Hybrid Cell Lines Research Articles

Development of a Diagnostic Kit to Detect Cryptosporidium parvum and Giardia lamblia

ObjectivesThis study aims to develop a high-sensitivity antibody diagnostic kit that will enable a rapid and accurate detection of Cryptospofidium parvum and Giardia lamblia in patients with diarrhea. MethodsThe cultivated C. parvum oocysts and G. lamblia cysts in each calf and dog were injected to mice to obtain antibodies, which were titrated. Spleen cells of the immunized mouse were separated and blended with myelomas to produce hybrid cell lines that form monoclonal antibodies. Using ELISA method, antibodies that specifically respond to C. parvum and G.lamblia were then selected. The cells were injected into the abdominal cavity of a BALB/c mouse to isolate hydrops abdominis containing high level of antibodies. The IgG antibody was purified using protein G gel. ResultsThe detection limit of monoclonal antibodies for Cryptosporidium parvum and Giardia lamblia was 125 oocysts/mL and 1250 cysts/mL, respectively. In addition, during testing they did not show cross-reactivity to viruses (n = 15), bacteria (n =17), and parasites (n = 9). ConclusionThe rapid diagnostic antibody kit developed in this study, which specifically responds to C. parvum and G. lamblia, will be useful in detecting and monitoring diarrheal infections.

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0°C and 37.0°C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0°C to 33.0°C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0°C. The highest maximum antibody concentration was obtained at 37.0°C and pH6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration

Alpha-motoneurons appear to be exceedingly affected in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Morphological and physiological degeneration of this neuronal phenotype is typically characterized by a marked decrease of neuronal markers and by alterations of cholinergic metabolism such as reduced choline acetyltransferase (ChAT) expression. The motoneuron-like cell line NSC-34 is a hybrid cell line produced by fusion of neuroblastoma with mouse motoneuron-enriched primary spinal cord cells. In order to further establish this cell line as a valid model system to investigate cholinergic neurodegeneration, NSC-34 cells were differentiated by serum deprivation and additional treatment with all-trans retinoic acid (atRA). Cell maturation was characterized by neurite outgrowth and increased expression of neuronal and cholinergic markers, including MAP2, GAP-43 and ChAT. Subsequently, we used differentiated NSC-34 cells to study early degenerative responses following exposure to various neurotoxins (H2O2, TNF-α, and glutamate). Susceptibility to toxin-induced cell death was determined by means of morphological changes, expression of neuronal marker proteins, and the ratio of pro-(Bax) to anti-(Bcl-2) apoptotic proteins. NSC-34 cells respond to low doses of neurotoxins with increased cell death of remaining undifferentiated cells with no obvious adverse effects on differentiated cells. Thus, the different vulnerability of differentiated and undifferentiated NSC-34 cells to neurotoxins is a key characteristic of NSC-34 cells and has to be considered in neurotoxic studies. Nonetheless, application of atRA induced differentiation of NSC-34 cells and provides a suitable model to investigate molecular events linked to neurodegeneration of differentiated neurons.

