Transformation-defective polyoma middle T antigen mutants defective in PLCgamma, PI-3, or src kinase activation enhance ERK2 activation and promote retinoic acid-induced, cell differentiation like wild-type middle T.

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCgamma, PI-3 kinase, or src-like kinase.

Discordant effects of rapamycin on proliferation and p70S6 kinase phosphorylation in normal and neoplastic rat chromaffin cells.

Normal adult rat chromaffin cells show a robust proliferative response in vitro to nerve growth factor (NGF) and other mitogens. In contrast, PC12 rat pheochromocytoma cells proliferate in the absence of exogenous mitogens and undergo neuronal differentiation in response to NGF. We demonstrate in this work that the antiproliferative drug rapamycin suppresses normal chromaffin cell proliferation. This effect is blocked by FK 506, indicating that it occurs via interaction of rapamycin with its intracellular binding protein, FKBP. Rapamycin must be added within 2 days of mitogen stimulation in order to be fully effective. PC12 cells are refractory to the antiproliferative effect of rapamycin, although rapamycin does exert its expected inhibitory effect in PC12 cells on both basal and NGF-stimulated activation of one of its biochemical targets, the 70-kDa S6 protein kinase (p70S6K). The discordant findings suggest that a proliferative signal normally requiring activation of p70S6K either is unnecessary in PC12 cells or is provided by a downstream or cross-communicating pathway. They also suggest that p70S6K does not participate in the morphological responses of PC12 cells to NGF. Determining the basis for rapamycin resistance in PC12 cells might help to identify signaling abnormalities involved in the pathogenesis of pheochromocytoma.

Bone marrow stromal cell-mediated gene therapy for hemophilia A: in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648.

To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for hemophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following transfection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10(6) cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (approximately 70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10(6) cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed > or = 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.

Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion.

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.

Polyoma middle T antigen in HL-60 cells accelerates hematopoietic myeloid and monocytic cell differentiation.

Expression of the polyoma virus middle T antigen in HL-60 cells accelerates their differentiation in response to both monocytic and granulocytic differentiation-inducing agents. Middle T-expressing cells treated with the granulocytic inducer retinoic acid or the monocytic inducer 1,25-dihydroxy vitamin D3 differentiated 24 h earlier than parental, mock-electroporated, or vector control cell lines. The rapid onset of differentiation correlated with an increase in the cellular level of the middle T protein as well as two known retinoic-acid-inducible markers in HL-60 cells: the paxillin and transglutaminase gene products. The accelerated functional differentiation response and expression of retinoic-acid-inducible markers indicate that middle T played a causal role in differentiation. Thus, expression of the polyoma middle T antigen in HL-60 cells enhanced a variety of molecular changes associated with cellular differentiation.

Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin.

The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method for routine expansion of canine bone marrow stromal cells, established from initial 10-20 ml marrow aspirates, to greater than 10(9) cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in morphology, and expand to only 10(8) cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1-10 ng/ml) in a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages, transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals.

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.

Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

Preservation of the bystander cytocidal effect of irradiated herpes simplex virus thymidine kinase (HSV-tk) modified tumor cells.

In vitro and animal experiments have demonstrated the potential efficacy of using the bystander effect alone in the treatment of brain tumors. A known problem in some in vitro and in vivo experiments is that a fraction of cells engineered to express the herpes simplex virus thymidine kinase (HSV-tk) gene survive ganciclovir (GCV) treatment and undergo cell division. To prevent the recurrent growth of HSV-tk+ cells in the presence of GCV we examined the potential use of lethal or sublethal irradiation of Walker 256 carcinosarcoma cells selected for expression of the HSV-tk gene (Walker-tk+). Western blot analysis of Walter-tk+ cells showed similar levels of HSV-tk protein expression at 0, 1, 3, 6 and 9 days after lethal gamma-irradiation. In vitro, there was no difference in the bystander effect exerted by non-irradiated, sublethally irradiated or lethally irradiated Walker-tk+ cells on wild-type Walker cells in the presence of GCV. In vivo experiments demonstrated long-term survival (100 days) in rats implanted intrathecally with sublethally or lethally irradiated Walker-tk+ cells with GCV treatments. Intrathecal implantation of irradiated Walker-tk+ cells either pre-mixed with Walker cells or used in in situ treatment of established Walker tumors resulted in prolonged animal survival compared to controls (p < 0.05). These experiments suggest that the bystander tumoricidal effect is preserved despite gamma-irradiation of the HSV-tk modified tumor cells and that irradiation could be an effective method to prevent long-term resistance to GCV in HSV-tk+ tumor cells.

A strategy for screening anti-tumor drugs utilizing oncogenes encoded in retroviral vectors.

