Redifferentiation and induction of tumor suppressors miR-122 and miR-375 by the PAX8/PPARγ fusion protein inhibits anaplastic thyroid cancer: a novel therapeutic strategy.

Anaplastic thyroid cancer (ATC) is an aggressive, fatal disease unresponsive to traditional therapies, generating a need to develop effective therapies. The PAX8/PPARγ fusion protein (PPFP) has been shown to favorably modulate tumor growth in follicular thyroid cancer, prompting our evaluation of its efficacy to inhibit ATC cell and tumor growth in vitro and in vivo. PPFP was constitutively expressed in five ATC cell lines: BHT-101, FRO, C-643, KTC-2 and KTC-3, and inhibited cell growth in four of five cell lines and xenograft tumor growth in four of four cell lines. PPFP-mediated growth inhibition involved multiple mechanisms, including upregulation of miR-122 and miR-375, associated with decreased angiogenesis and AKT pathway inactivation, respectively. Also, PPFP expression resulted in marked increase of thyroid-specific marker transcripts, including PAX8, thyroid peroxidase (TPO), sodium iodide symporter (NIS) and thyroglobulin, to varying degrees by activating their respective promoters, suggesting that PPFP induced cellular redifferentiation. Functional studies demonstrate that increased NIS messenger RNA is not associated with increased 125I uptake. However, ectopic expression of wild-type NIS-induced perchlorate-sensitive iodine uptake, suggesting that endogenous NIS in ATC cell lines is defective. As current treatment for ATC is only palliative, overexpression of PPFP may offer a novel therapeutic strategy for the treatment of ATC.

Preclinical efficacy of the oncolytic measles virus expressing the sodium iodide symporter in iodine non-avid anaplastic thyroid cancer: a novel therapeutic agent allowing noninvasive imaging and radioiodine therapy.

Anaplastic thyroid cancer is an extremely aggressive disease resistant to radioiodine treatment because of loss of sodium iodide symporter (NIS) expression. To enhance prognosis of this fatal cancer, we validated the preclinical efficacy of measles virus (MV)-NIS, the vaccine strain of the oncolytic MV (MV-Edm), modified to include the NIS gene. Western blotting analysis confirmed that a panel of eight anaplastic thyroid cancer (ATC)-derived cell lines do not express NIS protein, but do express CD46, the MV receptor. In vitro cell death assays and in vivo xenograft studies demonstrate the oncolytic efficacy of MV-NIS in BHT-101 and KTC-3, ATC-derived cell lines. Radioactive iodine uptake along with single-photon emission computed tomography (SPECT)-computed tomography imaging of KTC-3 xenografts after (99)Tc(m) administration confirmed NIS expression in vitro and in vivo, respectively, after virus treatment. Adjuvant administration of RAI, to MV-NIS-treated KTC-3 tumors showed a trend for increased tumor cell killing. As current treatment for ATC is only palliative, and MV-NIS is currently Food and Drug Administration approved for human clinical trials in myeloma, our data indicate that targeting ATC with MV-NIS could prove to be a novel therapeutic strategy for effective treatment of iodine-resistant ATC and will expedite its testing in clinical trials for this aggressive disease.

Antitumor Activity of VB-111, a Novel Antiangiogenic Virotherapeutic, in Thyroid Cancer Xenograft Mouse Models.

VB-111 is an engineered antiangiogenic adenovirus that expresses Fas-c in angiogenic blood vessels and has previously been shown to have significant antitumor activity in vitro and in vivo in Lewis lung carcinoma, melanoma, and glioblastoma models. To evaluate the efficacy of VB-111 in thyroid cancer, we conducted in vivo xenograft nude mouse studies using multiple thyroid cancer-derived cell lines models. VB-111 treatment resulted in 26.6% (P = 0.0596), 34.4% (P = 0.0046), and 37.6% (P = 0.0249) inhibition of tumor growth in follicular, papillary and anaplastic thyroid cancer models, respectively. No toxicity was observed in any model. All tumor types showed a consistent and significant reduction of CD-31 staining (P < 0.05), reflecting a reduction of angiogenic activity in the tumors, consistent with the intended targeting of the virus. A phase 2 clinical trial of VB-111 in patients with advanced differentiated thyroid cancer is ongoing.

Expression of the PAX8/PPARγ Fusion Protein Is Associated with Decreased Neovascularization In Vivo: Impact on Tumorigenesis and Disease Prognosis.

