Bystander Cytotoxicity Research Articles

PD6-1-5: Dose-response relationships in extensive small cell lung cancer and implications for resistance mechanisms.

Copyright © 2007 by the International Association for the Study of Lung Cancer S426 Restoration of the tumor suppressor genes P53, Retinoblastoma, FHIT and FUS1 had only minimal effect on cell death, although the tumor suppressors are functional defective in >80 % of SCLC. Modulating the oncogene activity of Bcl-2 was attempted by introducing a truncated version of Bax, with increased apoptotic activity. Inhibition of Hsp70-mediated cancer survival was attempted by the introduction of Hsp40 J domain, a dominant negative gene inhibiting Hsp70 activity. Although the gene products activated cellular death pathways in transfected cells, no significant cell death was observed. In contrast suicide gene strategies showed great potential for SCLC gene therapy. The suicide gene system herpes simplex virus thymidine kinase (HSVtk) with the prodrug ganciclovir (GCV) had already been tested and implemented at the laboratory showing >50 % cell death in vitro and tumor regression in vivo. HSVtk efficacy was improved by the replacement of GCV with penciclovir (PCV), which demonstrated significant less toxicity to untransfected cells. Another suicide gene system was implemented for testing: the yeast cytosine deaminase (YCD)gene either alone or fused with the yeast uracil phosphoribosyltransferase (YUPRT) gene and combined with the prodrug 5-FC. The YCD/YUPRT fusion gene showed superior efficacy, mediating 90 % SCLC cell death. This dual suicide gene strategy targets in contrast to HSVtk/GCV or PCV, both DNA and RNA synthesis, also causing cell death to non-proliferating, dormant cancer cells. The strategy demonstrated significant bystander cytotoxicity due to the diffusion of toxic metabolites to nearby cancer cells, showing potential to overcome the low transfection efficiency currently characterizing in vivo gene therapy. An update will be presented.

Novel Action of Paclitaxel against Cancer Cells: Bystander Effect Mediated by Reactive Oxygen Species

Generation of reactive oxygen species (ROS) has been observed in cancer cells treated with paclitaxel, but the underlying mechanisms and therapeutic implications remain unclear. In the present study, we showed that paclitaxel promoted ROS generation through enhancing the activity of NADPH oxidase (NOX) associated with plasma membranes. Treatment of breast cancer cells caused an increased translocation of Rac1, a positive regulatory protein of NOX, to the membrane fraction. The paclitaxel-induced ROS generation occurred rapidly within several hours of drug exposure, with O(2)(-) and H(2)O(2) accumulation mainly outside the cells while the intracellular ROS remained unchanged. Importantly, the increase in extracellular ROS caused lethal damage to the bystander cancer cells not exposed to paclitaxel, as shown by two different methods using coculture systems where the bystander cells were differentiated from the paclitaxel-treated cells by fluorescent or radioactive labeling. This cytotoxic bystander effect was also observed with other microtubule-targeted agents vincristine and taxotere but not with 5-fluorouracil or doxorubicin. This toxic bystander effect was enhanced by CuZnSOD that converts O(2)(-) to H(2)O(2) and was abolished by a catalase that eliminates H(2)O(2). Furthermore, paclitaxel was able to induce an almost complete inhibition of proliferation of the bystander cells in the coculture system. Our study revealed a novel mechanism by which paclitaxel induces toxic bystander effect through generation of extracellular H(2)O(2) from the membrane-associated NOX. This may contribute to the potent anticancer activity of paclitaxel and provide a novel basis to improve the clinical use of this important drug.

Bystander killing of breast cancer MCF‐7 cells by MDA‐MB‐231 cells exposed to 5‐fluorouracil is mediated via Fas

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10.

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G(2)/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27(Kip1). In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.

