Increased liposome-mediated gene transfer into haematopoietic cells grown in adhesion to stromal or fibroblast cell line monolayers.

We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells.

In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the beta-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMV beta plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration ot the cell nucleus.

IL-2-dependent expression of genes involved in cytoskeleton organization, oncogene regulation, and transcriptional control.

IL-2 induces growth, differentiation, and/or apoptosis of lymphoid cells. To study further the molecular basis of IL-2 function, we used a cDNA subtraction approach involving a cell line grown in IL-2 or IL-4. From the corresponding library, 66 nonredundant sequences were characterized; 16 of them encode identified proteins. The kinetics of in vitro expression of 8 selected sequences, the functions of which could be associated with IL-2-induced T cell activation/differentiation, was investigated using an IL-2-dependent T cell line. IL-2 increased the expression of cytoskeleton proteins (alpha-tubulin), oncogene-regulating proteins (CCCTC-binding factor, Jun inhibitor factor-1), and transcription factors (E2F-4, cyclic AMP-responsive element-binding protein, zhx-1). IL-2 also regulated the expression of genes coding for multifunctional proteins, e.g., beta-catenin and nucleolin. These results were verified using Con A-induced T cell blasts stimulated or not by IL-2. The in vivo expression of four of these genes was also analyzed in spleen and lymph node cells of IL-2-deficient and MRL/lpr mice, which both have high numbers of activated cells, but the latter have intact IL-2 expression. The expression of beta-catenin, CCCTC-binding factor, Jun inhibitor factor-1, and nucleolin was significantly higher in MRL/lpr animals. A similar analysis of thymocytes from IL-2-/- and IL-2+/- mice demonstrated the same expression patterns of the 4 sequences in these strains. The expression of the IL-2-induced genes described herein is similar to the regulatory pattern of IL-2R alpha. Taken together, our data provide additional evidence for the pleiotropic action of IL-2 in the periphery and IL-2 independence of molecular processes involved in thymocyte differentiation.

IL-2 receptor alpha-chain expression is independently regulated in primary and secondary lymphoid organs.

The IL-2R is composed of three chains: IL-2R alpha, IL-2R beta, and IL-2R gamma. In mice, IL-2Ra is critical and determines IL-2 binding to the tripartite IL-2R complex. To extend our previous studies, which demonstrated that IL-2 regulates IL-2R alpha expression in vitro, we have analyzed expression in IL-2-deficient mice in vivo. As in control animals, CD4- CD8- thymocytes and bone marrow-derived B220+ pre-B cells were IL-2R alpha positive. In contrast, activated lymph node and splenic CD4 T cells (CD4+ CD69+) were found to be IL-2R alpha negative, whereas approximately 20% of the same cell populations from the MLR/lpr strain, which also accumulate large numbers of CD4-activated T cells in the presence of intact IL-2, retained expression. A similar pattern of IL-2R alpha expression was found among splenic CD8 cells from IL-2(-/-) and IL-2(+/-) animals. These findings demonstrate that in primary lymphoid organs, IL-2 is not directly involved in IL-2R alpha expression. However, at the level of mature lymphocytes, and more specifically CD4 T cells, IL-2 remains in vivo, as in vitro, the most critical cytokine controlling both IL-2R alpha expression and sensitivity to IL-2.

Is LM7 a new human retrovirus or a mycoplasma virion?