The PAX8/PPARγ fusion protein (PPFP) occurs in 36% of human follicular thyroid carcinoma (FTC) and is associated with favorable prognosis. To elucidate the function of PPFP in FTC, we analyzed the consequences of PPFP expression in immortalized thyrocytes in vitro and in vivo via xenograft tumorigenesis. While PPFP-expressing cells exhibited oncogenic hallmarks, including increased growth and decreased apoptosis, in vitro, xenograft tumors were initiated but not sustained in vivo. PPFP xenograft tumors exhibited reduced CD31 staining and VEGF expression, suggesting that PPFP modulates neovascularization. Microarray analysis demonstrated increased expression of tissue inhibitor of metalloproteinase (TIMP-3), an inhibitor of angiogenesis, in PPFP cells and tumors, a finding confirmed by quantitative PCR and immunohistochemistry. Immunohistochemical staining of archival human thyroid tumors demonstrates a significant decrease in CD31 staining in all adenomas and carcinomas containing the PAX8/PPARγ rearrangement. Decreased angiogenesis in PPFP-containing tumors is directly correlated with our observations in the xenograft model and provides evidence for the first time that PPFP may impact FTC tumorigenesis by modulating angiogenesis in vivo.

ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice.

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1-2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials.

Culture and co-culture of DU145 prostate carcinoma, osteoblasts and HT-29 colon carcinoma cells on a fabricated type III collagen matrix.

DU145 prostate carcinoma cells cultured on type III collagen possessed a highly migratory potential which was twice as much as HT-29 colon carcinoma cells. Prior to attachment to collagen, DU145 cells were highly reactive for fibronectin and after attachment clear zones between cells and collagen suggested protease activity. HT-29 cells attached to type III collagen forming dome-like polyps, however, tight and/or gap junctions were not observed. hFob osteoblasts were co-cultured with DU145 to establish a prostate cancer-collagen matrix barrier-bone cell metastasis model. Osteoblasts maintained their differentiated osteoblastic characteristics on one side of the collagen barrier, demonstrating high alkaline phosphatase, osteocalcin and insulin growth factor (IGF) activities. hFob cell growth was prominent adjacent to demineralized bone matrix particles (BMPs) embedded in type III collagen. The collagen matrix was deteriorated on the DU145 side of the collagen barrier. The DU145-collagen III-hFob model will allow an evaluation of the influence of the matrix on prostate cancer-bone cell interaction and regulation by growth factors.

A monoclonal antibody against the X protein of hepatitis B virus: fine mapping of its epitope and application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

A HBx-specific mouse monoclonal antibody was developed and its epitope mapped to a hydrophilic segment 94HKRTLGL100 using the multipin peptide synthesis technique. A sensitive ELISA with a threshold of 5 to 10 ng was developed to identify the HBx-positive hepatitis B cases and measure the levels of HBx in sera. The same patient sera were also analyzed for the presence of anti-HBx using the purified recombinant antigen. HBx was present in 23% of the cases (15/65) whereas only 14% of the cases (9/65) were positive for anti-HBx. The mean value of HBx in acute hepatitis sera was higher (522 ng/ml) than in cirrhosis cases (48 ng/ml). PCR amplification of the S gene showed that all 15 HBx-positive cases were also positive for the viral DNA.

Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow.

Treatment of human bone marrow osteoprogenitors with osteogenin (BMP-3; at 1, 2.5 and 10 ng/ml) caused dose- and time-dependent inhibition of DNA synthesis and cell proliferation. Simultaneously, osteogenin stimulated type I collagen synthesis and cAMP production. Addition of osteogenin to the cell culture increased intracellular alkaline phosphatase activity and osteocalcin synthesis, with maximal stimulation at 2.5 ng/ml. Simultaneous addition of 2.5 ng/ml osteogenin and 1,25 dihydroxy vitamin D3 (10(-8) M) enhanced the stimulation observed in osteocalcin synthesis. The experiments reported here demonstrate the significant "in vitro" influence of osteogenin in the stimulation of osteogenic phenotype in osteoprogenitor cells which have been isolated from human bone marrow and cloned. These results support a reciprocal relationship between cell growth inhibition and expression of osteoblast differentiation.

Tissue transformation into bone in vivo. A potential practical application.

