Thymosin Beta-4 and Chronic Wound Healing
First human trial tests thymic hormone in pressure sores
About three million people in the U.S., primarily elderly and bedridden, suffer from chronic pressure sores (“bedsores”) that do not heal. These kinds of infections can result in loss of limbs or, in some cases, even death. Because they require such long-term care, the cost of treating just one pressure wound can range from $14,000 to $50,000.
When a chronic wound occurs, it is because the normal process of healing has been disrupted. Many factors may be responsible for such interruption, including infection, systemic causes such as diabetes, and the use of certain medications such as corticosteroids.
Standard treatments for pressure sores and diabetic ulcers include agents to debride them (remove dead tissue), topical agents such as antimicrobials and enzymes, and various types of dressings. Other options include treatment by Vacuum-Assisted Closure or surgery. But even with these advanced strategies, the recurrence rate for chronic wounds remains high. What is needed is a way to help the body heal once infections develop.
NewYork-Presbyterian Hospital/Columbia University Medical Center is addressing this challenge in a study on thymosin beta-4, a naturally occurring protein that can reduce inflammation and help wounds to heal. “Thymosin beta-4 is a major activator of actin, which improves the process of wound healing,” says Mark A. Hardy, MD. Dr. Hardy is Auchincloss Professor of Surgery at Columbia University College of Physicians and Surgeons, and Director of Islet Transplantation at NewYork-Presbyterian Hospital/ Columbia University Medical Center.
In collaboration with Alan Goldstein, PhD, Dr. Hardy was the first to test new synthesized thymic hormones in 1968. Since then the hormones have been studied by several groups in adult patients with cancer, children with mucocutaneous candidiasis, and now in the healing of chronic wounds. “As a surgeon, I have had a long-term interest in finding ways of helping to speed up wound healing,” says Dr. Hardy.
Although thymosin has not proven as successful in treating cancer as researchers had hoped, studies have found that it does improve the healing of chronic wounds. Initial tests on pressure sores in animal models found that thymosin is active in several wound healing processes, according to June K. Wu, MD, Assistant Professor of Clinical Surgery at Columbia University College of Physicians and Surgeons, and Co-Principal Investigator with Dr. Hardy. The next phase of the current study will be among the first to test thymosin beta-4 in people with pressure sores.
The thymosin beta-4 trial, sponsored by RegeneRx Biopharmaceuticals, Inc., is a randomized, double-blind, placebo-controlled study. About 20 patients will be enrolled at NewYork-Presbyterian/Columbia, and three-quarters of these will receive topical thymosin for their chronic wounds. The safety of three doses will be evaluated in this phase of study. NewYork-Presbyterian/Columbia is one among four participating institutions.
“Finding an agent that can promote healing of chronic wounds, reduce the time it takes for them to heal, or reduce the rate of recurrence of wounds, would have the potential to improve the lives of many disabled patients,” says Dr. Hardy.
Based on the results of this phase of the trial, the investigators plan to expand the study to examine the effect of thymosin beta-4 on the rate of postoperative wound healing, and to study the mechanism of its activity. This could lead to the clinical application of this novel hormonal approach to the recovery of surgical patients whose wound healing is impaired by various diseases.
Researchers Report that Thymosin beta 4 Improves Neurological Function after Stroke
ROCKVILLE, Md.–(BUSINESS WIRE)–Mar 5, 2010 – REGENERX BIOPHARMACEUTICALS, INC. (NYSE Alternext US:RGN) announced that a research team from the Henry Ford Hospital in Detroit, MI reported that Thymosin beta 4 (TÎ²4), administered to rats one day after embolic stroke, improved neurological functional outcome compared to control animals. Improvement in neurological function was measured at various time intervals over a seven week period and was statistically significant.
An increase in remyelination of axons (regeneration of the nerve sheath) was observed in rats receiving TÎ²4 compared to control animals, likely due to an increased mobilization of oligodendrocyte progenitors (stem cells surrounding axons) that differentiate into mature myelin-producing oligodendrocytes. In cell culture, TÎ²4 treated neuronal progenitor cells isolated from normal and stroke rats demonstrated increased mRNA levels of epidermal growth factor receptor. This receptor has previously been shown to be a regulator of oligoprogenitor cell expansion and tissue regeneration in response to brain injury and further supports the role of TÎ²4 in stem cell-mediated tissue repair.
