The RAG cell line defines a new complementation group of MHC class II deficiency

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN- ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding humanCIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Comparative characterization of a human large cell lung carcinoma cell line and the xenograft derived cell line.

The HK-1 cell line established from a human large cell lung carcinoma shows a high transformed phenotype and undifferentiated characteristics. This cell line grows as an adherent monolayer in fetal calf serum-supplemented medium, shows a high proliferation index, is able to grow in semi-solid agar and is tumorigenic in athymic nude rats. The cell line HK-2 derived from the HK-1 xenograft in athymic nude rats shows basically the same features found in the original HK-1 cell line, which include aneuploid nuclear DNA content, abnormal chromosomal number. rearranged marker chromosomes and abnormally localised nucleolar organizer regions. Cytokeratin and vimentin intermediary-sized filaments were found in both cell lines as well as in the original and induced tumour, while neither oncofetal antigens (alphafeto-protein, carcinoembryonic antigen, chorionic gonatropin and human placental lactogen) nor neural differentiation markers (neurofilament and neural specific enolase) were expressed. Analysis of sulphated glycosaminoglycans in the cell cultures and in the nude rat induced tumour showed high expression of chondroitin sulphate.     

MHC expression in a human adult stem cell line and its down-regulation by hCMV US gene transfection

Due to their unique capacity to self-renew and for multiple differentiation, stem cells are considered promising candidates for cell replacement therapy in many devastating diseases. However, studies on immune rejection, which is a major problem facing successful stem cell therapy, are rare. In this study, we examined MHC expression in the M13SV1 cell line, which has previously been shown to have stem cell properties and to be non-tumorigenic, in order to determine whether human adult stem cells might be rejected after transplantation. Our results show low expression levels of MHC class I molecules on the surface of these cells. An induction of MHC class I expression was observed when the cells were treated with IFN-gamma. Maximal induction of MHC class protein expression was observed at 48 h after treatment with concentrations above 5 ng/ml of IFN-gamma. Elevated MHC class I levels were sustained for 72 h after withdrawing IFN-gamma. Therefore, we introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into M13SV1 cells. Cells transfected with the hCMV US2, US3, US6 or US11 genes exhibited a reduction (40-60%) of MHC class I expression compared with mock-transfected cells. These results suggest that human adult stem cells are capable of expressing high levels of MHC class I proteins, and thus may be rejected on transplantation unless they are modified. In addition, viral stealth mechanisms can be exploited for stem cell transplantation.     

Metabolism of vitamin D in the human choriocarcinoma cell line JEG-3

Calcitriol is an antiproliferative prodifferentiating secosteroid that exerts a protective role for some kinds of cancer. Alterations in 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) activity have been found in some tumor cells, but there are no studies performed in human choriocarcinoma. In the present work, calcitriol production and CYP27B1 gene regulation were studied in the human choriocarcinoma cell line JEG-3, and compared with normal human syncytiotrophoblasts (hS) in culture. In JEG-3 cells, secretion of [(3)H]calcitriol was significantly less (P<0.001) than in hS (45+/-17fmol/mg protein versus 174+/-87fmol/mg protein, respectively; n=8). CYP27B1 mRNA was similar in both JEG-3 and hS cells; but the protein was detected only in hS extracts. In contrast to the hS, JEG-3 CYP27B1 gene expression was not regulated by calcitriol or by a cAMP analogue. Our results indicate that in JEG-3 cells calcitriol production is diminished due to CYP27B1 dysregulation and low protein content, and suggest that hyperproliferation could be a consequence of these alterations.     

Dissociation in expression of MB1/MT1 and DR1 alloantigens in mutants of a lymphoblastoid cell line

Cytotoxicity tests with alloantisera were used to study the expression of HLA-D region antigens in HLA-DR-null mutants of a human lymphoblastoid cell line. The initial cell line contained just one copy of the MHC as a haplotype that included DR1 and MB1/MT1. Gamma ray mutagenesis of the single haplotype cells followed by selection with complement and an anti-DR monoclonal antibody were then used to isolate DR-null mutants. Two categories of mutants were identified with a panel of alloantisera. Expressions of DR1 and MB1/MT1 were simultaneously lost in four mutants. Nine mutants still expressed MB1/MT1 but had lost the expression of DR1. The dissociated loss of expression of MB1/MT1 and DR1 antigens is evidence for separate genetic control of these alloantigens. The methods used exemplify a versatile approach for conveniently inducing separations of closely linked loci of the MHC.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, we identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate that 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the mu chain of immunoglobulin is B cell specific. Incubation of specific MoAb with cultures of Laz 221 cells at 37 degrees C reduces or modulates surface expression of five of these 22 antigens (p45, immunoglobulin mu chain, transferrin receptor, common ALL antigen (CD10), and p105). Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. For example, three of the antigens present on Laz 221 cells were only identified by MoAb raised to the Burkitt's cell line Ramos and vice-versa. Only one of these six shared antigens is present in greater amounts on the immunogenic cell population. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.     

Regulation of class II gene expression in a murine pre-B cell line

Class II major histocompatibility complex (MHC) gene expression has been studied in an Abelson virus-transformed pre-B cell line R8, and its Ia-negative variant R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of trans-acting factors that can regulate class II gene expression. Furthermore, the T cell-derived lymphokine B cell stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.     