The transformation of mesenchymal tissue, such as muscle, into cartilage and bone can be induced by the recently purified osteoinductive factor, osteogenin, and by its parent substratum, demineralized bone matrix. We investigated the possibility of transforming readily available muscle flaps into vascularized bone grafts of various shapes that could be used as skeletal replacement parts. In a rat experimental model, thigh adductor muscle island flaps were placed inside bivalved silicone rubber molds. Prior to closure of the mold, 18 flaps were injected with osteogenin and coated with demineralized bone matrix. Five flaps served as controls and were injected with the vehicle only, and not coated with demineralized bone matrix. The molds were implanted subcutaneously in the rats' flanks and reopened 10 days later. The control flaps consisted of intact muscle without any evidence of tissue transformation, whereas the flaps treated with osteogenin and demineralized bone matrix were entirely transformed into cancellous bone that matched the exact shape of the mold. Using tissue transformation, we were able to generate in vivo, autogenous, well-perfused bones in the shapes of femoral heads and mandibles.

New concepts in the management of ischaemic lower extremities.

Experience with a new operative procedure namely arterialization of popliteal vein (APV) on 182 cases with 80% success for ischaemic lower extremity due to chronic nonspecific arterial occlusive diseases (ChNAOD) not responding to conservative methods of management currently in vogue is described. Success of this new operation saves the limbs from amputation which was done when conservative methods failed. The main purpose of this article is to write about etiology-clinical features--laboratory--and angiographic findings of this ChNAOD and APV operation. Accidental discovery of gastrocnemious muscle circulation responsible for success of APV operation is mentioned.

Acceleration of cartilage and bone differentiation on collagenous substrata.

Chondroprogenitor cells of newborn murine mandibular condyles were cultured on top of collagen sponges for up to 18 days. After 24 h in culture, new chondroblasts developed which subsequently matured showing signs of hypertrophy, while the extracellular matrix revealed positive reactivity for type II collagen, cartilage proteoglycans and mineralization. Light and electron microscopy examinations showed signs of new osteoid formation, a feature that was preceded by positive immunohistochemical reaction for type I collagen, fibronectin and bone specific sialoprotein. A close temporal and spatial association was noted between the development of mature, mineralized cartilage and new osteoid. The differentiation of new cartilage and bone cells was linked to an increased activity of DNA synthesis and cellular proliferation. The de novo bone formation was accompanied by increasing rates of alkaline phosphatase activity and uptake of [45Ca] features that were found to be tightly correlated to each other. The collagen substrata appeared also to facilitate the migration of cells, their replication and their subsequent differentiation to their respective cellular lineage. Hence, collagen sponges in vitro appear to serve as a promising substrata for culture systems involved with the growth and differentiation of mineralizing tissues such as cartilage and bone.

Tumor cells stimulate in vivo periosteal bone formation.

It was demonstrated that in mice a variety of tumor cells of both viral (XC, MSVC) and non-viral origin (rat ameloblasts, Ehrlich ascites carcinoma, KB, WAMIB, WISH cell lines) produce, on transplantation in the adjacent bones, periosteal osteogenesis, and in addition can stimulate osteoclastic and tumor-mediated bone resorption. The production by the tumor cells of an osteoblast-activating factor, besides the osteoclast-activating factor described earlier by others, is postulated.

New bone formation and bone marrow differentiation induced in rats by extracellular bone matrix implantation: effect of local preirradiation on the process.

Subcutaneous implantation of demineralized diaphyseal rat bone matrix in ACI rats initiates a developmental cascade that results in the formation of new endochondral bone and an associated hematopoietic bone marrow differentiation. Irradiation (1500 rad, 60Co) of the implantation site 24 h prior to implantation suppresses the formation of endochondral bone and bone marrow. All phases of the developmental cascade, including chemotaxis, proliferation, and differentiation, are arrested by the irradiation. Simultaneous implantation with the extracellular matrix of bone marrow, bone, pieces of a four-day-old implant or of thoracic muscle--but not of brain, liver, or spleen tissue--results in the development of endochondral bone and bone marrow at the irradiated site. Concurrent implantation with the extracellular matrix of in vitro growing fibroblasts of marrow or ossicle origin does not restore the developmental cascade.

Heterotopically induced bone does not develop functional periosteal membrane.

Heterotopic bone induced in mice by implantation of demineralized rat bone matrix does not respond to Moloney sarcoma. The biochemical and morphological parameters of osteogenesis (alkaline and acid phosphatase activity, 45Ca-uptake and total calcium content) are similar in the sites of Moloney sarcoma as in the contralateral, unexposed to the sarcoma, side. In contrast, periosteum of implants of syngeneic costal bone in response to Moloney sarcoma proliferates and produces new bone in the same manner, as orthotopic bones at the sites of Moloney sarcoma do. It is concluded that ectopically induced bones do not develop a true periosteum.