“These data are compelling and are consistent with previously reported data in EAE mice (experimental models for multiple sclerosis) showing that TÎ²4 stimulates oligoprogenitor cells after injury. In this recent experiment, after an ischemic stroke, neurological function in the rat models was significantly improved, apparently by remyelination of neuronal axons induced by TÎ²4. The fact that TÎ²4 helps repair and regenerate tissue after a brain injury is not only remarkable, but strongly correlates with data previously published showing TÎ²4′s ability to regenerate cardiac tissue after an ischemic event,” stated Dr. Hynda Kleinman, chief of the Cell Biology Section at the National Institute of Dental and Craniofacial Research, NIH, and a consultant to RegeneRx.
“We are very pleased with these results, which provide a foundation to further explore TÎ²4 as a treatment for neurological injury,” commented Daniel C. Morris, MD, senior staff physician, Department of Emergency Medicine, Henry Ford Health Systems.
The research was presented by Dr. Morris, representing the Departments of Neurology and Emergency Medicine, Henry Ford Health System, Detroit, MI, at the International Stroke Conference, San Antonio, TX, February 23-26, 2010. The research was performed under a Material Transfer Agreement between RegeneRx Biopharmaceuticals, Inc. and the Henry Ford Health System.
About RegeneRx Biopharmaceuticals, Inc.
RegeneRx is focused on the discovery and development of novel peptides to accelerate tissue and organ repair. Currently, RegeneRx is developing three product candidates, RGN-137, RGN-259 and RGN-352 for dermal, ophthalmic, and cardiovascular tissue repair, respectively. These product candidates are based on TÎ²4, a synthetic copy of a 43-amino acid, naturally occurring peptide, in part, under an exclusive world-wide license from the National Institutes of Health. RegeneRx holds over 60 world-wide patents and patent applications related to novel peptides. It is currently in Phase 2 clinical development for dermal and ophthalmic wound healing and has now completed a Phase 1 clinical trial supporting delivery of RGN-352 for acute cardiovascular and other indications requiring systemic administration. RegeneRx is also developing novel peptides for the cosmeceutical industry based on its experience with TÎ²4 and its biological activities in the skin. It is currently in Phase 2 clinical development for dermal and ophthalmic wound healing and has now completed a Phase 1 clinical trial supporting delivery of RGN-352 for acute cardiovascular and other indications requiring systemic administration. RegeneRx is also developing novel peptides for the cosmeceutical industry based on its experience with TÎ²4 and its biological activities in the skin.
RegeneRx Technology Background
TÎ²4 is a synthetic version of a naturally occurring peptide present in virtually all human cells. It is a first-in-class, multi-faceted molecule that promotes endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, and collagen deposition, while down-regulating inflammation. RegeneRx has identified several molecular variations of TÎ²4 that may affect the aging of skin, among other properties, and could be important candidates as active ingredients in pharmaceutical and consumer products. Researchers at the National Institutes of Health, and at other academic institutions throughout the world, have published numerous scientific articles indicating TÎ²4′s in vitro and in vivo efficacy in accelerating wound healing and tissue protection under a variety of conditions. Abstracts of scientific papers related to TÎ²4′s mechanisms of action may be viewed at RegeneRx’s web page: Utilizing Thymosin Beta 4 (Tβ4) to develop drug candidates in three principal areas: dermal, ophthalmic, and cardiovascular wound healing. – RegeneRx
Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells
KM Malinda, AL Goldstein and HK Kleinman
National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.
Researchers Report Thymosin Beta 4 Significantly Reduces Damage From Traumatic Brain Injury And Improves Brain Function In Experimental Animals
RegeneRx Biopharmaceuticals, Inc. (NYSE Amex:RGN) announced today that in a preclinical research paper published in the May 2010 issue of the Journal of Neurosurgery, (online ahead of publication), scientists found that the systemic administration of thymosin beta 4, or Tβ4, significantly reduced brain tissue damage and improved brain function in rats with traumatic brain injury, or TBI. In the study, 10 rats were injected with Tβ4 one day following the inducement of TBI and four times thereafter over a 12-day period, while 9 rats were injected with a placebo or saline solution. In the group of rats treated with Tβ4, researchers observed reduced cell loss in the hippocampus, a part of the brain that plays an important role in long-term memory, as compared to the placebo group. The rats treated with Tβ4 also experienced growth of new blood vessels and neurons in the injured cerebral cortex, growth of brain cells known as oligodendrocytes in the CA3 field of the hippocampus, and recovery of sensory and motor functions as well as spatial learning. The researchers noted that the data for the first time demonstrate that delayed administration of Tβ4 significantly improves histological and functional outcomes in rats with TBI, indicating that Tβ4 has considerable therapeutic potential for patients with TBI.