A novel strategy for isolating potential anti-tumor drugs is presented. It is predicated on the idea that future anti-tumor drugs will be specific inhibitors of the signal-transduction pathways responsible for cell proliferation. Briefly, retroviral vectors are used to introduce focus-forming oncogenes into a test population of target cells, which are grown to confluence and treated with signal-transduction inhibitors. The inhibitors are screened for the ability to suppress the development of transformed foci without killing the confluent monolayer of non-transformed quiescent cells. For this work, a panel of inhibitors was first screened against the oncogene ras. The protein kinase C (PKC) inhibitor CGP 41251 and the protein tyrosine kinase (PTK) inhibitor CGP 45047 suppressed ras-induced focus formation and left a viable monolayer of quiescent cells. Focus inhibition was reversible; conversely, drug addition to developing foci retarded further expansion. CGP 41251 generally blocked proliferation of ras or control cells, suggesting that oncogenes cannot substitute for PKC. PTK inhibitors erbstatin and CGP 520 and phosphatase inhibitor okadaic acid failed to inhibit focus formation at concentrations toxic to the monolayer. Lavendustin A and CGP 47778A showed neither focus inhibition nor toxicity. In the complementary screen, a single inhibitor (CGP 41251) was tested against several oncogenes, including src, raf and polyomavirus middle T antigen. Focus formation by all oncogenes was suppressed. The strategy has several advantages over current drug-screening assays, and it can be adapted to large-scale screening with many drugs and many oncogenes.

Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin.

Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2. By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2. Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent.

The block of adipocyte differentiation by a C-terminally truncated, but not by full-length, simian virus 40 large tumor antigen is dependent on an intact retinoblastoma susceptibility protein family binding domain.

Simian virus 40 (SV40) can promote cell transformation and suppress differentiation. It does this partly by targeting tumor suppressors such as p53 and members of the retinoblastoma susceptibility protein (Rb) family. This work concentrates on mechanisms by which SV40 large tumor antigen (SVLT) suppresses adipocyte differentiation. We created cell lines derived from murine 3T3-L1 preadipocytes expressing different versions of SV40 early-region sequences. SVLT-expressing cells failed to exhibit adipocyte morphology, to induce glycerophosphate dehydrogenase activity, and to induce differentiation-dependent mRNA for adipocyte P2. SVLT alone was sufficient, in the absence of SV40 small tumor antigen, to inhibit differentiation. A truncated SVLT containing only the N-terminal 121 amino acids (SVLT1-121) blocked differentiation, thus mapping at least one differentiation blocking function to the N-terminal region. K1 (Glu-107-->Lys) point mutants of SVLT, which are unable to bind to the Rb protein family or induce neoplastic transformation, are defective for blocking differentiation in the case of SVLT1-121 but retain the ability to block differentiation in the case of full-length SVLT. This finding demonstrates that Rb family proteins are important in regulating adipocyte differentiation but that other functions of full-length SVLT can block adipocyte differentiation independently of RB family binding and transformation.

Tumor cells expressing the herpes simplex virus-thymidine kinase gene in the treatment of Walker 256 meningeal neoplasia in rats.

A promising strategy in the treatment of neoplastic meningitis involves the use of herpes simplex virus-thymidine kinase (HSV-tk)-modified cells. In these experiments the authors used cells expressing HSV-tk to treat meningeal carcinomatosis in the rat Walker 256 model. Intrathecal injection of 2 x 10(5) Walker cells resulted in a median survival time of 15 days. Up to 80% of animals implanted with HSV-tk-modified Walker cells (Walker-tk+) and treated with ganciclovir showed long-term survival (120 days or more), whereas the remaining animals died from tumor growth between 37 and 44 days after implantation. Tumor cells from an animal in which the treatment failed were cultured in vitro and were shown to be still sensitive to ganciclovir. However, continuous ganciclovir administration for 6 weeks rather than 2 weeks did not improve survival. Histopathological studies confirmed leptomeningeal infiltration in the untreated Walker or Walker-tk+ animals. Walker-tk+ cells were mixed with Walker cells in 1:1, 10:1, or 50:1 ratios, respectively, and implanted intrathecally; the animals were treated with ganciclovir. All groups of treated animals had long-term survivors, with 40% of the rats in the 10:1 and 50:1 groups demonstrating long-term survival and absence of microscopic tumors in the brain or spinal cord. Similarly, murine fibroblast HSV-tk virus-producer cells improved survival. Walker-tk+ cells were better than fibroblast-producer cells in improving the survival of animals with Walker tumors at low (1:1) but not at high (10:1) effector-to-target cell ratios. Repeated intrathecal administration of Walker-tk+ cells resulted in inhibition of established Walker tumors. The authors conclude that Walker-tk+ cells are at least as effective as murine virus-producer cells and could be used in the treatment of meningeal neoplasia.

