Characterization of Resistance to Rhabdovirus and Retrovirus Infection in a Human Myeloid Cell Line

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.     

Historical progress and the future of human cell culture research.

Progress in human cell culture research is discussed based primarily on our hematopoietic cell culture studies. The article includes a historical background of Burkitt lymphoma cell lines, discovery of EBV, normal B-lymphoblastoid cell lines with EBV, a variety of leukemia, lymphoma, and myeloma cell lines, clinical and theoretical contributions made by studies of T-cell leukemia cell lines, the discovery and clinical relevance of HTLV, HIV and HBLV, early attempts at adoptive immunotherapy of patients with cancer, and the future of human cell culture research. Despite the fact that current cell culture methods permit maintenance of only limited cell types of both normal and malignant origins, biotechnological advances such as hybridoma and recombinant DNA technologies should continue to provide unlimited research opportunities in all fields.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography.

Human placental trophoblasts, fibroblasts and the trophoblast-derived malignant cell JAR are potent producers of interferons (IFNs) when stimulated with Sendai virus. The three cell lines produced different levels and compositions of IFN-alpha subtypes and IFN-beta. Anti-IFN globulins, Cibacron Blue F3GA and Concanavalin A were covalently immobilized on pressure-stable, macroporous polymeric matrices derivatized with vinyl sulphone (HEMA-BIO 1000 VS and HEMA 1000 VS). These supports were packed in biocompatible PEEK columns and were coupled with switching valves, to develop a tandem high-performance affinity chromatographic (HPAC) method for the isolation, purification and biochemical characterization of the IFNs produced in Sendai virus-stimulated human placental trophoblasts, fibroblasts and trophoblast-derived malignant cell, JAR, cultures. Silver-stained SDS-PAGE and gel densitometric analysis revealed the purity of the purified proteins to be between 94 and 98%. Specific activities of the purified IFNs ranged between 0.37-2.76 x 10(8) IU/mg of protein with cumulative recoveries between 90 and 92.2%. The purified IFN components exhibited quantitatively different antiviral activities in human and bovine cell lines. The utility of the tandem method for the purification and characterization of human type 1 IFNs produced from other cell lines are also discussed.     

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.     

An antibody to de-N-acetyl sialic acid containing-polysialic acid identifies an intracellular antigen and induces apoptosis in human cancer cell lines

Polysialic acid (PSA), an α2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac), is developmentally regulated and its expression is thought to be restricted to a few tissues in adults. Recently, we showed that two human pathogens expressed a derivative of PSA containing de-N-acetyl sialic acid residues (NeuPSA). Here we show that an epitope identified by the anti-NeuPSA monoclonal antibody, SEAM 3 (SEAM 3-reactive antigen or S3RA), is expressed in human melanomas, and also intracellularly in a human melanoma cell line (SK-MEL-28), a human T cell leukemia cell line (Jurkat), and two neuroblastoma cell lines (CHP-134 and SH-SY5Y). SEAM 3 binding induced apoptosis in the four cell lines tested. The unusual intracellular distribution of S3RA was similar to that described for the PSA polysialyltransferases, STX and PST, which are also expressed in the four cell lines used here. Interestingly, suppression of PST mRNA expression by transfection of SK-MEL-28 cells with PST-specific short interfering RNA (siRNA) resulted in decreased SEAM 3 binding. The results suggest further studies of the utility of antibodies such as SEAM 3 as therapeutic agents for certain malignancies.     

Human leukemia-associated antigens: detection on cells of established lymphoblastoid lines.

Primate and rabbit antisera to different morphologic classes of human leukemia cells, after appropriate absorptions, detected leukemia-associated antigens present on cultured lymphoblastoid cell lines derived from leukemia patients. The primate antisera distinguished antigens on cells derived from myeloid leukemia patients from those on cells derived from lymphocytic leukemia patients. Of particular interest was the fact that antigens of myeloid leukemia, but not of lymphatic leukemia, were detected on lymphoid cell lines established from blood of patients with myeloid leukemia. One of four lymphoblastoid cell lines derived from normal donors expressed antigens of lymphatic leukemia. Leukemia-associated antigens were not found on the HRIK lymphoblastoid line derived from a Burkitt's lymphoma patient on skin fibroblasts or HeLa cells. Expression of these antigens on cultured cells derived from leukemia patients could not be related to the presence of the EB virus or the EB virus genome. Rabbit antisera detected antigens common to cells from patients with myeloid and lymphocytic leukemia. Absorption experiments demonstrated that the antigens detected on cell lines derived from leukemia patients are similar to those detected by the primate and rabbit antisera on fresh peripheral blood leukemic cells. The serologic detection of leukemia-associated antigens on lymphoblastoid cell lines indicates that some of these cultures contain cells with antigenic properties similar to those of human peripheral blood leukemic cells.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells(hESCs) can self-renew indefinitely and differentiate into all cell types in the human body.Therefore,they are valuable in regenerative medicine,human developmental biology and drug discovery.A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes.Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin.These hESCs express alkaline phosphatase and hE...     

Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.     

A high-resolution molecular-based panel of assays for identification and characterization of human embryonic stem cell lines

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies, particularly in view of the diverse methods for derivation and maintenance of these cell lines. However, characterization methods are generally not standardized and many currently used assays are subjective, making dependable and direct comparison of cell lines difficult. In order to address this problem, we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines, derived in two different laboratories using different derivation techniques, as pathogen free, genetically stable, and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control, a crucial element of successful hESC research and clinical translation.     

The effect of bone marrow stromal cells on the cloning of human leukemia/lymphoma cell lines

The cloning of established human leukemia and lymphoma cell lines at limiting dilutions in microwells and soft agar is described. Growth of most cell lines was improved by the addition of human AB serum and irradiated human bone marrow stromal cells. In general, the cloning efficiency in microwells was greater than in soft agar. The effect of bone marrow stromal cells appeared to be caused by a diffusable factor(s), but close cellular interaction could not be excluded since cloning in microwells produced consistent and optimal cell growth compared to growth in soft agar. It was concluded that cloning of leukemia and lymphoma cell lines in microwells was the preferred method and that similar techniques could improve the cloning of fresh leukemia and lymphoma cells.     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

Detection of the deletion of interferon-alpha and beta genes in lymphoblastoid cells by PCR]

The HuIFN-alpha and beta genes were examined by the PCR method in the 11 human lymphoblastoid cell lines. The results showed that the homozygous deletion of HuIFN-alpha and beta genes was detected in 5 of 11 cell lines and in 5 of 11 cell lines, respectively. The deletions of both the HuIFN-alpha and beta genes were observed in 4 of 11 cell lines. One T cell leukemia cell line deleted only HuIFN-alpha gene, while the other T cell leukemia line deleted only HuIFN-beta gene. This suggests that the deletions of HuIFN-alpha and beta genes may be related the development of leukemia or lymphoma.     

Human cell membrane components dominant in T cell lineage: identification and characterization of human TL-like antigens.

Cell membrane components that contain beta 2-microglobulin were purified from cells of a human T cell-type leukemia cell line, HPB-ALL. They contained membrane components that have the same molecular size and the same subunit structure as HLA(A,B,C) antigens but are separable from the typical beta 2-microglobulin-containing cell membrane components, i.e., the HLA (A,B,C) antigens, by xenoantibody reagents. A sensitive radioimmunoassay was constructed for detection of the T cell membrane components. The assay revealed that the cell membrane components are expressed exclusively on cells of T cell-type leukemia cell lines among the human lymphoid cell lines tested, predominantly in thymus, among the human organs and tissues tested. They were not present on cells of human B cell-type cell lines or on cells of nonlymphoid organs and tissues. No alloantibodies directed to the T cell membrane components, the putative human homologues of mouse TL antigens, were found in any of the human tissue typing sera tested.     

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells.     

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.     

Expression signatures of intragenic miRNAs and their corresponding host genes in myeloid leukemia cells

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in gene regulation. Approximately half of miRNAs are located within known genes and called intragenic miRNAs. 408 human intragenic miRNAs and their corresponding host genes were analyzed for genomic organization and functional characterization. Using quantitative real-time PCR, the expression levels of a subset of intragenic miRNAs and their host genes were examined in diverse myeloid leukemia cell lines, and their regulation in response to pharmacological stimuli was also evaluated. Expression of miR-22 strongly correlated with both myeloid leukemia subtypes and the expression of its host gene C17orf91 (p?<?0.05). The latter was absent in hematopoietic progenitors but abundant in erythroid and monocytic lineages. These results demonstrated that the expression signatures of miR-22 and C17orf91 are associated with developmental lineages and specific leukemia subtypes.     

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.