Characterization of a human stromal cell line supporting hematopoietic progenitor cell proliferation: Effect of HIV expression

Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ cell differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models.     

Abnormal stability of wild-type p53 protein in a human lung carcinoma cell line

We report here that ectopically expressed wild-type p53 protein showed more than 6 times longer half-life than normal human fibroblasts in NCl-H1299, a widely used cell line derived from non-small cell lung carcinoma lacking the expression of p53 protein. We found no abnormality in the phosphorylation and ubiquitination of p53, and the expression levels of MDM2. Although proteasome activity measured in vitro was not significantly different between the tumor cell line and normal human fibroblasts, proteasome inhibitors, ALLN, MG115, and MG132, did not accumulate p53 protein in the tumor cell line, but did accumulate p53 in normal human cells. These results provide a novel mechanism, by which p53 is stabilized in tumor cells, and they suggest that a mediator should exist between ubiquitinated p53 and proteasome, which may be defective in H1299 cells.     

Induced CD25 expression in a human B-lymphoma cell line transfected with the Epstein-Barr virus nuclear antigen 2 gene.

Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which plays a critical role in the EBV-induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co-transfected with the EBNA-2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA-2-expressing cell clones was analyzed by flowcytometry. None of the EBNA-2-expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA-2 in a different EBV-negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL-2R/Tac) on cell surface, however, was induced in two of six DG75-derived cell clones. One of the two CD25-induced cell clones was expressing EBNA-2 only, and the other was co-expressing EBNA-2 and LMP. The results suggest that EBNA-2 has a potential to up-regulate CD25 independently of CD23 on human B cells.     

The expression and activation of ERK/MAPK pathway in human esophageal cancer cell line EC9706

While there have been more and more studies concerning mitogen-activated protein kinases (MAPKs) signaling pathways, which control many cellular complex programmes, such as cell proliferation, differentiation, cell death and embryogenesis. However, few studies are carried out about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) in human esophageal cancer cell line. Therefore, in the present study, we investigated the expression and activation of ERK1/2 in human esophageal cancer cell line EC9706 and human normal esophageal epithelial cell line Heepic, which is as control. This study showed that ERK1/2 was transiently phosphorylated both in EC9706 and Heepic, the kinetics of which were slightly different. To further study the ERK/MAPK signaling pathway in EC9706 and Heepic cell line, U0126 a kind of specific inhibitor of MEK was used. This study showed that U0126 can block the phosphorylation of ERK1/2 in a short time, the complete inhibition concentration for EC9706 and Heepic cell line is 50 and 20 ??M, respectively. Incidentally, to further investigate the different roles of ERK1 and ERK2, vector-based short hairpin interference vectors targeted on ERK1/2 was constructed. Moreover, the effective interference target sequence was screened out in a transient transfection manner. MTT experiment showed that ERK2 is more important than ERK1 in the proliferation of EC9706 cells.     

Modification of the cell surface expression of histocompatibility antigens induced by the neurotoxin 2,5 hexanedione

Class I histocompatibility antigens (HLA) are expressed on the surface of almost all nucleated mammalian cells; the expression of this surface antigenic molecule may be changed or abrogated by several factors. In this paper, a modification in HLA expression in a human carcinoma cell line following exposure to the neurotoxicant 2,5 hexanedione is reported. This compound is known to produce a wide spectrum of subcellular pathological events; in this study, we describe an effect on the surface and cytoplasmic distribution of both light and heavy subunits of HLA antigens, demonstrated by immunocytochemical and immunoelectron microscopy techniques. Human carcinoma cells, which under normal growing conditions express the HLA, abrogate the surface expression of this glycoprotein after exposure to 2,5 hexanedione and an intracytoplasmic accumulation seems to occur. Several possibilities are discussed, such as an effect of the toxicant on the transport of the nascent glycoprotein.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - 2,5-HD 2,5 hexanedione - HLA Class I histocompatibility antigen - IU international unit     

Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line

The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.     

Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth

A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-gamma induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-beta receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Loss in transformed cells of cell cycle regulation of expression of a nuclear protein recognized by SLE patient antisera

The identification of cellular proteins involved in the control of cell proliferation in normal cells is essential for understanding the mechanism underlying growth regulation and cellular transformation. A nuclear protein termed Ki antigen with a relative mobility of 32,000 (Mr 32K) and which is recognized by SLE patient antisera has been identified in cells of human, bovine, and murine origin. Recently, cDNA clones for the bovine and human Ki antigens have been isolated using SLE patient antisera (T. Nikaido, et al., in preparation). The nucleotide sequence predicted a protein of 239 amino acids with a possible nuclear localization signal resembling that identified in SV40 T antigen and other nuclear proteins. Here we show that the expression of Ki antigen is regulated in the normal cell, but not in the transformed cell. Furthermore, in the K-ras temperature-sensitive mutant cell line, ts 371 normal rat kidney (NRK), Ki antigen expression increases several-fold at the permissive temperature relative to the nonpermissive temperature. These results suggest that expression of Ki antigen might be correlated with cellular transformation as well as with cell growth regulation.