Mouse Ltk Research Articles

In vitro Activation of B-CLL Cells.

Apart from surface Ig receptors, a variety of membrane molecules have now been described to deliver activation and progression signals to human B cells. Among them, CD40 antigen is likely to play a crucial role in the antigen-dependent maturation process. Recent studies performed in the laboratory have established that presentation of anti-CD40 mAbs in a crosslinked fashion by mouse Ltk(-) cells stably expressing human FcyRII/CDw32, allowed normal human B cells to enter into sustained proliferation. In their overwhelming majority, B-CLL cells are positive for CD40 expression. We have therefore examined the capacity of purified B-CLL cells to be stimulated by various cytokines for growth and differentiation, following crosslinking of slgs or CD40 antigen. In most B-CLL specimens studied, IL-2 was the sole factor, among a wide panel of cytokines tested, which reproducibly and significantly induced proliferation of leukemic B cells activated with anti-Ig reagents (SAC or anti-IgM antibodies). Unlike normal B cells, the great majority of anti-Ig activated B-CLL cells failed to proliferate in response to IL-4. In this activation system, IL-4 profoundly suppressed the IL-2 driven proliferation of B-CLL. An opposite pattern of growth-response was obtained following ligation of CD40 since IL-4 elicited proliferation of B-CLL whereas the growth-promoting effect of IL-2 was reduced. Under these culture conditions, IL-4 and IL-2 displayed additive effects on leukemic B cell growth. Surprisingly, IL-4 combined with anti-CD40 mAb allowed activation of certain leukemia specimens otherwise refractory to other stimulatory signals. Most B-CLL samples were induced for IgM synthesis upon SAC stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 mAb used alone or in combination with cytokines (IL1-IL6, IFNγ, TNFα, TGFβ) failed to induce Ig secretion from B-CLL. No evidence for Ig isotype switching was obtained with the cytokines listed above, whatever the mode of B-CLL activation. Taken together, our results suggest that B-CLL can be released in vitro from their apparent maturation block, by IL-2 and anti-Ig reagents or by IL-4 and immobilized anti-CD40 mAb. Additionally, the data reported here suggest that development of the agonistic and antagonistic activities of IL-4 on B cell growth and differentiation may depend upon the nature of the activation signal provided.

Expression of recombinant human insulin-like growth factor I in mammalian cells

In order to develop a suitable mammalian expression system for human insulin-like growth factors (hIGFs) and mutant IGFs, we have constructed several artificial IGF genes, based on a cDNA encoding the IGF-I precursor (153 amino acids). Transient expression experiments using mouse Ltk - cells revealed that the IGF-I gene constructs were efficiently expressed when placed under control of the SV40 Early promoter (SV40E). This resulted in the synthesis and secretion of IGF-I receptor-reactive products. Constructs encoding an IGF-I precursor with a truncated signal peptide of 25 amino acids under control of SV40E promoter or the inducible Drosophila heat shock hsp70 promoter, were used to establish stably transformed CHOdhfr - and mouse L cells. Clones secreting IGF-I were identified by an IGF-I-specific radioreceptor assay. Immunoblot analysis of conditioned media from these clones resulted in the specific precipitation of a protein of 7 kDa identical in size to native IGF-I purified from human serum. After optimization of the expression conditions, the stable cell lines secrete 0.5-2 μg/10 6 cells of IGF-I. The biological activity of the secreted recombinant IGF-I was shown by its ability to stimulate DNA synthesis in human MCF-7 cells. The results described in this paper indicate that a mammalian expression system, employing CHOdhfr - or L cells, is a useful system for the synthesis of biological active IGF-I.

Energy dependence of DNA-mediated gene transfer and expression

When cells are transfected with calcium phosphate-precipitated DNA, most of the DNA is internalized through active endocytosis but a small pool is postulated to be internalized by passive diffusion. The relative importance of these two DNA pools in gene transfer and expression has never been examined. We now show that mouse Ltk- fibroblasts internalize greater than 90% of radioactively labeled, calcium phosphate-precipitated DNA by a temperature- and energy-dependent process but a small pool of 2-3% of radioactively labeled DNA may be internalized even at 4 degrees C or under energy depletion conditions. The contribution of these two pools of DNA to gene expression was examined by transfecting Ltk- cells with DNA from a plasmid encoding marker for both transient expression (human growth hormone) and stable integration (neomycin resistance). When cells were depleted of ATP during transfection, transient expression of human growth hormone was reduced to only 2% of control values at day 4 posttransfection. Under the same condition, stable expression of neomycin resistance also was reduced to less than 1% of the control. Therefore, these data showed that only the pool of calcium phosphate-precipitated DNA internalized through an energy-dependent process is ultimately responsible for transient and stable expression of exogenous genes.

Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene

Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk- cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 microM hypoxanthine, 1 microM aminopterin, and 10 microM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk- recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB-1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10-fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no down-regulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility.

