MRI Study of Jellyfish Collagen, Hyaluronic Acid, and Cadaveric Dermis for Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Test a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice. STUDY DESIGN: Animal model. METHODS: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross-linked jellyfish collagen (MX-JC) and adipose derived stem cells (ADSCs), Group 2, MX-JC alone, Group 3, cross-linked hyaluronic acid (X-HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology. RESULTS: After 100 µL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 µL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 µL, Group 3: 27.4 ± 12 and 10.6 ± 8 µL, and Group 4: 37.5 ± 11 and 9.85 ± 1 µL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4. CONCLUSIONS: MX-JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX-JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX-JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2452-E2460, 2021.

Chondrogenic Predifferentiation Inhibits Vascular Endothelial Growth Factor Angiogenic Effect in Pericranium-Derived Spheroids.

Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or comorbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering, pluripotent cells can differentiate directly into bone cells. Does ability to recapitulate bone formation in vitro affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with preplanned craniotomy surgery and studied three-dimensional osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample, we investigated (i) continuous osteogenic differentiation (COD) and (ii) osteogenic differentiation preceded by chondrogenesis (CD → OD). The effect of vascular endothelial growth factor (VEGF) was compared to VEGF supplemented with fibroblast growth factor, interleukin (IL)-1, IL-6, platelet-derived growth factor, and tumor necrosis factor-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample, we observed no age- or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CD → OD spheroids were noted with regular media. In COD, however, VEGF statistically significantly increased angiogenesis compared to control media (p = 0.007). Also, in COD, both VEGF and VEGF + GFM increased osteogenesis (p = 0.047 and p = 0.038, respectively). By contrast, in CD → OD, neither VEGF nor VEGF + GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results, we characterized spheroid protein expression by nanoliquid chromatography coupled to tandem mass spectrometry. Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CD → OD: (i) endothelial markers JUP, PTGIS, PTGS2, and TYMP, (ii) tissue remodeling factors CHI3L1 and MMP14, and (iii) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, and WNT5A define a conserved angiogenic network and were >2-fold increased in VEGF compared to VEGF + GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD + VEGF conditions, PPF scaffolds loaded with pericranium-derived cells displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.

Time-course mass spectrometry data of adipose mesenchymal stem cells acquiring chondrogenic phenotype.

OBJECTIVES: Rabbit adipose mesenchymal stem cells were used for the purpose of studying acquisition of the chondrogenic phenotype over time at 1, 14 and 28 days after in vitro incubation with differentiation media, using nano-liquid chromatography electrospray ionization tandem mass spectrometry analysis. This was part of a preliminary study of the behavior of differentiated adipose stem cells for use in a rabbit model of laryngoplasty. DATA DESCRIPTION: The data comprise .MGF, .RAW, .MZID, and .XLSX, lists of peaks, peptides and proteins identified by nano-flow liquid chromatography electrospray ionization tandem mass spectrometry analysis upon incubation with non-differentiating (ND) or chondrogenic differentiating (CHD) media (ProteomeXchange ID PXD010236). XLSX files contain the following information: day 1 CT (control, N = 3499 proteins), day 14 ND (N = 3106 proteins), day 28 ND (N = 3116 proteins), day 14 CHD (N = 2901 proteins), and day 28 CHD (N = 2876 proteins). Proteins are characterized with respect to their - 10lgP value, percent coverage, number of total as well as unique peptides after trypsin digestion, derivatization method (carbamidomethylation, oxidation, or combined carbamidomethylation + oxidation), average mass, and include a full description.