Hydroxyurea Induces Bystander Cytotoxicity in Cocultures of Herpes Simplex Virus Thymidine Kinase–Expressing and Nonexpressing HeLa Cells Incubated with Ganciclovir

Suicide gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) cDNA and ganciclovir can elicit cytotoxicity to transgene-expressing and nonexpressing bystander cells via transfer of ganciclovir phosphates through gap junctions. HeLa cells do not exhibit bystander cytotoxicity, although we showed recently that they transfer low levels of ganciclovir phosphates to bystander cells. Here, we attempted to induce bystander cytotoxicity using hydroxyurea, an inhibitor of ribonucleotide reductase, to decrease the endogenous dGTP pool, which should lessen competition with ganciclovir triphosphate for DNA incorporation. Addition of hydroxyurea to cocultures of HSV-TK-expressing and bystander cells synergistically increased ganciclovir-mediated cytotoxicity to both cell populations while producing primarily an additive effect in cultures of 100% HSV-TK-expressing cells. Whereas HSV-TK-expressing cells in coculture were approximately 50-fold less sensitive to ganciclovir compared with cultures of 100% HSV-TK-expressing cells, addition of hydroxyurea restored ganciclovir sensitivity. Quantification of deoxynucleoside triphosphate pools showed that hydroxyurea decreased dGTP pools without significantly affecting ganciclovir triphosphate levels. Although hydroxyurea significantly increased the ganciclovir triphosphate:dGTP value for 12 to 24 hours in HSV-TK-expressing and bystander cells from coculture (1.4- to 4.9-fold), this value was increased for <12 hours (2.5-fold) in 100% HSV-TK-expressing cells. These data suggest that the prolonged increase in the ganciclovir triphosphate:dGTP value in cells in coculture resulted in synergistic cytotoxicity. Compared with enhancement of bystander cytotoxicity through modulation of gap junction intercellular communication, this strategy is superior because it increased cytotoxicity to both HSV-TK-expressing and bystander cells in coculture. This approach may improve clinical efficacy.

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.

In Vitro and in Vivo Enhancement of Ganciclovir-Mediated Bystander Cytotoxicity with Gemcitabine

To improve bystander cell killing with HSV-TK/GCV, we have utilized dFdCyd to reduce endogenous dGTP, which competes with GCVTP for incorporation into DNA. In this study we demonstrate the ability of dFdCyd to enhance GCV-mediated bystander cytotoxicity in cultured SW620 human colon carcinoma cells as well as in a murine xenograft model. In vitro, dFdCyd reduced cellular dGTP levels and produced a fourfold increase in the GCVTP:dGTP ratio. This elevated GCVTP:dGTP ratio resulted in a twofold increase in GCVMP incorporation into DNA in bystander cells cocultured with HSV-TK-expressing cells. The combination of GCV and dFdCyd was determined to be synergistic by isobologram analysis of bystander cytotoxicity. Tumors in mice treated with GCV and dFdCyd exhibited a significant growth delay requiring 40 days to obtain ∼10 times their initial size compared to tumors in PBS- or single-drug-treated animals, which grew rapidly, increasing to a similar size in just 19 to 24 days. In addition, complete tumor regression was observed only in animals treated with both drugs. Furthermore, dFdCyd alone or in combination with GCV produced no evidence of toxicity or significant weight loss. These data suggest that dFdCyd may improve the clinical efficacy of HSV-TK/GCV gene therapies.

807. Addition of HU to GCV Induces Bystander Cytotoxicity in Co-Cultures of HSVTK-Expressing and Non-Expressing HeLa Cells

