Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model.

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.

Subcutaneous or intrahepatic injection of suicide gene modified tumour cells induces a systemic antitumour response in a metastatic model of colon carcinoma in rats.

BACKGROUND: Suicide gene therapy consists of the transfer into tumour cells of a "suicide" gene that can convert a non-toxic compound into a lethal drug. Expression of the cytosine deaminase gene leads to the conversion of the non-toxic compound 5-fluorocytosine to 5-fluorouracil. We have recently shown that "suicide cell based vaccination" consisting of intrahepatic injection of cytosine deaminase expressing colon cancer cells followed by 5-fluorocytosine treatment induces regression of a distant wild-type liver tumour in rats. AIMS: This study was conducted to test if (i) a distant bystander effect on a liver tumour can be induced after subcutaneous suicide cell based vaccination and (ii) suicide cell based vaccination is efficient in limiting tumour dissemination to extrahepatic compartments. METHODS: An aggressive variant of rat colon carcinoma cells was selected after successive passages in vitro. Rats carrying an experimental liver "metastasis" generated by injection of these cells were vaccinated by subcapsular or subcutaneous injection of cytosine deaminase expressing cells followed by 5-fluorocytosine treatment. RESULTS: Subcutaneous and subcapsular vaccination induced 70% regression in the median volume of the pre-established liver tumour (p=0.001) and abolished tumour dissemination compared with control animals. CONCLUSIONS: This study has compared for the first time the efficiency of subcutaneous and intrahepatic suicide cell based vaccination in a metastatic colorectal carcinoma model in rats. The results indicate that both modes of vaccination are equally efficient in inducing a systemic antitumour response, suggesting that this strategy is a powerful approach against the development and dissemination of metastatic colon carcinoma.

[Vaccination by suicide gene therapy against a model of hepatic metastasis from colon cancer in the rat]. / Vaccination par thérapie génique suicide contre un modèle de métastases hépatiques de cancer colique chez le rat.

UNLABELLED: Suicide gene therapy consists of transferring into tumor cells a viral or bacterial gene encoding for an enzyme which converts a non-toxic product into a lethal drug. STUDY AIM: To analyze the therapeutic potential of vaccination with tumor cells expressing the bacterial cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) treatment in a rat liver metastasis model. MATERIAL AND METHOD: We used a rat colon carcinoma cell line which, after subcapsular or intraportal injection in syngenic animals, generates single or multiple experimental liver metastases, respectively. We have shown that introduction of a vector expressing the CD gene in this colon carcinoma cell line results in 5-FC sensitivity (PRObCD). RESULTS: Intrahepatic subcapsular injection of PRObCD tumor cells, followed by 5-FC treatment, induces total regression of a wild-type tumor pre-established in the contralateral liver lobe in 45% of animals with a 96% decrease in mean volume (p < 0.0001), demonstrating the existence of a distant bystander effect. This vaccination significantly increased the survival of rats with single (log-rank p < 0.0001) or multiple (log-rank p = 0.01) liver metastasis CONCLUSIONS: These results suggest that suicide gene-modified tumor cells can act as potent therapeutic vaccines against liver metastasis from colon carcinoma.

Src-regulated extracellular signal-related kinase and Syk-regulated c-Jun N-terminal kinase pathways act in conjunction to induce IL-1 synthesis in response to microtubule disruption in HL60 cells.

A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.

Binding of anti-CD23 monoclonal antibody to the leucine zipper motif of FcepsilonRII/CD23 on B cell membrane promotes its proteolytic cleavage. Evidence for an effect on the oligomer/monomer equilibrium.