Histone Variants as Stem Cell Biomarkers for Long-Term Injection Medialization Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Vocal fold (VF) paralysis by sectioning the recurrent laryngeal nerve dramatically impacts the life of thyroidectomy patients. Volume-expanding materials can temporarily restore VF medialization. To prolong this benefit, adipose mesenchymal stem cells (ADSCs) and micronized acellular dermis (MACD) were co-injected in a rabbit model of injection medialization laryngoplasty. Biomarkers of in situ proliferation were identified by mass spectrometry proteomics and pathway analysis to guide future efforts to increase the length of benefit. METHODS: ADSCs were expanded and/or differentiated into chondrocytes (CHON) as collagen microspheres. After VF paralysis rabbits received MACD, MACD + undifferentiated ADSC, or MACD+CHON, ADSCs differentiated into chondrocytes. After 12 weeks, animals were sacrificed and 5-µm paraffin-embedded cryosections were prepared from larynges for hematoxylin and eosin visualization and nanoflow liquid chromatography electrospray-ionization tandem-mass spectrometry analysis of tissue collections. Validated proteins were processed by Venn subtraction and gene ontology (GO) overrepresentation analysis to identify unique pathways and biomarkers. RESULTS: Confirmed proteins numbered 147 (MACD), 1,243 (MACD+ADSC), and 1,033 (MACD+CHON). Totally, 333 proteins were uniquely found in the MACD+ADSC group, including mesenchymal surface markers CD9, CD44, fibronectin, and vimentin. Over 70% of proteins belonged to catalytic activity and binding GO categories, with the histone (H) family being overrepresented (P < 0.05). Histone variants H3.3, H2A.V, and H2A.Z (associated with open chromatin states) were overrepresented in the MACD+ADSC group, whereas structural histones H2A, H2B, and H4 were not. CONCLUSION: Biomarkers, including atypical histones, are associated with in vivo proliferation of ADSCs and an expanded VF medialization volume. LEVEL OF EVIDENCE: NA Laryngoscope, 128:E402-E408, 2018.

Preliminary results of tissue-engineered injection laryngoplasty material in a rabbit model.

OBJECTIVES/HYPOTHESIS: Design and test a novel biomaterial for injection laryngoplasty aimed to increase the duration of effectiveness of micronized acellular dermis. STUDY DESIGN: Animal model. METHODS: Injection laryngoplasty was performed in three groups (n = 5) of New Zealand White rabbits. Acellular dermis was either used alone as a control (group 1), was combined with undifferentiated stem cells (group 2), or with predifferentiated chondrocytic cells (group 3). Groups 2 and 3 were supplemented with growth factors. Animals were sacrificed 4 and 12 weeks after laryngoplasty and histologic analysis was completed. The major outcome measure was volume of tissue remaining. RESULTS: After 4 weeks, the mean volume of tissue remaining was 341 ± 89 mm3 , 295 ± 102 mm3 , and 133 ± 15 mm3 , for groups 1 to 3, respectively. At the 12-week time point, volumes were 62 ± 62 mm3 , 235 ± 35 mm3 , and 107 ± 99 mm3 . After 12 weeks, there was a significantly higher volume in group 2 compared to group 1 or 3 (P = .01, P = .04). Volumes between week 4 and week 12 were significantly lower in group 1 (P = .02), but not significantly different for groups 2 and 3 (P = .38, P = .74). Histologic evaluation revealed a robust lymphocytic infiltration in all cases as well as morphologic and immunophenotypic features suggestive of chondrogenic differentiation in a single animal. CONCLUSIONS: Micronized acellular dermis combined with stem cells and growth factors showed significantly less resorption 12 weeks after injection laryngoplasty compared to micronized acellular dermis alone. Groups using novel tissue-engineered biomaterial showed a lower resorption rate over time compared with acellular dermis alone. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:160-167, 2018.

Correction: COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo.

[This corrects the article DOI: 10.18632/oncotarget.9133.].

Human Adipose-Derived Mesenchymal Stem Cells for Osseous Rehabilitation of Induced Osteoradionecrosis: A Rodent Model.

Objective Human adipose-derived mesenchymal stem cells (ADSCs) were used to rehabilitate bone damaged by osteoradionecrosis (ORN) in an established animal model. Study Design Prospective animal study. Setting Academic department laboratory. Subjects and Methods After institutional review board and Institutional Animal Care and Use Committee approval, 24 athymic nude rats were divided into 5 groups: 4 groups irradiated (20 Gy) by brachytherapy catheter placed at the left hemimandible and 1 mock irradiation control (n = 4). For all groups, ORN was initiated by extraction of the central molar 1 week later. After 28 days, animals (n = 5/group) received injection at the extraction site with saline (SAL), ADSCs, platelet-rich plasma and collagen (PRP/COL), or ADSCs + PRP/COL. Rats were sacrificed 28 days later and their mandibles harvested for histopathology analysis (osteoblasts, osteoclasts, and fibrosis) and bone volume measurement using 3-dimensional micro-computed tomography. Results All but 1 rat survived the experiment period (23/24). Radiographic and histological analysis revealed 60% bone loss in the SAL group compared with the nonirradiated control. Injection of ADSCs increased jaw region bone volume by up to 36% ( P < .01). All experimental groups (ADSC, PRP/COL, and ADSC + PRP/COL) showed dramatically decreased osteoclast counts ( P < .001) while injection of PRP/COL with or without ADSCs increased osteoblasts. Increased fibrosis was observed after ADSC injection ( P < .05). Conclusion The application of human ADSCs to an induced mandibular osteoradionecrosis model in athymic rats results in increased deposition or preservation of bone, demonstrated both histologically and radiographically. This offers an encouraging possible treatment option for translational research in this difficult disease.