“We believe these results are very encouraging. The fact that Tβ4 was administered beginning one day following injury and was still able to improve outcomes is significant,” added Dr. Allan L. Goldstein, professor of biochemistry and molecular biology at the George Washington University Medical School, and chairman of the board of directors and chief scientific advisor for RegeneRx. “We have now seen compelling data using Tβ4 in three different animal models – an EAE mouse model for multiple sclerosis, a rat model for embolic stroke, and this new study in traumatic brain injury – that have each demonstrated Tβ4′s ability to promote angiogenesis, regenerate neuronal tissue, and improve functional outcome. These data are also consistent with previously published studies showing regeneration of heart tissue after ischemic injuries to the myocardium.”
Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes
A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.
Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells
Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.
Thymosin β4 (Tβ4) is a polypeptide involved in cellular proliferation, differentiation, and migration, over-expressed in several tumor entities. We evaluated its expression and function in 298 newly diagnosed multiple myeloma patients and the murine 5TMM model. Mean Tβ4 expression was significantly lower in myeloma cells compared to normal plasma cells (P<0.001). The same observation can be made in the 5TMM-mouse model by qRT-PCR and ELISA. Here, Tβ4 overexpression by lentiviral transduction of 5T33MMvt-cells led to significantly decreased proliferative and migratory capacities and increased sensitivity to apoptosis-induction. Mice injected with Tβ4 over-expressing myeloma cells showed a longer survival compared to mice injected with controls (88,9 vs. 65,9 days, P<0.05). In 209 MM patients treated with high-dose therapy and autologous stem cell transplantation, expression of Tβ4 below the median was associated with a significantly shorter event free survival (37.6 vs. 26.2 months, P<0.05). In conclusion, our results indicate a possible tumor suppressive function of Tβ4.
β-thymosins are a family of small peptides that were originally proposed to be thymic hormones.1 They were identified as actin monomer binding proteins, controlling the availability of actin for polymerization. They may, therefore, have a crucial role in regulating cellular functions involving actin polymerization/depolymerization cycles. Currently, 15 β-thymosins have been identified and characterized as highly conservative 5-kDa peptides containing 40 to 44 amino acid residues. In most mammalian tissues, thymosin-β 4 (Tβ4), the most abundant thymosin peptide, Tβ10 and Tβ15, have been studied as important members of the β-thymosin family.2 Several studies reported that these genes are over-expressed in solid tumors, which could be correlated to the angiogenic and metastatic potential of the studied tumors.3
Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells (PC) in the bone marrow (BM). MM cell biology can be dissected into the interactions of MM cells with their surrounding stroma (matrix proteins, cytokines and BM cells) and in the acquisition of essential changes in cell behavior, such as self-sufficiency in growth signals, evasion of apoptosis and acquisition of invasive and spreading capacities.4 Earlier reports indicated that Tβ4 was down-regulated in RNA from primary human MM cells and cell lines.5
This observation is in contrast to the results obtained in most solid tumors where an upregulation is seen in malignant cells compared to their normal counterparts. Cha et al. showed that overexpression of Tβ4 resulted in an increased metastatic capacity of lung cancer cells and increased angiogenic response.6
Since migration, invasion and associated angiogenesis are key features in MM biology, we were interested in studying Tβ4 expression in a large panel of MM patients and its functionality in the murine 5TMM model.
Gene expression analysis on human myeloma cells
Tβ4 expression was analyzed in purified PCs from BM samples obtained from 14 healthy donors, 11 patients with monoclonal gammopathy of unknown significance (MGUS) and 298 previously untreated multiple MM patients at the University Hospitals of Heidelberg or Montpellier.7 Of these, 209 MM patients were treated by high-dose therapy and autologous stem cell transplantation (ASCT). Biotinylated complementary RNA (cRNA) was amplified according to the Affymetrix labeling protocol (Affymetrix, Santa Clara, CA, USA). cRNA from a first group of patients (7 normal donors, 7 MGUS and 65 MM patients) was hybridized to the human U133 A and B. This group will be referred to as the HM1-group. A second independent validation group of patients (7 normal donors, 16 MGUS and 233 MM patients) was named the HM2 group. For these patients, the U133 2.0 GeneChip was used. These micro-array data had been previously used for several analyses, but thymosin β4 expression had never been analyzed before.8,9 HM2 data were corrected for batch effect due to the usage of different labeling kits according to Johnson et al.10 Expression data were gcrma-normalized and analyzed by the bioconductor packages for R. For patients’ characteristic see Online Supplementary Table S1.