In the present study we have compared the binding of two monoclonal antibodies to CD23, EBVCS1 and mAb25, which recognize the stalk and the lectin domain, respectively, on the CD23 molecule. At 4 degreesC, EBVCS1 binds to about 10% of the receptors recognized by mAb25 on the B cell surface. At 37 degreesC, whereas mAb25 reaches its maximal binding within a few seconds, EBVCS1 requires 60 min to bind to the same extent. Stabilization of the oligomeric structure of CD23 with IgE strongly affects in a dose-dependent fashion the number of binding sites seen by EBVCS1 but not the t1/2 to reach them, suggesting that EBVCS1 binds to the coiled coil region through an allosteric mechanism. EBVCS1 rapidly down-modulates the membrane CD23 expression with a coincident increase of CD23-soluble fragments in the culture medium, an effect that is inhibited by IgE. In contrast, mAb25, as well as IgE, protects CD23 from proteolytic cleavage and stimulates its endocytosis. These results suggest that EBVCS1 unravels the coiled coil structure of CD23, rendering it more susceptible to proteolytic attack. This supports the oligomeric model proposed previously (Gould, H., Sutton, B., Edmeades, R., and Beavil, A. (1991) Monogr. Allergy 29, 28-49). The biological significance of these observations is discussed.

CD20 monoclonal antibodies decrease interleukin-4-stimulated expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) in human B cells by increasing the extent of its cleavage.

CD20 monoclonal antibody (mAb) B1 is known to inhibit B cell proliferation. We show that B1 reduced both anti-mu + interleukin-4 (IL-4)-induced DNA synthesis and the concomitant expression of CD23 at the surface of human tonsillar B cells. B1 mAb had no effect on CD23 mRNA levels. The disappearance of CD23 molecule from the surface correlates with an increase of soluble CD23 fragments in the culture medium, indicating that CD20 mAb B1 stimulated the cleavage of the molecule. B1 also inhibits IgE production by peripheral blood mononuclear cells cultured in the presence of IL-4. Suppression of IgE synthesis and enhancement of CD23 cleavage are concomitant but appear not to be functionally related.

CD20 monoclonal antibodies stimulate extracellular cleavage of the low affinity receptor for IgE (Fc epsilon RII/CD23) in Epstein-Barr-transformed B cells.

This study demonstrates that monoclonal antibodies to the B cell-specific CD20 molecule down-regulate both constitutive and interleukin-4-induced CD23 expression on Epstein-Barr-transformed B cells. This effect of CD20 antibody B1 does not take place at the transcriptional level as shown by the lack of effect on the CD23 mRNA level. Incorporation of 35S-labeled amino acids into CD23 polypeptide chain is not affected either. In cycloheximide-treated cells, B1 increases the decline of CD23 from the cell surface. The disappearance of CD23 molecule correlates with an increase of soluble CD23 fragments detected in the culture medium. Taken collectively, these results indicate that CD20 mAb B1 stimulates the cleavage of the CD23 molecule at the surface of B cells.

CD20 monoclonal antibodies down-regulate IgM at the surface of B cells.

The CD20 molecule is a phosphoprotein expressed on the surface of B lymphocytes that plays a role in the regulation of B cell proliferation and differentiation. In this study it was found that monoclonal antibodies (mAb) directed to CD20 decrease the expression of IgM at the surface of normal human B lymphocytes and B cell lines. This effect was time-dependent with a half-time of about 5 h. Incubation of B cells with CD20 mAb B1 did not affect the steady-state level of IgM mRNA, suggesting that it acts at a nontranscriptional stage. Phorbol esters also produced inhibitory effect on surface IgM expression. Staurosporine reversed both the phorbol ester- and the CD20-induced down-regulation. Genistein did not reverse the down-regulation induced by the CD20 mAb B1. CD20 most likely triggers a protein kinase C-dependent pathway to down-regulate sIgM. CD20 mAb also counteracted the interleukin-4 (IL-4)-induced up-regulation of sIgM. The ability of anti-IgM to mobilize intracellular calcium was reduced in sIgM down-regulated cells, suggesting that B cells activation through the antigen receptor may be negatively regulated by CD20 and positively by IL-4.