Assessment of Scaffolding Properties for Chondrogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells in Nasal Reconstruction.

IMPORTANCE: Nasal reconstruction in patients who are missing a significant amount of structural nasal support remains a difficult challenge. One challenge is the deficiency of cartilage left within the nose as a consequence of rhinectomy or a midline destructive disease. Historically, the standard donor source for large quantities of native cartilage has been costal cartilage. OBJECTIVE: To enable the development of protocols for new mesenchymal stem cell technologies as alternative procedures with reduced donor site morbidity, risk of infection and extrusion. DESIGN, SETTING, AND MATERIALS: We examined 6 popular scaffold materials in current practice in terms of their biodegradability in tissue culture, effect on adipose-derived mesenchymal stem cell growth, and chondrogenic fate commitment. Various biomaterials of matching size, porosity, and fiber alignment were synthesized by electrospinning and overlaid with rabbit adipose-derived mesenchymal cells in media supplemented or not with chondrogenic factors. Experiments were performed in vitro using as end points biomarkers for cell growth and chondrogenic differentiation. Polydioxanone (PDO), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), PHBV-polycaprolactone, poly(L-lactide-co-caprolactone), poly(lactic-co-glycolic acid), and polystyrene scaffolds of 60% to 70% porosity and random fiber alignment were coated with poly(L)-lysine/laminin to promote cell adhesion and incubated for 28 days with 2.5 to 3.5 × 105 rabbit adipose mesenchymal cells. MAIN OUTCOMES AND MEASURES: Cell growth was measured by fluorometric DNA quantitation and chondrogenic differentiation of stem cells by spectrophotometric sulfated glycosaminoglycan (sGAG) assay. Microscopic visualization of cell growth and matrix deposition on formalin-fixed, paraffin-embedded tissue sections was performed, respectively, with nuclear fast red and Alcian blue. RESULTS: Of 6 scaffold materials tested using rabbit apidose mesenchymal cells, uncoated scaffolds promoted limited cell adhesion but coating with poly(L)-lysine/laminin enabled efficient cell saturation of scaffold surfaces, albeit with limited involvement of scaffold interiors. Similar growth rates were observed under these conditions, based on DNA content analysis. However, PDO and PHBV/PCL scaffolds supported chondrogenic fate commitment better than other materials, based on soluble sGAG analysis and microscopic observation of chondrogenic matrix deposition. The mean (SD) sGAG scaffold values expressed as fold increase over control were PDO, 2.26 (0.88), PHBV/PCL, 2.09 (0.83), PLCL, 1.36 (0.39), PLGA, 1.34 (0.77), PHBV, 1.07 (0.31), and PS, 0.38 (0.14). CONCLUSIONS AND RELEVANCE: These results establish materials, reagents, and protocols for tissue engineering for nasal reconstruction using single-layer, chondrogenically differentiated, adipose-derived mesenchymal stem cells. Stackable, scaffold-supported, multisheet bioengineered tissue may be generated using these protocols. LEVEL OF EVIDENCE: NA.

Preclinical Models for Investigation of Herbal Medicines in Liver Diseases: Update and Perspective.

Liver disease results from a dynamic pathological process associated with cellular and genetic alterations, which may progress stepwise to liver dysfunction. Commonly, liver disease begins with hepatocyte injury, followed by persistent episodes of cellular regeneration, inflammation, and hepatocyte death that may ultimately lead to nonreversible liver failure. For centuries, herbal remedies have been used for a variety of liver diseases and recent studies have identified the active compounds that may interact with liver disease-associated targets. Further study on the herbal remedies may lead to the formulation of next generation medicines with hepatoprotective, antifibrotic, and anticancer properties. Still, the pharmacological actions of vast majority of herbal remedies remain unknown; thus, extensive preclinical studies are important. In this review, we summarize progress made over the last five years of the most commonly used preclinical models of liver diseases that are used to screen for curative herbal medicines for nonalcoholic fatty liver disease, liver fibrosis/cirrhosis, and liver. We also summarize the proposed mechanisms associated with the observed liver-protective, antifibrotic, and anticancer actions of several promising herbal medicines and discuss the challenges faced in this research field.

Adipose-Derived Mesenchymal Stem Cell Features in Patients with a History of Head and Neck Radiation.

