A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation.

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

Systemic inhibition of tumor growth and angiogenesis by thrombospondin-2 using cell-based antiangiogenic gene therapy.

Recent studies indicate that continuous administration improves the antitumoral efficacy of angiogenesis inhibitors, as compared with intermittent dosing, suggesting a potential role of gene therapy in antiangiogenic tumor therapy. We established a tissue-engineered implant system for the continuous in vivo production of thrombospondin-2 (TSP-2), a potent endogenous inhibitor of tumor growth and angiogenesis. Fibroblasts were retrovirally transduced to overexpress TSP-2 and were seeded onto biodegradable polymer scaffolds. After transplantation into the peritoneal cavity of nude mice, bioimplants maintained high levels of TSP-2 secretion over extended time periods, resulting in increased levels of circulating TSP-2. Bioimplant-generated TSP-2 potently inhibited tumor growth and angiogenesis of human squamous cell carcinomas, malignant melanomas, and Lewis lung carcinomas that were implanted at a distant site. These results provide the first proof-of-principle for the feasibility and therapeutic efficiency of systemic, cell-based antiangiogenic gene therapy using biodegradable polymer grafts for the treatment of cancer.

Overexpression of H-Ryk in mouse fibroblasts confers transforming ability in vitro and in vivo: correlation with up-regulation in epithelial ovarian cancer.

Abnormalities in the function of receptor tyrosine kinases (RTKs) have been demonstrated to be important in the pathogenesis of cancer. H-Ryk, a new member of the RTK family, is an unusual RTK in that it is catalytically inactive because of amino acid substitutions of conserved residues in the catalytic domain. We show by immunohistochemistry that it is expressed in the epithelium, stroma, and blood vessels of normal tissues. Evaluation of a panel of 33 primary ovarian tumors (2 benign, 8 borderline, and 23 malignant) was performed. H-Ryk was overexpressed in borderline and malignant ovarian tumors. In serous and clear cell subtypes, there was increased expression in the epithelium, stroma, and blood vessels. Consistent with this observation, overexpression of H-Ryk in the mouse fibroblast cell line NIH3T3 induces anchorage-independent growth and tumorigenicity in nude mice. This implies that overexpression of the receptor can be transforming and may therefore be significant in the pathogenesis of ovarian cancer.

Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells.

Ewing's sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of five ETS proteins. While the majority of tumors express EWS/FLI1 fusions, some Ewing's tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural differences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its C-terminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors differ greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural differences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.

Role of the cAMP and MAPK pathways in the transformation of mouse 3T3 fibroblasts by a TSHR gene constitutively activated by point mutation.

Constitutive activating mutations of the TSHR gene, have been detected in about 30 per cent of hyperfunctioning human thyroid adenomas and in a minority of differentiated thyroid carcinomas. The mutations activating the TSHR gene(s) in the thyroid carcinomas, were located at the codon 623 changing an Ala to a Ser (GCC-->TCC) or in codon 632 changing a Thr to Ala or Ile (ACC-->GCC or ACC-->ATC). In order to study if the constitutively activated TSHR gene(s) has played a role in the determination of the malignant phenotype presented by these tumors, we investigated: (1) the transforming capacity after transfection of mouse 3T3 cells, of a TSHR cDNA activated by an Ala-->Ser mutation in codon 623 or an Thr-->Ile mutation in codon 632 and (2) the pathway(s) eventually responsible(s) for the malignant phenotype of the cells transformed by these constitutively activated TSHR cDNAs. Our results show that (1) the TSHR(M623) or (M632) cDNAs give rise to 3T3 clones presenting a fully neoplastic phenotype (growth in agar and nude mouse tumorigenesis); this phenotype was weaker in the cells transformed by the 632 cDNA; (2) suggest that the fully transformed phenotype of our 3T3 cells, may be the consequence of the additive effect of the activation of at least two different pathways: the cAMP pathway through G(alpha)s and the Ras dependent MAPK pathway through G(beta)gamma and PI3K and (3) show that the PI3K isoform playing a key role as an effector in the MAPK pathway activation in our 3T3-transformed cells is PI3Kgamma. Signaling from PI3Kgamma to MAPK appears to require in our murine cellular system a tyrosine kinase (still not characterized), Shc, Grb2, Sos, Ras and Raf. It is proposed that the constitutively activated TSHR genes detected in the thyroid carcinomas, may have played an oncogenic role, participating in their development through these two pathways.

Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways.

The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.

IRF-1 reverts the transformed phenotype of oncogenically transformed cells in vitro and in vivo.

The expression of the transcriptional activator and tumor suppressor IRF-1 induces multiple effects that counteract the growth of tumor cells in vitro and in vivo. These include the inhibition of cell proliferation, the secretion of interferon-beta (IFN-beta), the induction of apoptosis specifically in certain cell types and the induction of a strong T-cell response. Here, we show that apart from its immune-activating properties, IRF-1 expression leads to a reversion of the tumorigenic phenotype of NIH3T3 cells transformed by different oncogenes. This was analysed in detail in a cell line in which the expression of c-Ha-ras and c-myc is under the control of a doxycycline-regulated promoter allowing to switch between the normal and oncogenic cell status. In the same cells, a beta-estradiol activatable IRF-1 fusion protein is expressed. After IRF-1 activation the oncogene-mediated acceleration of the cell cycle is reverted. Further, a complete IRF-1-mediated reversion of the oncogenic phenotype is observed in soft-agar growth assays. IRF-1 activation induces IFN-beta secretion; however, the observed effects are not mediated by IFN-beta. Inhibition of tumor growth is observed in nude mice as long as IRF-1 is active, indicating that neither B- nor T-cells must become activated for tumor growth suppression.

Gene transfer of anti-gp41 antibody and CD4 immunoadhesin strongly reduces the HIV-1 load in humanized severe combined immunodeficient mice.

OBJECTIVE: To study the anti-HIV-1 effects of the delivery of anti-gp41 monoclonal antibody (mAb) and soluble CD4 (sCD4) immunoadhesin by genetically modified cells in HIV-1-infected, humanized severe combined immunodeficient (SCID) mice. DESIGN: The complementary DNA of mAb 2F5, an anti-HIV-1 gp41 antibody, and of sCD4-IgG chimeric immunoadhesin were transferred into 3T3 cells using Moloney murine leukaemia virus vectors. The cells were then incorporated into a collagen structure called the neo-organ, which allowed the continuous production of the therapeutic molecules. METHODS: The antiviral effects in vivo of 2F5 or sCD4-IgG or both compounds were evaluated in neo-organ-implanted SCID mice that were grafted with human CD4 CEM T cells and challenged with HIV-1 Lai or MN. RESULTS: In SCID mice implanted with 2F5 neo-organs, antibody plasma levels reached 500-2000 ng/ml. Viral loads after HIV-1 challenge were significantly reduced in neo-organ-implanted HIV-infected mice. Although 29 x 10(7) and 13 x 10(8) HIV-1-RNA copies/ml were detected at 12 days in the controls (mice injected with Lai and MN, respectively) less than 16.5 x 10(3) HIV-1-RNA copies/ml were observed in all implanted mice injected with either Lai or MN. The intracellular viral load was also reduced in CD4 cells recovered from the implanted mice. Comparable antiviral effects were obtained with CD4-IgG neo-organs. CONCLUSION: Our results confirm the anti-HIV properties of 2F5 and sCD4-IgG continuously produced in vivo after ex-vivo gene therapy in SCID mice.

Enhancement of interleukin-4-mediated tumor regression in athymic mice by in situ retroviral gene transfer.