Production and characterization of WEG-1, an epidermal growth factor/transforming growth factor-alpha-responsive mouse uterine epithelial cell line.

Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.

A more potent bystander cytocidal effect elicited by tumor cells expressing the herpes simplex virus-thymidine kinase gene than by fibroblast virus-producer cells in vitro.

Retrovirus-mediated herpes simplex virus-thymidine kinase (HSV-tk) gene therapy is a promising approach in the treatment of brain tumors. Previous in vitro and in vivo studies have demonstrated a bystander effect in which nonmodified tumor cells in proximity to HSV-tk-modified tumor cells are killed with the modified cells in the presence of ganciclovir. In the present study the authors assessed the contribution of infectious HSV-tk retrovirus made by producer cells to the bystander cytocidal effect in tissue culture using Walker 256 rat breast carcinosarcoma cells, which represent an established model for carcinomatous meningitis. The authors observed ganciclovir-dependent growth inhibition even when only one HSV-tk-positive Walker cell was mixed with 1000 HSV-tk-negative Walker cells and showed that the bystander cytocidal effect is not mediated by toxic cell lysis products. Walker cells engineered to produce HSV-tk retrovirus with titers ranging from 10(3) to 10(5) colony-forming units/ml exert no greater cytocidal effect than nonviral producer HSV-tk-positive Walker cells in vitro. Murine fibroblast-producer cells with viral titers ranging from 10(6) to 10(7) colony-forming units/ml exerted a stronger cytocidal effect than nonviral producer HSV-tk-positive murine fibroblasts. Despite the high viral titers of fibroblast producer cells, HSV-tk-modified Walker cells performed better than fibroblast producer cells in their cytotoxic effect on wild-type Walker tumor cells. Given that HSV-tk-modified tumor cells can become ganciclovir resistant, we tested gamma-irradiation as a means to overcome resistance. Lethal gamma-irradiation of the HSV-tk-positive Walker cells did not abolish their bystander effect on Walker HSV-tk-negative cells. One can infer from these results that HSV-tk-modified tumor cells, irradiated or not, may be a better alternative to murine fibroblast producer cells in the treatment of central nervous system neoplasia.

Rapid regeneration of virus from cells infected with a retroviral vector.

Recombinant retroviral vectors usually encode the genes of interest in place of the viral structural genes, which must be provided in trans. These viruses are therefore defective for replication: infected cells cannot produce progeny virus. However, in some cases it may be desirable to generate virus from an infected cell clone displaying a phenotype of interest. We describe a rapid method for producing virus, which involves fusing the infected cells to fresh packaging cells. Stable producer lines are generated after fusion by co-selecting for the Ecogpt+ marker in the packaging cells and the G418 resistance (neor) marker in the infected cells. We have used this method to develop cell lines that produce retroviruses encoding a Leu 61-activated c-Ha-ras oncogene as well as a neor gene. The viruses confer oncogenic transformation on 95%-100% of infected target cells as assayed by altered morphology, focus formation and soft agar growth.

Bystander tumoricidal effect in the treatment of experimental brain tumors.

The retrovirus-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene into tumor cells renders them sensitive to the cytocidal effect of the antiviral drug ganciclovir. This method has shown promising results as a treatment for experimental brain tumors. These experiments indicate that a major mechanism for the effectiveness of HSV-tk retroviral gene therapy may be the bystander tumoricidal effect. The bystander effect was hypothesized to explain tumor eradication, given that the efficacy of in vivo gene transfer to tumor cells was less than 100%. We demonstrate, in this report, that the bystander tumoricidal effect is a major contributor to the tumoricidal effect of ganciclovir in cell culture experiments using the mouse K1735 C19 cerebral melanoma line, thereby expanding the observation of the bystander phenomenon to a broader range of tumor types. The bystander effect was studied in vitro by coculturing wild-type C19 melanoma cells with HSV-tk-expressing C19 (C19-STK) cells. A maximal tumoricidal effect was seen when only 1 in 10 tumor cells expressed the HSV-tk gene. This suggests that in effect, 1 tumor cell with the HSV-tk gene, when given ganciclovir, will destroy 10 neighboring or bystander cells. The destruction of bystander cells does not appear to be mediated by a soluble factor(s) released into the media but, rather, requires close cell proximity or cell contact. In addition, HSV-tk-expressing C19 cells can exert an antitumoral effect not only on wild-type C19 cells but also on cells from a variety of different tumor cell lines, including a human glioblastoma multiforme cell line, indicating that the bystander effect is not a cell line-specific phenomenon. Finally, we observed that the bystander tumoricidal effect could be harnessed directly without using retrovirus-producing cells to increase survival in the mouse C19 brain tumor model. The potential implications of our findings in treating human brain tumors are discussed.

Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.