791 PROSTATE CANCER PROMOTING MUTATIONS OF THE COI GENE ASSOCIATED WITH G PROTEIN DYSREGULATION

You have accessJournal of UrologyProstate Cancer: Basic Research (IV)1 Apr 2013791 PROSTATE CANCER PROMOTING MUTATIONS OF THE COI GENE ASSOCIATED WITH G PROTEIN DYSREGULATION Qian Sun, Rebecca S. Arnold, and John A. Petros Qian SunQian Sun Atlanta, GA More articles by this author , Rebecca S. ArnoldRebecca S. Arnold Atlanta, GA More articles by this author , and John A. PetrosJohn A. Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.355AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inherited missense mutations of the mitochondrially encoded cytochrome oxidase 1 (COI) gene are present in 25% of prostate cancer patients. Laboratory analysis of one such prostate cancer ûassociated mutation (T6124C; Met74Thr) demonstrated that the mutation caused increased cell proliferation and in vivo tumor growth. To better understand the cell signaling events that this mutation produces we performed whole genome expression profiling of mutant and wild type cancer cell lines. METHODS The T6124C mutation of the COI gene was transferred from a prostate cancer patient's peripheral blood cells to a strong cancer nucleus (134B cells) that were devoid of their native mtDNA in a cytoplasmic transfer protocol to make cytoplasmic hybrid (cybrid) cell lines that contained either pure mutant or pure wild type mitochondria. RNA was prepared followed by fluorescent RT-PCR and hybridization to Agilent whole genome expression chips. Differences in gene expression were determined by averaging signal intensity for the two wild type cell lines and comparing to the average of the two mutant cell lines. D-score, q-value and fold change were used to determine significant findings. RESULTS The most highly induced gene was RGS1 (regulator of G-protein signaling 1) that was three logs higher in mutant cells. RGS1 is highly expressed in a broad variety of cancers including cervical, melanoma, renal cell and T-cell leukemias. Also upregulated (55 fold) was P2RY8 (purinergic receptor P2Y, G protein coupled 8) a single gene transforming agent overexpressed in leukemia. Amongst the most significantly downregulated (26-fold) transcripts was GPSM 2 (G protein signaling modulator 2) which is essential to maintenance of cell polarity. CONCLUSIONS Inherited missense mutations of the COI gene increase the risk for the development of prostate cancer and are present in 25% of all men with prostate cancer. One such prostate cancer patient-derived mutation has been studied extensively and found to increase cell proliferation and in vivo tumor growth and induces the RGS1 gene known to be overexpressed in a wide variety of human cancers as well as P2RY8, another G protein coupled protein that is potent in transformation. The loss of the G protein signal modulator GPSM2 suggest disruption of cell polarity, an essential characteristic of malignant transformation. We therefore propose that inherited mtDNA mutations predispose individuals to the development of prostate cancer in part by dysregulation of G-protein signaling thereby promoting transformation while disrupting cell polarity. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e326 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qian Sun Atlanta, GA More articles by this author Rebecca S. Arnold Atlanta, GA More articles by this author John A. Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Varicella-Zoster Viruses Associated with Post-Herpetic Neuralgia Induce Sodium Current Density Increases in the ND7-23 Nav-1.8 Neuroblastoma Cell Line

Post-herpetic neuralgia (PHN) is the most significant complication of herpes zoster caused by reactivation of latent Varicella-Zoster virus (VZV). We undertook a heterologous infection in vitro study to determine whether PHN-associated VZV isolates induce changes in sodium ion channel currents known to be associated with neuropathic pain. Twenty VZV isolates were studied blind from 11 PHN and 9 non-PHN subjects. Viruses were propagated in the MeWo cell line from which cell-free virus was harvested and applied to the ND7/23-Nav1.8 rat DRG x mouse neuroblastoma hybrid cell line which showed constitutive expression of the exogenous Nav 1.8, and endogenous expression of Nav 1.6 and Nav 1.7 genes all encoding sodium ion channels the dysregulation of which is associated with a range of neuropathic pain syndromes. After 72 hrs all three classes of VZV gene transcripts were detected in the absence of infectious virus. Single cell sodium ion channel recording was performed after 72 hr by voltage-clamping. PHN-associated VZV significantly increased sodium current amplitude in the cell line when compared with non-PHN VZV, wild-type (Dumas) or vaccine VZV strains ((POka, Merck and GSK). These sodium current increases were unaffected by acyclovir pre-treatment but were abolished by exposure to Tetrodotoxin (TTX) which blocks the TTX-sensitive fast Nav 1.6 and Nav 1.7 channels but not the TTX-resistant slow Nav 1.8 channel. PHN-associated VZV sodium current increases were therefore mediated in part by the Nav 1.6 and Nav 1.7 sodium ion channels. An additional observation was a modest increase in message levels of both Nav1.6 and Nav1.7 mRNA but not Nav 1.8 in PHN virally infected cells.

Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells

Cancerous cell lines have traditionally shown low sensitivity to laboratory or pharmaceutical preparations of botulinum neurotoxin. The work presented here demonstrates that the mouse neuroblastoma/rat glioma hybrid cell line NG108-15 is capable of more sensitively detecting BoNT/A1 than any cell line previously described. This cell line has previously been described to have motor neuron like characteristics, therefore making it a good model to study BoNTs. Differentiation of NG108-15 cells in serum-free medium containing retinoic acid and purmorphamine dramatically increased sensitivity of the neurons to BoNT/A (EC50=∼16LD50U). Additional pre-treatment with triasialoganglioside GT1B prior to toxin exposure reduced the EC50 further to ∼11LD50U. Co-culture of the neurons with C2C12 myotubes also significantly increased BoNT/A sensitivity of NG108-15 cells (EC50=26U) in the absence of differentiation factors.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

The role of the alternative coreceptor GPR15 in SIV tropism for human cells

Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4+, CD8+ and CD19+ peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15+ PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4+, GPR15+ cells is not responsible for the well documented ability of SIV to infect CCR5− blood cells.

Cryptic fate of gliadin epitopes: implication for celiac disease (P730) (106.26)

Abstract Epitopes of an intact antigen that elicit potent T cell activation are classified as immunodominant epitopes. By contrast, epitopes that fail to trigger a T cell response, but can bind to class II, are called cryptic epitopes. HLA-DM (DM), an important regulator of antigen presentation, determines the immunodominant and cryptic fate of epitopes. Notably, however, we previously reported that DM interacts weakly with HLA-DQ2 (DQ2), a genetic risk factor for celiac disease (CD). Here, using the T x B hybrid cell line, T2, transfected with DQ2 with or without higher than physiological levels of DM, we have investigated the contribution of DM in the presentation of different gliadin epitopes (VII). The presentation of digested gluten requires internal processing and was dramatically inhibited when APCs were fixed. Cells lacking DM efficiently presented all four epitopes to gliadin-specific T cells. Interestingly, cells expressing DM were significantly compromised in their ability to display the above epitopes despite the higher level of surface DQ2 in DM+ cells. We conclude that all fourgliadin peptides were DM-suppressed epitopes. We hypothesize that in vivo the inefficient interaction between DQ2 and DM influences the fate of these epitopes, changing them from cryptic to immunodominant, and inducing a gliadin-specific immune response in CD patients expressing the DQ2 allele.

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal‐survival of motor neuron

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA.

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

BackgroundAlthough p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.ResultsThree monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.ConclusionThe ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

Novel human partner cell line for immortalisation of rare antigen-specific B cells in mAb development

It is well-documented that post-translational modification (PTM) events, such as glycosylation, play an important role in antibody-dependent cell-mediated cytotoxicity (ADCC) [1,2]. In current technological processes, monoclonal antibody (mAb) production is widely achieved using heterologous hybridoma systems or genetic engineering using various non-human cell lines as expression host. As a consequence, PTMs generated from non-human cell lines may differ from their human counterparts, resulting in diminished antibody efficacy, aberrant folding and adverse immunogenic response. Therefore, the use of human partner cell lines to generate “fully human” mAbs is beneficial as it circumvents functional complications associated with non-human cell lines. A human cross-lineage hybrid cell line was developed in our laboratory as a candidate partner for immortalisation of rare primary human antigen-specific B lymphocytes using binary electrical cell hybridisation technique which has been developed in house. This novel partner cell line is a tri-hybrid of IL-4 secreting Th2 lymphoblast derived from a patient with acute lymphoblastic leukaemia (T), CD20+ B lymphoblast, also derived from a patient with acute lymphoblastic leukaemia (W), and IL-6 secreting peripheral blood-derived CD14+ monocyte (M). The selection of cell phenotypes used to create the tri-hybrid was based on factors known to maintain and promote antibody production. The resulting tri-hybrid (WTM) displayed characteristics of mixed CD phenotypes with the majority of cells being CD20+ (95%) with co-expression of CD4+ (54%) and CD14+ (24%). It secreted IL-4, IL-6, IL-8 and GM-CSF but was negative for IL-1A, IL-1B, IL-2, IL-5, IL-10, IL-12, IL-13 and IL-17. The cell line did not express the tumour suppressor protein, p53, and neither did it secrete immunoglobulins (Ig)/ Ig chains nor were they expressed on the surface. WTM cells were then used as a fusion partner with primary antigen-experienced CD19+ B cells which had been isolated from peripheral blood, activated in vitro, and the resultant hybrids were sorted for IgM+ and IgG+ expression. 100% hybridisation success rate was achieved using a binary electrical cell hybridisation technique and the number of resulting stable hybrids varied from 48% to 78% depending on the phenotype of B lymphocytes used in experiments. 23% to 68% of those stable hybrids secreted Ig with production ranging between 0.2 to 1.2 ig/106 cells (Table ​(Table1).1). Cytokine screening of some of the Ig producing hybrids revealed a cytokine profile which was inherently different to that of the WTM partner cell line. The Ig producing hybrids concurrently expressed IL-10 and GM-CSF but not IL-4, IL-6 or IL-8. These hybrids were also positive for TGF-â, RANTES, MIP, MCP and MDC. Table 1 Success rate of stable hybrid generation and Ig producing hybrids In conclusion, major advantages of our method involve the rapid generation of stable Ig producing hybrids from a small B lymphocyte population size (50 cells) and the elimination of conventional laborious screening methods for hybrids and Ig producing clones. Thus, when the number of rare antigen-specific B cells available is a limiting factor in generating hybridoma, EBV transfection or direct sequencing, binary electrical B lymphocyte hybridisation with WTM cells can provide a very attractive approach for the generation of stable hybrid cell lines producing monoclonal antibodies.

Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization

Swertia tetraptera Maxim. is an important source of secoiridoid glucosides. To produce these pharmacologically valuable compounds heterologously in somatic hybrid cell lines, S. tetraptera protoplasts were irradiated with various doses of UV light and fused with protoplasts from a long-term cell line of Bupleurum scorzonerifolium Willd. This recipient was chosen as the cell line is cytogenetically stable and fast growing; furthermore, protoplasts isolated from the cell line are readily regenerable. From a set of 86 putative hybrid calli, only two were able to regenerate viable green plants. The hybridity of the 19 of the 86 selections was revealed by a combined isozyme and RAPD analysis, supported by a karyotypic study based on genomic in situ hybridization (GISH). Clone I-3 contained 0.014% swertiamarin while the regenerants had 0.069% swertiamarin and 0.409% gentiopicroside while the III-4 plants contained only 0.015% gentiopicroside.

The behavior of Bupleurum scorzonerifolium as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm2 UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm2 for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.

Xylosyltransferase 2, a protein encoded on chromosome 17q, is involved in HTLV entry

Early studies mapped the entry receptor for HTLV-1 and HTLV-2 to the long arm of chromosome 17. However, later observations that the titer of HTLV Env-pseudotyped viruses was low on some mouse-human hybrid cell lines containing human chromosome 17q led to the belief that this gene was not involved in HTLV infection. Recent studies showing that efficient HTLV-I infection requires three molecules (GLUT-1, NRP-1, and HSPGs), prompted us to examine whether a gene on chromosome 17 is related to this receptor complex. Since NRP-1 or GLUT-1 map to other chromosomes, we examined chromosome 17q for genes involved in the synthesis of HSPGs. A gene at 17q21.33, XYLT2, encodes Xylosyltransferase 2 (XT-II), one of two isoforms of an enzyme involved in glycosaminoglycan-chain modifications on proteoglycans. Studies revealed that both the titer of HTLV-1 pseudotyped virus and the level of binding of soluble HTLV-1 SU proteins were dramatically lower on a cell line lacking functional xylosyltransferase (CHOK1-745) than on the parental line (CHOK1). Expression of transgenic XT-II in CHOK1-745 cells increased the levels of HTLV-1 SU binding and the titer of HTLV-1 Env pseudotypes to levels similar to those in wild-type cells. The role of XT-II in HTLV-2 and HTLV-3 Env-mediated entry is being investigated, as well as the role of xylosyltransferases in the infection of T and dendritic cells by HTLVs. These data provide molecular evidence for the previously reported association of a chromosome 17-encoded product in HTLV-1 entry and confirm the critical role of proteoglycans in this process.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Isolation and Characterization of a Metastatic Hybrid Cell Line Generated by ER Negative and ER Positive Breast Cancer Cells in Mouse Bone Marrow

BackgroundThe origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior.MethodsER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.ResultsThe presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.ConclusionsSpontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.