Intratumoral grafting of genetically engineered cells that produce interleukin-4 (IL-4) has been shown to produce tumor regression as well as prolong survival of mice harboring intracerebral gliomas. We sought to determine whether retroviral-mediated gene delivery into tumor cells in situ resulted in enhanced tumor regression by IL-4. Two mouse fibroblast lines were obtained: they both secreted similar levels of IL-4 but one produced a retrovirus vector bearing the IL-4 gene (CRE-MFG-IL-4 cells), whereas the other did not (NIH3T3-IL-4 cells). In mixed transplantation assays in the subcutaneous flanks of athymic mice, CRE-MFG, IL-4 cells were more effective than NIH3T3-IL-4 cells in inhibiting the growth of rat C6 glioma cells (p < 0.005, ANOVA). Subcutaneous tumors injected with fibroblasts that produced a control retrovirus vector without producing IL-4 (CRE-MFG-LacZ cells) did not inhibit subcutaneous tumor growth. An intracranial assay was used to evaluate survival of athymic mice harboring intracranial gliomas. Three days after implanting rat C6 glioma cells into the right frontal lobes of athymic mice, NIH3T3-IL-4 cells (n = 10) or CRE-MFG-IL-4 cells (n = 10) were stereotactically inoculated into the tumor bed. The average survival of mice treated with CRE-MFG-IL-4 cells was 38 days (+/- 2.4, SE), whereas that of mice treated with NIH3T3-IL-4 cells was 31 days (+/- 0.8, SE) (p < 0.005, ANOVA; p < 0.001, log-rank analysis).(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained production of human transferrin by transduced fibroblasts implanted into athymic mice: a model for somatic gene therapy.

Somatic gene therapy has been proposed as a means of treating inherited diseases involving defective or absent plasma proteins, viral diseases, and cancer. Introduction of the gene of interest into fibroblasts and implantation of these genetically modified fibroblasts using a skin equivalent system may be an attractive model for gene therapy because skin fibroblasts are easily obtained and propagated in culture. This study evaluated expression of the gene for human transferrin (hTf) by genetically modified fibroblasts in vitro and in vivo. NIH 3T3 fibroblasts, which form non-metastasizing tumors in athymic mice, were transduced with a retroviral vector encoding hTf. The transduced cells were cloned by limiting dilution and hTf production by the cloned cells measured. Two clones of cells producing high levels of hTf were used to seed collagen-coated nylon matrices, which were maintained in culture for up to 53 d. The rate of synthesis of hTf by the seeded matrices was constant after 22 d in vitro. Matrices seeded with cloned, transduced cells were implanted subcutaneously into seven athymic mice, and plasma levels of hTf were assessed biweekly. In all animals, the plasma level of hTf was detectable at week 6 after implantation. Levels of hTf remained elevated in the animals until the implants were removed at week 12. At week 10, the level of hTf in the plasma correlated with tumor volume in tumors less than 2000 mm3 in size. The half-life of hTf in the mice was 39.5 h. In this model, gene expression did not decline for the 12-week observation period.

Toxicity studies and distribution dynamics of retroviral vectors following intrathecal administration of retroviral vector-producer cells.

The use of intrathecal, retroviral-mediated transfer of the herpes simplex thymidine kinase (HStk) gene and subsequent ganciclovir (GCV) administration has recently been shown to improve survival in a rat model of leptomeningeal carcinomatosis. Clinical application of this approach is attractive because access to the cerebrospinal fluid (CSF) space is relatively noninvasive and distribution of producer cells and vectors may be facilitated by circulation of CSF, overcoming distribution problems inherent in solid tumors. However, meningeal inflammation, transduction and injury to normal CNS tissue, proliferation of the xenogeneic producer cells in the subarachnoid space, immune-mediated injury, and development of hydrocephalus are possible complications of intraventricular or intrathecal administration of vector-producer cells. In addition, the dynamics of producer cell and vector distribution in the CSF are unknown. To address these issues, we evaluated the safety of this approach for gene delivery and assessed the dynamics of distribution of producer cells and retroviral vectors in rats and non-human primates. In rats, transduction of normal central nervous system (CNS) structures surrounding the subarachnoid space was evaluated after intrathecal and intraventricular injections of beta-galactosidase and HStk vector-producer cells, with and without GCV. In primates, beta-galactosidase and HStk vector-producer cells were injected intraventricularly and GCV was administered either intrathecally or intravenously. Toxicity was evaluated by neurologic examination, serial gadolinium-enhanced MRI scans of the brain, and blood and CSF profiles. A subgroup of monkeys received repeated intraventricular injection of vector-producer cells and intravenous GCV. The titer of retroviral-vector was measured in cisternal and lumbar CSF samples after repeated producer cell injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated recovery of irradiation-induced bone marrow depression by fibroblast-mediated interleukin 6 gene therapy in combination with bone marrow transplantation in mice.

