Proteasomal targeting of a viral oncogene abrogates oncogenic phenotype and enhances immunogenicity.

The ability of viral or mutated cellular oncogenes to initiate neoplastic events and their poor immunogenicity have considerably undermined their potential use as immunotherapeutic tools for the treatment of human cancers. Using an Epstein-Barr virus-encoded oncogene, latent membrane protein 1 (LMP1), as a model, we report a novel strategy that both deactivates cellular signaling pathways associated with the oncogenic phenotype and reverses poor immunogenicity. We show that cotranslational ubiquitination combined with N-end rule targeting of LMP1 enhanced the intracellular degradation of LMP1 and total blockade of LMP1-mediated nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription (STAT) activation in human cells. In addition, although murine cells expressing LMP1 were uniformly tumorigenic, this oncogenicity was completely abrogated by covalent linkage of LMP1 with ubiquitin, while an enhanced CD8+ T cell response to a model epitope fused to the C-terminus of LMP1 was observed following immunization with ubiquitinated LMP1. These observations suggest that proteasomal targeting of tumor-associated oncogenes could be exploited therapeutically by either gene therapy or vaccination.

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.

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.

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.

Increased expression of IgE-dependent histamine-releasing factor in endometriotic implants.

A complex network of cytokines mediates the immunoregulatory responses leading to endometriosis. Recent intensive studies suggest that monocyte and T cell chemoattractants contribute to the inflammatory environment of endometriotic implants. The relationship between the inflammation present during endometriosis and the development of endometriotic implants in the peritoneal cavity remains unclear. On the other hand, the association between endometriosis and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) exposure has been discussed in recent years, and our previous results revealed that IgE-dependent histamine-releasing factor (HRF) is inducible by TCDD. The present study aimed to clarify the expression, localization, and function of HRF in endometriosis. Northern blot analysis demonstrated that HRF is overexpressed in endometriotic implants. RT-PCR with Southern blot analysis, however, showed that HRF overexpression was not always accompanied by CYP1A1 induction in endometriotic implants, suggesting that HRF is inducible in endometriosis without exposure to TCDD. HRF is also inducible by macrophage colony-stimulating factor (M-CSF). Immunohistochemistry showed CD68-positive macrophages in the stroma of endometriotic implants, adjacent to regions with prominent HRF accumulation. HRF-overexpressing cells exhibited high implantation efficiency in comparison to control cells when the cells were injected into the peritoneal cavities of nude mice. These results suggest that the accumulation of macrophages in endometriotic implants induces HRF; the overexpression of HRF accelerates the growth of endometriotic implants.

[Construction and tumorigenic study on a novel fusion gene AML1-MTG16].

OBJECTIVE: To test whether splicing overlapping extension(SOE) method can be a tool for obtaining rare fusion gene's transcripts and to study the tumorigenic capacity of a novel fusion gene AML1-MTG16. METHODS: SOE method was used to obtain AML1- MTG16 fusion gene's transcripts. MTG16, AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 segment were inserted into pEGFP- C1,pDsRed-N1 vector respectively,then transfected NIH3T3 cell line by lipofection. Forty-eight hours later, the transfected cells were examined by laser-scanning confocal microscopy. Stable transfected cells were obtained by G418 500ug/ul selection for one month. Growth curve, soft agar colonies formation tumorigenesis in nude mice were done to compare the difference between stable transfected cells. RESULTS: Recombined AML1-MTG16 by SOE contained its CDS. NIH3T3 expressing AML1-MTG16 had a faster proliferation in medium, colony growth in soft agar. AML1-MTG16 expression cells also induced tumors formation following injection into nude mouse. MTG16,AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 were colocalized in the nucleus of cotransfected NIH3T3 cells under the examination of laser-scanning confocal microscope. CONCLUSION: SOE is an effective method to get rare fusion gene's transcripts. AML1-MTG16 plays an important role in leukemogenesis. MTG16 may also have a carcinogenic property within the AML1-MTG16 fusion gene. Carcinogenic property of AML1-MTG16 is restricted to its localization in the nuclear matrix. N terminal of MTG16 may play an important part in the carcinogenic activity of AML1-MTG16.

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.

Human adrenocortical cell xenotransplantation: model of cotransplantation of human adrenocortical cells and 3T3 cells in scid mice to form vascularized functional tissue and prevent adrenal insufficiency.