The 5T2MM and 5T33MM murine models of myeloma
The 5TMM models originated in elderly C57Bl/KaLwRij mice.11 The 5T33MMvivo (5T33MMvv) cells grow in vitro stroma-dependently with a limited survival while the 5T33MMvitro (5T33MMvt) cell line is a clonally identical variant that originated from an in vitro culture of 5T33MMvv cells, growing BM stroma-independently in RPMI-1640 supplemented with 10% bovine serum 1% natriumpyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (all from Biowhittaker, Verviers, Belgium).12
Quantification of intracellular protein levels of Tβ4 and F-Actin G-Actin
Enzyme-Linked Immunosorbent Assays (ELISA) for measuring Tβ4 concentrations were performed according to the manufacturer’s instructions (Immundiagnostik, Bensheim, Germany). Cells (107) were lyzed in a phosphate buffer containing 0.14 M NaCl, 2.6 M KCl, 8 mM Na2HPO4, 1.4 M KH2PO4 and 1% Triton X100 and sonicated with an ultrasound finger. protein levels and ratios between F-Actin and G-Actin were determined using the G-actin/F-actin in vivo assay kit (Cytoskeleton Inc, Denver, USA).
Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7700 Sequence Detection System. For the detection of both human and mouse Tβ4 mRNA and the endogenous reference gene GUS, Assays on Demand (Applied Biosystems) were used. To verify the results obtained with the microarrays studies, Tβ4 expression was measured in 3 cell lines and in 3 patient samples and their correlations statistically verified using a Spearman correlation test.
Generation of 5T33MMvt cells over-expressing Tβ4
A lentiviral transferplasmid encoding mouse Tβ4 (m Tβ4) was constructed. The mTβ4 gene was obtained from HJ Cha (NIDCR, NIH, Bethesda, USA)6 and inserted into the transferplasmid pHR’tripCMV-IRES-tNGFR-SIN.13 mTβ4-encoding lentiviral vector particles were produced in 293T cells, collected, ultracentrifugated and their viral titer determined.14 After transduction, 5T33MMvt cells were surface stained using an in-house biotinylated anti-tNGFR antibody and purified by FACS sorting into a 6-well plate (Becton Dickinson, FACSVantage). Next, they were analyzed for Tβ4 expression by RT-PCR. The 5T33MMvt cells over-expressing Tβ4 will be referred to as 5T33MMvtTβ4+.
In vitro and in vivo effects of Tβ4 overexpression
In vitro proliferation was assessed by measuring DNA synthesis using a 3H-thymidine incorporation assay, as described earlier.15 Apoptosis sensitivity of the MM cells was analyzed by staining with FITC labeled-annexin V and propidium iodide according to the manufacturers’ instructions (BD Biosciences, Erembodegem, Belgium). In vitro migration studies were performed using Transwell chambers and 10% fetal calf serum as chemoattractant and were quantified through flow cytometry. To determine the effect of Tβ4 overexpression on survival, groups of 10 C57BLKaLwRij mice were intravenously injected with either 5×105 5T33MMvtTβ4+ or wild-type 5T33MMvt cells. Animals were sacrificed when they showed signs of morbidity, namely hind limb paralysis. Kaplan-Meier analysis was used to determine a difference in the survival.