BACKGROUND: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated. METHODS: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice. RESULTS: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT. CONCLUSIONS: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.

Enhanced antitumor effects of bone marrow transplantation in combination with fibroblast-mediated IL-2 and IL-3 gene therapy.

BACKGROUND: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy. METHODS: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice. RESULTS: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy. CONCLUSIONS: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.

Phenotypic properties of 3T3 cells transformed in vitro with polyoma virus and passaged once in syngeneic animals.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.

Enteric glia promote functional recovery of CTM reflex after dorsal root transection.

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

Alginate-encapsulated producer cells: a potential new approach for the treatment of malignant brain tumors.

In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells. However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells. These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate. This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation. This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells. The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry. The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture. The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks. The production of the bacterial E. coli beta-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged beta-galactosidase activity. H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined. The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro. To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells. The migration in vitro was totally inhibited in the presence of H528 encapsulated cells. Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry. It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments. Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors. Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors.

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor.

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.

Intradermal cell transplantation in soluble collagen.

Intradermal, as opposed to subcutaneous, cell transplantation was previously shown to be advantageous for tumor cell growth, but this site has not been used for transplantation of normal nonneoplastic cells. In preliminary experiments we found that it was difficult to control the size and shape of transplants when we injected dissociated cells intradermally. This problem was solved by placing cells in nongelled, pepsin-solubilized collagen prior to injection. This technique permitted the successful transplantation of normal bovine adrenocortical cells and of neoplastic cells (3T3 cells secreting FGF) in scid mice. Primary bovine adrenocortical cells formed functional vascularized tissue and the transplants rescued the animals from the lethal effects of adrenalectomy. The histological structure of transplant tissues resembled that previously observed when cells were transplanted in the subrenal capsule space. We also used a line of 3T3 cells that has been genetically modified to secrete a form of acidic FGF. When transplanted intradermally in collagen, they formed rapidly enlarging masses of cells that could easily be palpated beneath the skin of the animal. Intradermal injection of cells in pepsin-solubilized collagen is a simple and reliable technique for transplanting normal primary cells and preneoplastic cells. The ability to grow both types of cells in an easily accessible site allows less invasive monitoring of growth, angiogenesis, and other features of the transplant.

High frequency of RAS oncogene mutation in chronic myeloid leukemia patients with myeloblastoma.

To determine the role of the mutated RAS oncogene during development into the blast phase, we sequentially analysed RAS oncogene mutations in the bone marrow of 27 patients with chronic myeloid leukemia (CML). DNA from CML patients in chronic and blast phases and nude mouse tumor DNA formed by a tumorigenicity assay (in vivo selection assay) were subjected to the polymerase chain reaction (PCR) and oligonucleotide hybridization. In addition, one patient in the chronic phase and five in the blast phase were also analysed. PCR analysis of DNA from the leukemic patients revealed that 3.6% (1 of 28) and 15.6% (5 of 32) of the patients in the chronic and blast phases, respectively, had RAS mutations. N- or K-RAS oncogene mutations were found mostly in the blast phase (4 of the 5 patients with the RAS oncogene mutation). Of the 5 patients with the RAS oncogene mutation, three developed myeloblastoma, a myeloblast cell tumor, in the blast phase. None of the 28 patients without the RAS mutation developed myeloblastoma. These results suggest that the RAS oncogene mutation occurred in the late stage of the disease and contributed to transformation to the blast phase in some CML patients. The findings also indicate an association between the presence of the RAS mutation and the formation of myeloblastoma.