Spontaneous and ligand-induced endocytosis of CD23 (Fc epsilon receptor II) from the surface of B lymphocytes generates a 16-kDa intracellular fragment.

It has been reported that the 45-kDa low-affinity Fc epsilon receptor (Fc epsilon RII) on B cells is cleaved spontaneously from the cell surface to release soluble fragments. This study demonstrates an additional fate of the Fc epsilon RII. 125I-labeled CD23+ B cells were cultured for 24 h at 37 degrees C. After lysis, cell extracts were immunoprecipitated with CD23 monoclonal antibodies. Using this methodology, we demonstrated that an increasing amount of the labeled Fc epsilon RII becomes progressively resistant to externally applied trypsin, indicating that a fraction of the cell surface receptors are internalized. In parallel, a labeled 16-kDa material, recognized by CD23 monoclonal antibodies directed to the lectin-like domain of the Fc epsilon-RII appears inside the cells. Chloroquine does not affect internalization of the Fc epsilon RII, but completely abolishes the formation of the intracellular fragment, suggesting that the receptor is processed by proteolytic cleavage in acidic organelle. In addition, the internalization is enhanced in the presence of CD23 monoclonal antibodies. These data demonstrate that Fc epsilon RII can be internalized by ligand-induced endocytosis and subsequently cleaved in an intracellular compartment. These results also support the view that the Fc epsilon RII is involved in antigen focusing and antigen presentation.

Pertussis toxin-induced mitogenesis in human T lymphocytes.

Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.

Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.

Molecular association between major histocompatibility complex class I antigens and insulin receptors in mouse liver membranes.

Molecular association between major histocompatibility complex (MHC) antigens and cellular proteins are thought to be involved in various immunological and nonimmunological functions of MHC antigens, including hormone signaling. The existence of physical interactions between insulin receptors and MHC class I antigens was investigated in liver plasma membranes from congenic H-2k mice. Insulin receptors were specifically labeled with a 125I-labeled photoreactive insulin analogue, and cellular proteins were solubilized and incubated with various monoclonal antibodies. Immunoprecipitates were analyzed by polyacrylamide gel electrophoresis followed by autoradiography. Antibodies reacting with distinct epitopes on H-2k class I antigens were all able to precipitate up to 25% of the labeled insulin receptors in H-2k mouse liver membranes, whereas no insulin receptors were precipitated in H-2b mouse liver membranes. Sequential immunoprecipitations showed that insulin receptors and H-2 antigens were coprecipitated and that no cross-reactivity occurred. The specificity of the interaction between insulin receptors and H-2 antigens was demonstrated after double labeling of membrane proteins by photoreactive insulin and lactoperoxidase-catalyzed iodination. These results thus show that, in mouse liver membranes, insulin receptors are physically associated to class I antigens of the MHC.

The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens.

Insulin receptors were labeled with 125I-photoreactive insulin (specifically labeling alpha-subunits) and by insulin-stimulated autophosphorylation (specifically labeling beta-subunits). The results show that the insulin receptor exists under different free and disulfide-linked combinations of alpha and beta subunits. Moreover, the insulin receptor is closely associated to class I antigens of the major histocompatibility complex to form a high molecular weight multi-molecular membrane complex.

Interaction between insulin receptors and major histocompatibility complex antigens in mouse liver membranes.

Insulin receptors from H-2k mouse liver membranes were specifically labelled using a photoreactive insulin analogue coupled to the alpha-subunit (Mr 130,000) of the receptor by ultraviolet-irradiation. Up to 25% of the labelled insulin receptors recovered after membrane solubilization and precipitable by anti-insulin receptor antibodies could be immunoprecipitated by various monoclonal antibodies reacting with H-2Kk antigens. None of these monoclonal antibodies inhibited insulin binding to its receptor. These results show that insulin receptors and H-2Kk antigens interact in liver plasma membranes to form non-covalent complexes. This association between insulin receptors and major histocompatibility complex antigens could be involved in the pathogenesis of certain forms of insulin resistance.