A putative retrovirus called LM7 was recently isolated from a patient with MS. This retrovirus was detected in LM7 and LM711 cultured human leptomeningeal cells. In the present work, nucleic acids from LM711 cell culture supernatants were purified and subjected to avian myeloblastosis viral (AMV) reverse transcriptase and to random polymerase chain reaction (rPCR) in order to characterize the genomic material of LM7 virions. Analysis of reverse transcription products allowed the detection of an approximately 14 kb ribonucleic acid in all LM711 cell culture supernatants. However, sequencing of rPCR-amplified molecules as well as RNA blotting data showed essentially that all tested cells producing LM7 particles were infected with mycoplasma. Moreover, purification of LM7 particles onto a linear sucrose density gradient established that the 14 kb nucleic acid was always associated with the 1.19-1.21 g ml-1 sucrose fractions, which are known to correspond to the buoyant density of mycoplasma. In addition, no viral genomic RNA was detected in the 1.17 g ml-1 sucrose fraction containing the low reverse transcription activity. These results strongly suggest that microscopic images and serological data could be related to mycoplasma and/or to a virion associated with the bacteria. The LM7 particle might be a new and additional enveloped virus able to infect Mycoplasma hyorhinis hosts. Thus, for instance, it would be presumptuous to assert, with our current understanding, that the LM7 virion is one of the causal agents of MS in humans.

HIV-1 env, nef, and gag-specific T-cell immunity in mice: conserved epitopes in nef p27 and gag p25 proteins.

Cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (HIV-1) was studied in mice immunized with vaccinia virus recombinants. Proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. No specific activity was observed against gag p18 protein. Env, nef, and gag-specific T-cell lines were generated by repeated stimulation of immune spleen cells with recombinant HIV-1 proteins. They were CD4 positive, proliferative, and also cytotoxic against HIV-transfected target cells. Specificity of the T-cell response against nef and gag protein was analyzed with synthetic peptides. Peptides nef 15, nef 16, and gag AM-30 were, respectively, reactive in nef- and gag-specific proliferative and cytolytic assays. The three peptides described have a relatively conserved amino acid sequence among HIV isolates and appear broadly immunoreactive among species.

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.

Induction of mouse p55 interleukin-2 receptor gene expression by IL-2 and IL-4 and characterization of its transcription initiation sites.

Two murine T cell lines (C30.1 and Line 1) were used to study the expression of the p55 interleukin-2 receptor gene. C30.1 is an IL-2-dependent T cell line that can be stimulated for a short period of time by IL-4. Line 1 cells are propagated in IL-4 but they also proliferate in response to IL-2. In both cell lines stimulation by IL-2 leads to a strong induction of p55 IL-2 receptor mRNA while stimulation by IL-4 leads only to a very moderate increase in expression of this mRNA. The induction of p55 IL-2 receptor mRNA by IL-4 is comparable to that of beta-actin mRNA. These data confirm that IL-2 upregulates p55 IL-2 receptor gene expression while IL-4, which also activates T cells, does not lead to specific induction of this gene. We have also determined the transcription initiation sites utilized by the p55 IL-2 receptor gene in C30.1 and Line 1 cells. Seven sites were identified, one of which predominates. Resting cells, or cells stimulated with IL-2 or IL-4, display the same pattern of transcription site utilization.

Transient nuclear location of the IL-2 receptor during T cell activation.

Binding of interleukin-2 (IL-2) to its membrane receptor (IL-2R) on target cells is followed by internalization of the IL-2R. The subsequent intracellular fate of IL-2R is not known. This paper describes the intracellular location of the p55 subunit of the IL-2R during IL-2 mediated T cell activation and growth of two mouse T helper clones. IL-2R was visualized by immunohistochemistry using two rat monoclonal antibodies (5A2 and 7D4). Immunostaining shows that the p55 subunit of the IL-2R is transiently present in the nucleus of activated T cells. The intranuclear location of the IL-2R suggests that the p55 subunit, either alone or in conjunction with the IL-2 or the p70 subunit, may be implicated in the regulation of gene expression involved in T cell proliferation.

p55 IL-2 receptor mRNA precursors in murine T lymphocyte nuclei.

An unusual family of cDNA clones homologous to human p55 IL-2R sequences was isolated from the murine HT-2 Th cell line. These clones were mapped, partially sequenced, and compared with previously published human and mouse IL-2R sequences. They appear to consist of various combinations of exons and introns, suggesting that they are derived from p55 IL-2R mRNA precursors. The configuration of exons in the splicing intermediates indicates that the murine and human gene organizations are similar and that the 3' end of intron 3 is well conserved between the two species. RNA mapping experiments using nuclear, cytoplasmic, and total RNA and probes derived from various parts of the p55 IL-2R gene support and extend the sequence data. They indicate that detectable amounts of immature p55 IL-2R mRNA are found specifically in the cell nucleus of the HT-2 cell line. Similar data were obtained for the Th cell clone 52.3 and the cytotoxic T cell line CTLL. All these results indicate that the T cell nucleus contains significant amounts of immature p55 IL-2R mRNA.

