developed from outbred NIH Swiss mice, a syngeneic recipient for this cell line does not exist. However, on the basis of the determined antigenic phenotype the host most compatible to NIH 3T3 cells can be selected for in vivo experiments, where an immunocompetent recipient is required. Two inbred mouse strains identical with NIH 3T3 cells in the antigens examined in this study, B10.Q and DBA/1, are of potential use for transplantation with NIH 3T3 cells.     

Molecular properties of F9 embryoglycan recognized by a unique antibody in sera from patients with germ cell tumors

Human antibody against an embryoglycan present on a mouse teratocarcinoma cell line F9 was found in sera from 16 of 29 patients with embryonal carcinoma, yolk sac tumor, immature teratoma, and choriocarcinoma of gonadal and extragonadal origins by Farr assay. In contrast, none of the sera from patients (77 cases) with dysgerminoma, seminoma, germinoma, and mature teratoma or from patients (118 cases) with nongerm cell types of ovarian tumors contained this antibody. The antigenic embryoglycan was of high molecular weight (Mr greater than 70,000) on Sephacryl S300 column chromatography and carried binding sites for Grifonia simplicifolia agglutinin-1. The antigenic embryoglycan was also found in F9 cell-cultured medium. Absorption of patients' sera with synthetic Blood Group B trisaccharides failed to remove the antibody against F9 embryoglycan. None of these patients' sera showed higher hemagglutination titer to rabbit erythrocytes than the normal range. In contrast, alpha-galactosyl carbohydrates obtained from Ehrlich ascites tumor cells effectively inhibited the binding of patients' sera with F9 embryoglycan. These results indicate that the human antibody against F9 embryoglycan recognizes alpha-galactosyl structures that are distinct from B blood group antigen, but are cross-reactive with alpha-galactosyl structures on Ehrlich ascites cells.     

MUC2 expression is regulated by histone H3 modification and DNA methylation in pancreatic cancer

Mucins are highly glycosylated proteins that play important roles in carcinogenesis. In pancreatic neoplasia, MUC2 mucin has been demonstrated as a tumor suppressor and we have reported that MUC2 is a favorable prognostic factor. Regulation of MUC2 gene expression is known to be controlled by DNA methylation, but the role of histone modification for MUC2 gene expression has yet to be clarified. Herein, we provide the first report that the histone H3 modification of the MUC2 promoter region regulates MUC2 gene expression. To investigate the histone modification and DNA methylation of the promoter region of the MUC2 gene, we treated 2 human pancreatic cancer cell lines, PANC1 (MUC2-negative) and BxPC3 (MUC2-positive) with the DNA methyltransferase inhibitor 5-azacytidine (5-aza), the histone deacetylase inhibitor trichostatin A (TSA), and a combination of these agents. The DNA methylation level of PANC1 cells was decreased by all 3 treatments, whereas histone H3-K4/K9 methylation and H3-K9/K27 acetylation in PANC1 cells was changed to the level in BxPC3 cells by treatment with TSA alone and with the 5-aza/TSA combination. The expression level of MUC2 mRNA in PANC1 cells exhibited a definite increase when treated with TSA and 5-aza/TSA, whereas 5-aza alone induced only a slight increase. Our results suggest that histone H3 modification in the 5' flanking region play an important role in MUC2 gene expression, possibly affecting DNA methylation. An understanding of these intimately correlated epigenetic changes may be of importance for predicting the outcome of patients with pancreatic neoplasms.     

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Background:

Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2.

Methods:

We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC.

Results:

Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells.

Conclusions:

Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells.     

Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. I. Virus replication in lymphocytes infected with Friend virus and cultures in diffusion chambers in vivo.

A new technique for infection of mature lymphocytes with murine leukemia virus (Friend) MuLV-F) is described. Spleen cells for normal, non-infected donors were placed into diffusion chambers (constructed with 0.22 mum por size Millipore filters) which were then implanted into the peritoneal cavities of normal syngeneic recipient mice. The cells were infected with an injection of MuLV-F into the peritoneal cavity and, in some instances, also by placing virus into the chambers. Cells were recovered by treating the chamber content with elastase and collagenase. The infection was determined in two ways: (1) cells with replicating MuLV were enumerated as infection centers (IC) on S+L- indicator cells; and (2) virus-related cell membrane antigen (MA) was detected by immunofluorescence. Cells recovered from chambers after 2-3 weeks of culture represented about 10% of the original inoculum; viability was approximately 90%. The number of IC in MuLV-F-infected chambers was about 10 times higher than obtained by infection and cultivation of spleen cells in vitro. The kinetics of IC and MA in chamber-cultured. MuLV-F-infected spleen cells was similar to that in the spleen of infected mice during the first 10 days after infection. Later on, the process of infection within the chambers slowed down, reaching about 50% MA-positive and about 10% IC-positive cells, whereas the number of both IC- and MA-positive cells in the spleen reached 80% or more. The infection of splenic lymphocytes in diffusion chambers occurred equally well when chambers were implanted into: (1) syngeneic, virus susceptible hosts; (2) syngeneic, lethally irradiated hosts; and (3) allogeneic, virus-resistant hosts, suggesting that the process within the chamber is independent of MuLV replication in the tissues of the chamber-bearing mouse. The diffusion chamber technique seems to provide an environment in which various types of isolated lymphocytes of different mouse strains can interact with MuLV almost as efficiently as in vivo.