Co-transfer of restriction endonucleases and plasmid DNA into mammalian cells by electroporation: Effects on stable transformation

We co-transferred cloned HSV thymidine kinase gene and restriction endonucleases, HindIII, XbaI, or XhoI, into thymidine kinase-deficient mouse Ltk − cells by electroporation. Stimulation of the transformation efficiency was observed with HindIII or XbaI, while little effect was observed with XhoI. The stimulation was observed for various forms of exogenous DNA (linear or circular plasmid DNA and single-stranded phage DNA) and correlated with the number of strand breaks in the host-cell DNA. These results suggest that the presence of DNA double-strand breaks stimulates the integration of exogenous DNA into host cell genomes.

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation)

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.

Mouse major (γ) satellite DNA is highly conserved and organized into extremely long tandem arrays: Implications for recombination between nonhomologous chromosomes

We have isolated and sequenced 30 independent clones derived from MnlI digestion of purified mouse major (γ) satellite DNA. These clones contained between 0.9 and 1.1 γ monomeric units derived presumably from random chromosomal sources. Individual clones showed a mean deviation from the mouse consensus satellite sequence of 3.9%, with a range of 0.9–9.1%. Cleavage of total mouse LTK cell genomic DNA with three different restriction enzymes ( HindIII, BglII, BamHI) that do not cut within satellite monomers, followed by Southern and pulsed-field gel electrophoretic analyses, showed that the majority of monomers were organized into largely uninterrupted arrays that varied from a minimum of 240 kb to greater than 2000 kb in length. We suggest that the high degree of conservation of the mouse γ-satellite sequences throughout the mouse genome results from frequent recombinational exchange between nonhomologous chromosomes. Further, this same process, facilitated by the all-acrocentric constitution of the typical mouse karyotype, and the extremely long and homologous γ-satellite arrays, may be related to the common occurrence of Robertsonian translocation in mouse.

A novel interferon-γ regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-γ (IFN-γ) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk − cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-γ was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

The effect of cell synchronization on the efficiency of stable gene transfer by electroporation

We synchronized thymidine kinase deficient mouse Ltk − cells by two different methods, hydroxyurea double-block treatment or aphidicolin single-block treatment and transformed them with the cloned herpes simplex virus thymidine kinase gene at various time intervals by the electroporation technique. Marked enhancement of stable transformation efficiency was observed at the time corresponding to the peak of G 2/M phase. These results suggest that the G 2/M phase is the most efficient period for stable gene transfer by electroporation.

A Highly Inducible System of Gene Expression by Positive Feedback Production of Glucocorticoid Receptors

To investigate the function of a cloned gene, inducible enhancer/promoters are preferred for controlling the level of expression experimentally. Highly inducible gene expression systems with both low basal levels and enhanced maximal levels of expression are especially sought. We have developed a new expression system using a positive feedback mechanism. Glucocorticoid receptor cDNA was fused to the enhancer/promoter of mouse mammary tumor virus (MTV) to induce receptor production. Simultaneous introduction of this receptor plasmid into mouse Ltk- cells augmented the expression of the reporter gene linked with the MTV enhancer/promoter by 10- to 100-fold in an induced state, but did not significantly change the expression of that in an uninduced state. This novel system of gene expression is a good candidate for an efficient genetic switch.

In vivotranscription of a cloned prosimian primate SINE sequence

SINE transcription was studied by introducing a galago Monomer family member (gal39), into the mouse Ltk- cell line. Cells transiently transfected with gal39 produce gal39-specific RNA polymerase III-directed transcripts. In permanent cell lines gal39 expression was largely shut off. Genomic environment, copy number and tandem repetition of integrated SINE sequences influenced whether or not RNA polymerase III-directed gal39 transcripts were detectable. These transcripts differ in length from the observed endogenous transcript in cultured galago cells which hybridizes to this repetitive DNA family.

High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase.

Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.

The Escherichia coli LexA repressor-operator system works in mammalian cells.

We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.

Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology.

Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk) from each of three different strains (F, MP, and 101) of herpes simplex virus type one and a complete herpes simplex virus type 1 (strain F) tk gene containing an 8-bp insertion mutation was studied. The pairs of tk sequences resided as closely linked repeats within the genome of mouse LTK- cells. The frequency of recombination between sequences exhibiting 232 bp of uninterrupted homology and containing no mismatches other than the insertion mutation was comparable to the frequency of recombination between two sequences exhibiting four additional nucleotide mismatches distributed in such a way to preserve the 232-bp stretch of contiguous homology. In contrast, the placement of only two single-nucleotide mismatches (in addition to the insertion mutation) in such a manner to reduce the longest uninterrupted homology to 134 bp resulted in a 20-fold reduction in recombination. We conclude that the rate of intrachromosomal recombination in mammalian cells is determined by the amount of uninterrupted homology available and not by the total number of mismatches within a given interval of DNA. Furthermore, efficient recombination appears to require between 134 and 232 bp of uninterrupted homology; single-nucleotide heterologies are most likely sufficient to disrupt the minimal efficient recombination target. We also observed that if recombination was allowed to initiate within sequences exhibiting perfect homology, the event could propagate through and terminate within adjacent sequences exhibiting 19% base pair mismatch. We interpret this to mean that heterology exerts most of its impact on early rather than late steps of intrachromosomal recombination in mammalian cells.

Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology.

Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk) from each of three different strains (F, MP, and 101) of herpes simplex virus type one and a complete herpes simplex virus type 1 (strain F) tk gene containing an 8-bp insertion mutation was studied. The pairs of tk sequences resided as closely linked repeats within the genome of mouse LTK- cells. The frequency of recombination between sequences exhibiting 232 bp of uninterrupted homology and containing no mismatches other than the insertion mutation was comparable to the frequency of recombination between two sequences exhibiting four additional nucleotide mismatches distributed in such a way to preserve the 232-bp stretch of contiguous homology. In contrast, the placement of only two single-nucleotide mismatches (in addition to the insertion mutation) in such a manner to reduce the longest uninterrupted homology to 134 bp resulted in a 20-fold reduction in recombination. We conclude that the rate of intrachromosomal recombination in mammalian cells is determined by the amount of uninterrupted homology available and not by the total number of mismatches within a given interval of DNA. Furthermore, efficient recombination appears to require between 134 and 232 bp of uninterrupted homology; single-nucleotide heterologies are most likely sufficient to disrupt the minimal efficient recombination target. We also observed that if recombination was allowed to initiate within sequences exhibiting perfect homology, the event could propagate through and terminate within adjacent sequences exhibiting 19% base pair mismatch. We interpret this to mean that heterology exerts most of its impact on early rather than late steps of intrachromosomal recombination in mammalian cells.

Extinction of JC virus tumor-antigen expression in glial cell--fibroblast hybrids.

JC virus (JCV) is a ubiquitous human papovavirus that shares sequence and structural homology with simian virus 40 (SV40). In contrast to SV40, expression of JCV is restricted to a small number of cell types, including human fetal glial cells, uroepithelial cells, amnion cells, and some endothelial cells. To study the control of JCV early region expression, we made heterokaryons and stable hybrids between JCV-transformed hamster glial cells and mouse fibroblasts. Binucleate heterokaryons exhibited extinction of large tumor antigen expression in the hamster nuclei as assayed by indirect immunofluorescence. This extinction was both time and dose dependent: extinction reached maximal levels at 24-36 hr after fusion and was dependent on the ratio of glial cell to fibroblast nuclei in multinucleated heterokaryons. Extinction also was observed in stable hybrids between the glial cells and mouse Ltk- cells. Southern blot analysis showed that the extinguished hybrids contained viral sequences. Reexpression of large tumor antigen was observed in several subclones, suggesting that extinction was correlated with the loss of murine fibroblast chromosomes from these hybrids. The cis-acting region that mediates extinction resides within the viral regulatory region, which contains two 98-base-pair repeats that have enhancer activity. These data demonstrate that cellular factors that negatively regulate viral gene expression contribute to the restricted cell-type specificity of this virus.

Cloned and expressed human Fc receptor for IgG mediates anti-CD3-dependent lymphoproliferation.

We have utilized gene transfer experiments to investigate the role of a human monocyte receptor for IgG (Fc gamma RII) in mouse IgG1 anti-CD3 (Leu 4)-induced lymphoproliferation in vitro. Mouse Ltk- cells expressing human Fc gamma RII or a mutant of Fc gamma RII lacking the entire cytoplasmic domain of the receptor mediate anti-CD3-induced lymphoproliferation in cultures of adherent cell-depleted human PBMC. Expression of an Fc gamma RII mutant lacking transmembrane and cytoplasmic domains (soluble Fc gamma RII) in COS7 cells yielded a secreted receptor which retained affinity for IgG, even in the absence of the mutant receptor's N-linked oligosaccharides. Soluble Fc gamma RII inhibits rosette formation by human IgG-sensitized RBC and the Fc gamma RII-bearing cell line K562, but does not sitmulate anti-CD3-induced lymphoproliferation under the conditions tested.

Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter.

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, we have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect "TATA box" at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk- fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene.

Gene transfer of the ly‐3 chain gene of the mouse cd8 molecular complex: co‐transfer with the ly‐2 polypeptide gene results in detectable cell surface expression of the ly‐3 antigenic determinants

The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the alpha-subunit of an avian sodium pump.

cDNA encoding the alpha-subunit of the (Na+ + K+)-ATPase was cloned from a chicken kidney cDNA library and the nucleotide sequence determined. The deduced amino acid sequence showed 92% sequence homology with the alpha-subunit of the sheep kidney (Na+ + K+)-ATPase, and high cross-species homologies were found among nucleotide sequences both in the 5'- and 3'-untranslated regions of the "kidney-type" alpha-subunit mRNAs. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk- cells. Expression of the avian alpha-sub-unit could be activated by culture of the cells in 10 mM butyrate. Cells expressing avian alpha-subunits displayed high-affinity ouabain binding (KD = 2.6 +/- 0.7 x 10(-7) M) and ouabain-sensitive 86Rb+ uptake, characteristic of avian cells.