Nuclear location of the p55 subunit of the IL-2 receptor following activation of the HT-2 T helper cell line.

After mitogenic or antigenic stimulation, T cells express interleukin-2 receptors (IL-2R). The mechanism and control of signal transduction following binding of IL-2 to IL-2R are poorly understood. Using two rat monoclonal antibodies (5A2 and 7D4) specific for two distinct epitopes of the p55 subunit of mouse IL-2R, we have studied the cellular localization of this molecule by immunocytochemistry during the IL-2-mediated activation of mouse T helper cell clone HT-2. During the activation cycle, nuclear staining for the p55 subunit of the IL-2 receptor was transiently observed. It is suggested that the transient nuclear location of the IL-2R may play a critical role in the control of T-cell activation, proliferation and/or differentiation.

Effects of butyric acid on cell cycle regulation and induction of histone H1(0) in mouse cells and tissue culture. Inducibility of H1 (0)in the late S-G2 phase of the cell cycle.

We have examined the regulation of the synthesis of histone H1(0) in cultured mammalian cells treated with butyric acid. Treatment of cells with the inducer results in the arrest of synthesis of DNA and the other histones, while increasing the synthesis of H1(0) by a factor of 11. The induction of H1(0) by butyric acid occurs in a pulse with a peak at six hours, followed by a decrease to negligible levels. This pulse-like induction appears to be due to the fact that the cells are inducible for H1(0) only in the late S or G2 phases of the cell cycle. This, coupled with the fact that butyric acid blocks cells in G1, results in the burst of H1(0) synthesis after addition of the inducer. The G1 block provoked by butyric acid does not appear to result from the accumulation of H1(0). Removal of butyric acid from G1-blocked cells resulted in the resumption of cellular proliferation without prior loss of H1(0), demonstrating that the presence of this histone is not sufficient to prevent cellular proliferation.

Histone variant composition of normal and leukaemic human lymphocytes: analysis by gel electrophoresis of whole cells and nuclei.

The histone variant composition of normal and leukaemic lymphocytes was analysed using a method which circumvented endogenous cellular proteolysis. Whole cells were solubilized in buffer containing protamine which released the histones from the DNA and allowed their immediate analysis by Triton X-100-acetic acid-urea gel electrophoresis. The results were comparable to those obtained with histones which had been acid-extracted from cells, nuclei or chromatin; however, the new method was more reproducible since proteolysis was controlled. By histone variant analysis, chronic lymphocytic leukaemia patients could be separated into two groups. One group had lymphocytes with a histone H2a variant ratio of about 1.0; this finding resembled lymphocytes (75-85% T cells) from normal individuals. The larger group had lymphocytes with a reproducibly lower histone H2a variant ratio; these cells had a relative increase in the second variant, H2a.2. The groups of patients could not be distinguished by clinical disease state. No other differences were noted in the histone variant composition of lymphocytes from normal and leukaemic individuals.

Vitamin C preferential toxicity for malignant melanoma cells.

Vitamin C has been suggested and disputed as an anti-cancer agent. For cells in culture, no preferential effect against any type of cancer has yet been demonstrated. Our aim here is to show that vitamin C is selectively toxic to at least one type of malignant cell--a melanoma--at concentrations that might be attained in humans. Copper ions react with ascorbate and generate free radicals in solution. Ascorbate when combined with copper rapidly reduces the viscosity of DNA solutions and has exhibited some carcinostatic effects on transplanted sarcoma 180 tumours in mice. We reasoned that the elevated copper concentration in melanoma could result in a more selective toxicity for ascorbate.