To establish an experimental model for replacement of endocrine organ function by xenotransplantation, human adrenocortical cells from postnatal donors were transplanted beneath the kidney capsule of adrenalectomized scidmice together with mitomycin C-treated 3T3 cells that secrete FGF. Adrenocortical cells from seven donors, male and female, ranging from 6-50 years of age, were used. 12 of 13 animals survived > 16 days following surgery. After 50 days they were sacrified to allow assessment of the histology and ultrastructure of tissue formed from the transplanted cells. Only 1 of 23 adrenalectomized sham-operated animals survived > 16 days. In all surviving animals, vascularized adrenocortical tissue formed at the site of transplantation. Cortisol, the normal human glucocorticoid, was present in the plasma of these animals, replacing corticosterone, the mouse glucocorticoid. Some animals, but not most, had measurable aldosterone. The tissue formed from the transplanted cells showed histological and ultrastructural features of normal adrenal cortex. Mitochondria had tubulo-vesicular cristae and there were prominent microvilli between cells. Tissues had a well-developed vasculature, sometimes with large sinusoidal vessels. Proliferation in the transplant tissues was very low. These results show that tissue formed from transplanted human adrenocortical cells is able to replace the essential functions of the adrenal gland in scid mice. This demonstrates that transplanted human endocrine cells can functionally replace a surgically removed endocrine organ in a host animal.

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.

Angiostatin gene transfer as an effective treatment strategy in murine collagen-induced arthritis.

OBJECTIVE: To determine the efficacy of local therapy with human angiostatin gene in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with bovine type II collagen. Before the onset of arthritis, NIH3T3 fibroblasts, transduced with angiostatin-expressing retroviral vectors or control vectors, were transplanted into the knee cavity. The incidence of arthritis in the knee joints was evaluated histologically based on pannus formation and cartilage destruction. Paws were evaluated macroscopically for redness, swelling, and deformities and immunologically for levels of interleukin-1 beta. Angiogenesis in paws and knee joints was studied by immunohistochemistry using anti-CD31 antibody and measurement of von Willebrand factor levels. RESULTS: Pannus formation and cartilage erosion were dramatically reduced in knees transplanted with angiostatin-expressing cells. In addition, the onset of CIA in the ipsilateral paws below the knees injected with the angiostatin gene was significantly prevented. Furthermore, angiostatin gene transfer inhibited arthritis-associated angiogenesis. CONCLUSION: Local production of angiostatin in the knee was able to prevent the onset of CIA not only in the knee injected with genetically engineered cells, but also in the uninjected ipsilateral paw. This suggests that transfer of the angiostatin gene, and potentially also its protein, may provide a new, effective approach to the treatment of rheumatoid arthritis.

Cell-interactive alginate hydrogels for bone tissue engineering.

There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.

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.

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.

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.

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.

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.

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro.

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.

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.

Frozen allogeneic human epidermal cultured sheets for the cure of complicated leg ulcers.

BACKGROUND: Skin ulcers due to venous stasis or diabetes are common among the elderly and are difficult to treat. Repeated applications of cell-based products have been reported to result in cure or improvement of leg ulcers of small size in a fraction of patients. OBJECTIVE: To examine the effects of frozen human allogeneic epidermal cultures for the treatment of acute and chronic ulcers. METHODS: We treated a series of 10 consecutive patients with leg ulcers of different etiology and duration with frozen human allogeneic epidermal cultures stored frozen and thawed for 5-10 minutes at room temperature before application. Three patients had ulcers with exposed Achilles or extensor tendon. The ulcers treated were as large as 160 cm2 in area and of up to 20-years' duration. After preliminary preparation of the wounds by debridement to remove necrotic tissue and application of silver sulfadiazine to control infection, thawed cultures were applied biweekly from 2 to 15 times depending on the size and complexity of the ulcer. RESULTS: All ulcers healed, including those with tendon exposure. After the first few applications, granulation tissue formed in the ulcer bed and on exposed tendons, and epidermal healing took place through proliferation and migration of cells from the margins of the wound. The time required for complete healing ranged from 1 to 31 weeks after the first application. CONCLUSION: The use of frozen human allogeneic epidermal cultures is a safe and effective treatment for venous or diabetic ulcers, even those with tendon exposure. It seems possible that any leg ulcer will be amenable to successful treatment by this method.