OBJECTIVES/HYPOTHESIS: Radiation plays a prominent role in advanced stage head and neck tumors. Often, the radiated area includes adjacent nonmalignant mesenchymal tissue, which contains a mixture of cells that has been shown to accelerate wound healing. The purpose of this study is to determine the long-term effect of radiation on the expansion potential of adipose-derived stromal/stem cell (ADSC) tissue and on the ability of resident stem cells in this fraction to undergo phenotypic differentiation. Study Design/Methods: After institutional review board approval, 12 patients with a history of head and neck radiation and pending surgery were enrolled. Adipose tissue was collected from irradiated tissue (XRT) and nonirradiated tissue (NRT) sites. Mesenchymal stem cells were isolated from these populations, with subsequent assessment of cellular kinetics and differentiation potential between harvest sites. RESULTS: Adipose-derived stromal/stem cells could not be isolated from XRT in six patients due to lack of in vitro cell proliferation. For the remaining six patients, overall cumulative population-doubling time was longer for XRT relative to NRT (29.3 vs. 11.5 days; P = 0.02). However, no significant differences were observed in cell generation time or viability. When XRT and NRT ADSC fractions were grown to standardized concentrations and incubated under conditions that induce phenotypic differentiation of resident stem cells, no significant changes in chondrogenic, adipogenic, or osteogenic differentiation were observed. CONCLUSION: These preliminary observations suggest that irradiated ADSCs close to the surgical site undergo long-term changes in proliferative capacity. The potential for phenotypic differentiation is retained, however, in ADSCs that survive the irradiation process. LEVEL OF EVIDENCE: 2b.

Suspected leukemia oncoproteins CREB1 and LYL1 regulate Op18/STMN1 expression.

Stathmin (STMN1) is a microtubule destabilizing protein with a key role in cell cycle progression and cell migration that is up-regulated in several cancers and may contribute to the malignant phenotype. However, the factors that regulate its expression are not well understood. Loss as well as gain-of-function p53 mutations up-regulate STMN1 and in acute myelogenous leukemia where p53 is predominantly wild-type, STMN1 is also over-expressed. Here we show regulatory control of STMN1 expression by the leucine zipper transcription factor (TF) CREB1 and the basic helix-loop-helix TF LYL1. By ChIP-chip experiments we demonstrate in vivo the presence of LYL1 and CREB1 in close proximity on the STMN1 promoter and using promoter assays we reveal co-regulation of STMN1 by CREB1 and LYL1. By contrast, TAL1, another suspected oncoprotein in leukemia and close relative of LYL1, exerts no regulatory effect on the STMN1 promoter. NLI, LMO2 and GATA2 are previously described co-activators of Tal1/Lyl1-E47 transcriptional complexes and potentiate Lyl1 activation of the STMN1 promoter while having no effect on TAL1 transactivation. Promoter mutations that abrogate CREB1 proximal binding or mutations of the DNA-binding domain of CREB1 abolish LYL1 transcriptional activation. These results show that CRE and Ebox sites function as coordinated units and support previous evidence of joint CREB1-and LYL1 transcription events activating an aberrant subset of promoters in leukemia. CREB1 or LYL1 shRNA knock-down down-regulate STMN1 expression. Because down-regulation of STMN1 has been shown to have anti-proliferative effects, while CREB1 and LYL1 are suspected oncoproteins, interference with CREB1-LYL1 interactions may complement standard chemotherapy and yield additional beneficial effects.

Wilms' tumor 1 suppressor gene mediates antiestrogen resistance via down-regulation of estrogen receptor-alpha expression in breast cancer cells.

The antiestrogen tamoxifen has been used in the treatment of hormone-responsive breast cancer for over a decade. The loss of estrogen receptor (ER) expression is the most common mechanism for de novo antiestrogen resistance. Wilms' tumor 1 suppressor gene (WT1) is a clinically useful marker that is associated with poor prognosis in breast cancer patients; its high level expression is frequently observed in cases of breast cancer that are estrogen and progesterone receptor negative. The lack of expression of these receptors is characteristic of tumor cells that are not responsive to hormonal manipulation. To determine whether there is a linkage between WT1 expression and antiestrogen resistance in breast cancer cells, we studied the effect of WT1 on tamoxifen responsiveness in ERalpha-positive MCF-7 cells. We found that overexpression of WT1 in MCF-7 markedly abrogated tamoxifen-induced cell apoptosis and 17beta-estradiol (E(2))-mediated cell proliferation. The expressions of ERalpha and its downstream target genes were significantly repressed following overexpression of WT1, whereas the down-regulation of WT1 by WT1 shRNA could enhance ERalpha expression and the sensitivity to tamoxifen treatment in ERalpha-negative MDA468 and HCC1954 cells that express high levels of WT1. Furthermore, we have confirmed that the WT1 protein can bind to endogenous WT1 consensus sites in the proximal promoter of ERalpha and thus inhibit the transcriptional activity of the ERalpha promoter in a WT1 site sequence-specific manner. Our study clearly implicates WT1 as a mediator of antiestrogen resistance in breast cancer through down-regulation of ERalpha expression and supports the development of WT1 inhibitors as a potential means of restoring antiestrogen responsiveness in breast cancer therapy.