Expression of the herpes simplex virus type I thymidine kinase (HSVTK) gene in tumor cells sensitizes them to the antiviral agent ganciclovir (GCV). Incubation with GCV results not only in cytotoxicity from cells that express the transgene, but neighboring bystander cells as well. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSVTK-expressing to non-expressing cells through protein channels known as gap junctions. We have previously reported good bystander cytotoxicity in the U251 glioblastoma and SW620 colon carcinoma cell lines, which have 84% and 39% gap junction intercellular communication (GJIC), respectively. However, in the HeLa cell line (23% GJIC), no bystander cytotoxicity resulted from incubating a co-culture of HSVTK-expressing and bystander HeLa cells with GCV. Further analysis revealed that lack of bystander cytotoxicity in these cells was not due to an inability to transfer phosphorylated GCV, but resulted from a low level of transfer along with a rapid half-life of GCVTP in bystander cells (3.1 hrs) when compared to the HSVTK-expressing cells (12.3 hrs). We hypothesized that reduction of the endogenous competitor for GCVTP incorporation into DNA, dGTP, would increase levels of GCV incorporation into DNA resulting in the induction of bystander cytotoxicity. To that end, the effect of hydroxyurea (HU), a ribonucleotide reductase inhibitor, on GCV cytotoxicity in cultures of 100% HSVTK-expressing HeLa cells and co-cultures of HSVTK-expressing and bystander HeLa cells was examined. Isobologram analysis of the interaction between GCV and HU demonstrated an additive-antagonistic effect on 100% cultures of HSVTK-expressing HeLa cells, while exhibiting a synergistic effect on the HSVTK-expressing and bystander cells in a co-culture. To determine the mechanism for the synergistic cytotoxicity demonstrated in each of the populations from the co-culture when incubated with GCV and HU, GCVTP and dNTP pools were quantified and the results were compared to 100% cultures of HSVTK-expressing HeLa cells. The results demonstrated a significant increase in the GCVTP:dGTP values in cultures of 100% HSVTK-expressing cells (>2.5 fold) and in co-cultures of HSVTK-expressing cells (>1.4 fold) and bystander cells (>1.8 fold) at 4 hours. However, at 12 hours the GCVTP:dGTP value for 100% cultures of HSVTK-expressing HeLa cells was at control levels (1.3 0.5 fold) while the values for HSVTK-expressing cells (>2.7 fold) and bystander cells (>4.9 fold) in co-culture were still significantly higher. These data suggest that the prolonged increase in the GCVTP:dGTP value in co-cultures incubated with GCV and HU leads to an increase in the levels of GCVTP incorporated into DNA resulting in synergistic cytotoxicity.

Resistance to Activation-Induced Cell Death and Bystander Cytotoxicity Via the Fas/Fas Ligand Pathway Are Implicated in the Pathogenesis of Cutaneous T Cell Lymphomas

By engaging Fas, Fas ligand (FasL) on activated T lymphocytes induces activation-induced cell death (AICD), and also triggers apoptosis of target cells during immune downregulation. We previously showed that within cutaneous T cell lymphoma (CTCL) lesions, malignant CD4(+) T cells expressing FasL accumulated, and were inversely distributed with CD8(+) T cells. We thus determined the responses of human CTCL cells to AICD and their cytotoxic to Fas(+) target T cells in vitro. CTCL cells expressing Fas were resistant to AICD following activation by CD3 monoclonal antibody (mAb) whereas still undergoing apoptosis if Fas was ligated to Fas mAb. CTCL cell lines, as well as Sezary Syndrome patients' peripheral blood lymphocytes, exhibited ratio-dependent cytotoxicity to Fas(+) Jurkat cells. The kinetic study showed that FasL surface expression was absent before activation, and its expression was low and/or delayed after activation. We therefore hypothesize that CTCL cells express functional FasL possibly contributing to bystander cytotoxicity within tumor infiltrates. In addition, decreased and/or delayed FasL surface expression following activation may in part contribute to their resistance to AICD. Both bystander cytotoxicity and resistance to AICD are likely to contribute to the loss of cytotoxic anti-tumor CD8(+) T cells as well as the accumulation of malignant T cells in CTCL.

GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity

The role of gap junctional intercellular communication (GJIC) in bystander killing with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) was evaluated in U251 cells expressing a dominant-negative connexin 43 cDNA (DN14), and in HeLa cells, reportedly devoid of connexin protein. These cell lines both exhibited 0% GJIC when assayed by Lucifer Yellow fluorescent dye microinjection. Bystander cytotoxicity was still apparent in 50:50 cocultures of DN14 and HSV-TK-expressing U251 cells, but not in 50:50 cocultures of HeLa cells. However, the sensitivity of HeLa HSV-TK-expressing cells to GCV decreased nearly 100-fold (IC90=109 microM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC90=1.2 microM). A more sensitive flow cytometry technique to measure GJIC over 24 h revealed that the DN14 and HeLa cells exhibited detectable levels of communication (29 and 23%, respectively). Transfer of phosphorylated GCV to HeLa bystander cells occurred within 4 h after drug addition, and GCV triphosphate (GCVTP) accumulated to 213+/-84 pmol/10(6) cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867+/-33 pmol/10(6) cells) compared to 100% cultures of HSV-TK-expressing cells (1773+/-188 pmol/10(6) cells). The half-life of GCVTP in the HSV-TK-expressing cells was approximately four times that measured in the bystander cells (12.3 and 3.1 h, respectively). These data suggest that the lack of bystander cytotoxicity in HeLa cocultures is due to low transfer of phosphorylated GCV and a rapid half-life of GCVTP in the bystander cells. Thus, GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolism.