Different studies indicated a pivotal role of Tβ4 in the metastatic process of solid tumors.16,17 An adenoviral-based overexpression of Tβ4 was applied in a colon cancer and melanoma model showing increased growth, motility and invasive capacities in vitro and a larger tumor load in vivo.18,19 Since proliferation, migration and invasion are part of the hallmarks of the biology of MM, we were interested in investigating an involvement of Tβ4 in this disease. We first investigated the Tβ4 expression pattern in 298 primary MM-cell samples and 14 normal plasma cell samples from healthy donors. Tβ4 expression is significantly lower in MM cells of the HM1 group (P<0.05) and HM2 group (P<0.001) compared to normal plasma cells. This holds true for a significantly lower Tβ4 expression in its pre-malignant stage (MGUS), its early (Durie Salmon stage I) or late stage (Durie Salmon II and III) in both HM1 and HM2 groups (P<0.001) (Figure 1A). No relevant correlation could be found between Tβ4 expression and percentage of plasma cell infiltration in the bone marrow smear. Gene expression assessed by DNA-microarray correlates well with qRT-PCR performed on MM patient samples (coefficient of correlation r=0.993, P<0.001). These data are in agreement with results from Gondo et al. showing a decrease in Tβ4 expression in a small number of MM samples by Northern blot analysis.5
To assess the functional involvement of differential Tβ4 expression we used the 5T33MMvt and 5T33MMvtTβ4+ cell lines. In a 3H thymidine assay, 5T33MMvtTβ4+ cells showed a significant decrease in DNA synthesis compared to control cells (P<0.05). 5T33MMvtTβ4+ cells showed a significantly increased sensitivity to vinca-alkaloids (vinblastin) and bortezomib (Figure 2B; P<0.001 for both bortezomib and vinblastin).
Likewise, bortezomib induced apoptosis was higher in 5T33MMvtTβ4+ compared with 5T33MMvt cells (P<0.05; Figure 2C). In addition to affect survival pathways, Tβ4 overexpression reduced migratory capacities of 5T3MM cells; the percentages of cells that migrated in basal conditions and in 10%FCI was significantly lower in 5T33MMvtTβ4+ compared to control cells (P<0.05; Figure 2D). The relative increase after stimulation (compared to basal conditions) was, however, similar in both populations. We further examined the effects of Tβ4 expression on tumor development and survival of diseased mice by injecting mice intravenously with 5T33MMvtTβ4+ or control cells. In this study, the mean survival of mice injected with control cells was significantly shorter 65.9 days (SD 6.6 days), compared to 88.9 days (SD 9.3 days) for mice injected with 5T33MMvtTβ4+ cells (P<0.05; Figure 2F). These in vivo results confirm data obtained using the in vitro proliferation and apoptosis assays.
In solid tumors, Tβ4 expression is frequently upregulated in malignant and metastatic cells. In these cancers, higher Tβ4 expression resulted in increased metastatic and invasive capacities of tumor cells, while proliferation remained unaffected.6 In hematologic disorders, malignant plasma cell disorders, such as plasma cell leukemia and MM were the rare disorders that showed a decreased Tβ4 expression.5,20 In contrast to solid tumors, publications on the function of Tβ4 in hematologic conditions are scanty but indicate some inhibitory activity. Tβ4 was initially isolated and purified from a thymic protein preparation, called thymosin fraction-5. Addition of this protein fraction to different leukemic cell lines resulted in a decrease in growth responses.21 Similar inhibitory effects were recently described for Tβ4 on hematopoietic stem cells,22 bone marrow derived mast cells23 and human promyelocytic leukemia cells,24 in agreement with the results presented here. Whereas a mechanistic explanation of this discrepancy is beyond the scope of this paper, further investigations are clearly merited.
Since Tβ4 has been shown to bind G-actin in a 1:1 manner and thus affects the polymerization of G-Actin into F-Actin, we analyzed in a semi-quantitative way, intracellular G-actin and F-Actin. This quantification showed a lowered G-Actin-F-Actin ratio after Tβ4 overexpression (Figure 2E). F-Actin is of particular importance in cytoskeleton changes involved in cellular migration and in microtubuli organization controlling the mitotic spindle.25,26 In line with these results, vinca-alkaloids (e.g. vinblastine used here) with micro-tubulin (polymerization) inhibitory activity, had more affect on the proliferation capacities of 5T33MMvtTβ4+ cells than on control cells (Figure 2B). Since immunohistochemical studies also showed a nuclear staining of Tβ4 in 5TMM cells (results not shown), involvement of other pathways might also be implicated. Supervized gene analysis comparing Tβ4high with Tβ4low found different groups of genes differently expressed, including genes involved in cytoskeleton organization, nuclear homeostasis, lymphocyte differentiation and protein metabolism, which might indicate that the role of Tβ4 is more complicated than initially supposed.
In conclusion, our results propose a tumor suppressive function of Tβ4 expression in MM with impact on survival. Tβ4 was down-regulated in MM cells of patients compared to the normal BM plasma cells and studies with the murine 5T33MM model show a decreased in vitro and in vivo tumor growth for cells over-expressing the Tβ4 gene.