Lyl1 interacts with CREB1 and alters expression of CREB1 target genes.

The basic helix-loop-helix (bHLH) transcription factor family contains key regulators of cellular proliferation and differentiation as well as the suspected oncoproteins Tal1 and Lyl1. Tal1 and Lyl1 are aberrantly over-expressed in leukemia as a result of chromosomal translocations, or other genetic or epigenetic events. Protein-protein and protein-DNA interactions described so far are mediated by their highly homologous bHLH domains, while little is known about the function of other protein domains. Hetero-dimers of Tal1 and Lyl1 with E2A or HEB, decrease the rate of E2A or HEB homo-dimer formation and are poor activators of transcription. In vitro, these hetero-dimers also recognize different binding sites from homo-dimer complexes, which may also lead to inappropriate activation or repression of promoters in vivo. Both mechanisms are thought to contribute to the oncogenic potential of Tal1 and Lyl1. Despite their bHLH structural similarity, accumulating evidence suggests that Tal1 and Lyl1 target different genes. This raises the possibility that domains flanking the bHLH region, which are distinct in the two proteins, may participate in target recognition. Here we report that CREB1, a widely-expressed transcription factor and a suspected oncogene in acute myelogenous leukemia (AML) was identified as a binding partner for Lyl1 but not for Tal1. The interaction between Lyl1 and CREB1 involves the N terminal domain of Lyl1 and the Q2 and KID domains of CREB1. The histone acetyl-transferases p300 and CBP are recruited to these complexes in the absence of CREB1 Ser 133 phosphorylation. In the Id1 promoter, Lyl1 complexes direct transcriptional activation. We also found that in addition to Id1, over-expressed Lyl1 can activate other CREB1 target promoters such as Id3, cyclin D3, Brca1, Btg2 and Egr1. Moreover, approximately 50% of all gene promoters identified by ChIP-chip experiments were jointly occupied by CREB1 and Lyl1, further strengthening the association of Lyl1 with Cre binding sites. Given the newly recognized importance of CREB1 in AML, the ability of Lyl1 to modulate promoter responses to CREB1 suggests that it plays a role in the malignant phenotype by occupying different promoters than Tal1.

The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells.

INTRODUCTION: There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein. METHODS: Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture. RESULTS: Transfection with WT1-ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1-ZF form lacking the lysine-threonine-serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1-ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1-ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells. CONCLUSION: Our observations strongly argue that the WT1-ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1-ZF - KTS provides a potential candidate of gene therapy for breast cancer.

Transcriptional activation of c-myc proto-oncogene by WT1 protein.

The Wilms' tumor 1 gene (WT1) plays an essential role in urogenital development and malignancy. Through DNA binding, WT1 can either enhance or repress transcription depending on the context of the DNA-binding sites or the cell type in which it is expressed. WT1 is overexpressed in a variety of human cancers, including leukemia and breast cancer; in these diseases, the expression of WT1 is associated with a poor prognosis. To determine how WT1 affects c-myc expression in the context of breast cancer cells, we have examined the ability of both endogenous and exogenous WT1 proteins in breast cancer cells to bind to the c-myc promoter in vivo. Using c-myc-promoter-driven luciferase constructs, we found that different forms of WT1 could enhance the expression of the reporter. Unlike other studies where WT1 is reported to be a negative regulator of c-myc, we found that both the - and + KTS forms of WT1 could act to enhance c-myc expression, depending on the cell type. The WT1-binding site near the second major transcription start site of the c-myc promoter was confirmed to be involved in upregulation of human c-myc by WT1. Finally, we demonstrated that overexpression of WT1 induced a significant increase in the abundance of endogenous c-myc protein in breast cancer cells, consistent with the upregulation of c-myc transcription following WT1 induction. These observations strongly argue that in the case of breast cancer WT1 is functioning as an oncogene in part by stimulating the expression of c-myc.