Bystander activity of Ad-mda7: Human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism

The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.

IL-13 receptor-targeted cytotoxin cancer therapy leads to complete eradication of tumors with the aid of phagocytic cells in nude mice model of human cancer.

Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Ralpha2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Ralpha2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Ralpha2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.

CYTOCHROME P450-BASED CANCER GENE THERAPY: RECENT ADVANCES AND FUTURE PROSPECTS

Cytochrome P450-based cancer gene therapy is a novel prodrug activation strategy for cancer treatment that has substantial potential for improving the safety and efficacy of cancer chemotherapeutics. The primary goal of this strategy is to selectively increase tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating P450 enzyme. This strategy has been exemplified for the alkylating agents cyclophosphamide and ifosfamide, which are bioactivated by select P450 enzymes whose expression is generally high in liver and deficient in tumor cells. Transduction of tumors with a prodrug-activating P450 gene, followed by prodrug treatment, greatly increases intratumoral formation of activated drug metabolites. This leads to more efficient killing of the transduced tumor cells without a significant increase in host toxicity. P450 gene therapy is accompanied by substantial bystander cytotoxicity which greatly enhances the therapeutic effect by extending it to nearby tumor cells not transduced with the therapeutic P450 gene. Although endogenous P450 reductase is not expected to be a limiting factor in prodrug activation in tumor cells that express moderate levels of an exogenous P450 gene, P450 reductase transduction has recently been found to substantially enhance intratumoral prodrug activation and its associated therapeutic effects. Using this gene combination, an overall 50- to 100-fold increase in tumor cell kill in vivo over that provided by hepatic drug activation alone has been observed. Striking improvements in therapeutic effects can thus be achieved using an established anticancer drug in an intratumoral prodrug activation strategy based on the combination of a cytochrome P450 gene with the gene encoding NADPH–P450 reductase. This strategy is readily extendable to several other widely used P450-activated cancer chemotherapeutic prodrugs, as well as to prodrugs that undergo P450 reductase-dependent bioreductive activation and which may exhibit synergy when combined with P450-activated prodrugs in a P450/P450 reductase-based cancer gene therapeutic regimen.

Gene therapy of neoplastic meningosis.

Gene therapy of neoplastic meningosis is a promising new approach that relies on introduction of 'suicide' genes into cancer cells. The most commonly used gene has been the herpes virus thymidine kinase gene (HSV-tk) which has been delivered to cancer cells via retroviral or adenoviral vectors. A bystander cytocidal effect to non-transduced tumor cells has been documented and is dependent upon intercellular communication via gap junctions. A variety of gene therapy approaches using the HSV-tk system have been used in experimental models of neoplastic meningosis with promising results. Optimizing vector design and bystander cytotoxicity is a prerequisite for successful gene therapy in patients with neoplastic meningosis.

Gene-directed enzyme prodrug therapy: quantitative bystander cytotoxicity and DNA damage induced by CB1954 in cells expressing bacterial nitroreductase.

Clones of human colon carcinoma (WiDr), ovarian carcinoma (SK-OV-3), and Chinese hamster V79 cells expressing the nitroreductase enzyme (NR) from E. coli B were 52-, 225- and 177-fold respectively more sensitive to a 24-h incubation with the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) than the parent lines. The IC50s of non-NR-expressing bystander cells were measured in the presence of differing proportions of NR-expressing cells. The shift in IC50 was used to calculate a value for the bystander effect, termed the transmission efficiency (TE), which is the decrease in IC50 due to bystander effect as a percentage of the maximum decrease possible. The percentage of NR-expressing cells for which the TE was 50%, (the TE50) is a single datum of bystander efficacy. WiDr and V79 cell lines, had a similar TE50 of approximately 2%. SK-OV-3 gave a lower value of 0.3%. These TE50 correlate with concentrations of cytosolic NR activity, which is distinguished from endogenous DT diaphorase activity by kinetic differences. A novel method is described which enables both DNA crosslinks and drug-induced single-strand breaks to be simultaneously quantified in a sedimentation assay. Using this technique, bystander DNA damage was demonstrated in V79 cells, of approximately 50% of that in activator cells.

Tumor-specific T-bodies: towards clinical application.

Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. The T-body approach combines antibody recognition and T cells effector function. It is based on T cells expressing chimeric receptors composed of antibody-derived Fv or scFv as their extracellular recognition elements joined to lymphocyte triggering molecules. This receptors can redirect the specificity of T cells in an MHC independent manner. Upon encountering their target cells, T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in model systems both in vitro and in vivo. T cells expressing chimeric receptors made of antitumor antibodies are able to discriminate between a tumor and normal cell with negligible bystander cytotoxicity. Further studies should be carried out to evaluate and optimize the persistence, homing patterns and reactivation potential of T-bodies in the body.

The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions.

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk+) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk+ cells) exerted bystander cytotoxicity on gap junction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiments also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk+ cells. In conclusion, these results indicate that in several cellular environments the bystander effect is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.

The T-body approach: potential for cancer immunotherapy.

Chimeric receptors containing antibody-derived Fv or scFv as their extracellular recognition elements can redirect the specificity of T cells in an MHC-independent manner. Upon encountering their target cells, such T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in both in vitro and in vivo model systems. T cells expressing chimeric receptors are able to discriminate between antigen-expressing and normal cells, with negligible bystander cytotoxicity. Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. Further in vivo studies should be carried out to evaluate and optimize the persistence, homing patterns, and reactivation potential of T-bodies in vivo. Several technical obstacles must be overcome before this approach may be applied clinically. A most urgent problem is the low efficiency of T cell transfection techniques and the particular difficulty of transducing primary T cell populations. While retroviral-mediated gene transfer is more efficient than conventional techniques such as electroporation, the proportion of transfected cells remains low, necessitating an enrichment step. In addition, antibodies with improved discrimination between cell-bound and soluble forms of tumor antigens must be obtained to expand the repertoire of tumor antigens which may be targeted. For each antigen-antibody system, the optimal design of the scFv must be determined. In the future application of this technology, the recognition element used for chimeric TCR is not limited to antibody-derived fragments [27]. Various ligands may be coupled to a T cell-triggering molecule in an attempt to redirect cytotoxic function towards target cells expressing a particular receptor molecule. Although still experimental, we feel that with fine tuning, the T-body approach shows promise as an efficient and broad-spectrum modality for tumor immunotherapy.

The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro.

Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that retroviral transduction of tumor cells is not required for bystander cytotoxicity.

Immobilized IgG Immune Complex Induces Secretion of Tumor Necrosis Factor-α by Porcine Alveolar Macrophages

Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory mediator produced by activated monocytes and macrophages. We have previously shown that porcine alveolar macrophages (PAM) mediate bystander cytotoxicity through hydrogen peroxide production following activation with immobilized IgG immune complex (IIC) (J. Immunol. 1983; 131:1438-1442). In this report, we have investigated whether IIC induces TNF-alpha secretion by PAM. Isolated PAM from Minnesota miniature swine were cultured for 18 h with and without recombinant human interferon-gamma (rhIFN-gamma). Cultured PAM were then incubated with IIC or IgG immune complex in suspension (SIC). The supernatants generated were assessed for cytotoxic activity using a TNF-alpha-sensitive WEHI-164 cell line. Anti-recombinant human TNF-alpha (rhTNF-alpha) monoclonal antibody neutralized the observed cytotoxicity of IIC-activated PAM supernatant completely, indicating that this cytotoxicity is mediated by TNF-alpha. IIC induced TNF-alpha secretion by PAM after 3 h of incubation, reaching a plateau from 6 to 12 h and decreasing thereafter. TNF-alpha release was enhanced by pretreatment of PAM with rhIFN-gamma. SIC did not induce significant levels of TNF-alpha secretion by PAM; however, SIC with cytochalasin B-pretreated PAM induced equivalent levels of TNF-alpha secretion as IIC-activated PAM. We conclude that IIC or SIC with cytochalasin B pretreatment, both of which prevent internalization of IgG immune complex-bound Fc receptor (FcR), provide a signal for PAM to generate TNF-alpha through FcR modulation. This suggests that in vivo, deposited (immobilized) IgG immune complexes-bound FcR may be a stimulus for activation of PAM to generate TNF-alpha rather than circulating (mobilized) immune complexes, which may contribute to the pathogenesis of diffuse interstitial fibrosis of the lung, especially in idiopathic pulmonary fibrosis.