Gerald R. Hankins

In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.

Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat.

Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague–Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 × 105 PFU/well of BMP viral vector was 4890 × 10−12 U/well for ADCMVBMP-9, 302 × 10−12 U/well for ADCMVBMP-4, 220 × 10−12 U/well for ADCMVBMP-6, 45 × 10−12 U/well for ADCMVBMP-2, and 0.43 × 10−12 U/well for ADCMVBMP-7. The average volume of new bone induced by 107 PFU of BMP vector in athymic nude rats was 0.37±0.03 cm3 for ADCMVBMP-2, 0.89±0.07 cm3 for ADCMVBMP-4, 1.02±0.07 cm3 for ADCMVBMP-6, 0.24±0.05 cm3 for ADCMVBMP-7, and 0.63±0.07 cm3 for ADCMVBMP-9. In immunocompetent Sprague–Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 108 PFU induced 0.10±0.03 cm3 of new bone, whereas ADCMVBMP-9 at a lower viral dose of 107 PFU induced more bone, with an average volume of 0.29±0.01 cm3.

Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system

Abstract. Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II–III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents.

OBJECTIVE Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the &bgr;-galactosidase (Ad-&bgr;-gal) gene. Ad-&bgr;-gal served as the control. Each animal received a percutaneous, paraspinal injection of 106 hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-&bgr;-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-&bgr;-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.

Allelic loss on chromosome 6Q in primary prostate cancer

Molecular genetic analyses of human prostate cancer (CaP) has revealed frequent loss of specific chromosome regions suggesting the presence of putative tumor suppressor gene(s) (TSG) on these chromosome loci whose inactivation may play a role in prostate tumorigenesis. To understand the role of 6q alterations in CaP, we have undertaken a comprehensive analysis of proximal 6q. Genomic DNA from tumor and normal prostate tissues from radical prostatectomy specimens of 38 patients were analyzed by polymerase chain reaction (PCR) for 13 polymorphic microsatellite loci on 6q. Allelic losses of 1 or more polymorphic loci were detected in 11 of 38 patients (29%). Six of 11 tumors showing any 6q deletion were found to have allelic losses at D6S1056 and D6S300 loci. Our results revealed a 1.5 megabase interval between D6S1056 and D6S300 at 6q16.3‐21 as the minimal region of deletion, which may contain the putative TSG involved in prostate tumorigenesis. One of the tumor samples demonstrated homozygous deletion at a distal location D6S314 (6q23‐24), suggesting another locus potentially associated with CaP. Although the relationship of 6q loss of heterozygosity (LOH) with various clinico‐pathologic variables, i.e., cancer recurrence or pathologic stage, did not reveal a statistically significant association, the risk for 6q LOH to non‐organ confined (pT3) disease was 5‐fold higher than for organ confined disease. Int. J. Cancer (Pred. Oncol.) 84:331–335, 1999.

Human Mesenchymal Stem Cells Transduced with Recombinant Bone Morphogenetic Protein-9 Adenovirus Promote Osteogenesis in Rodents

The present study was undertaken to determine whether ex vivo bone morphogenetic protein-9 (BMP-9) gene therapy using human mesenchymal stem cells (hMSCs) can induce endochondral bone formation in athymic nude rats. An in vitro study was initially performed on hMSCs to evaluate morphological changes and osteoblastic differentiation induced by replication-defective adenovirus type 5 with the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or beta-galactosidase (Ad-beta-gal) gene. In vivo, athymic nude rats received an injection (10(6) hMSCs transduced with recombinant adenovirus at 50 PFU/cell) into the anterior thigh musculature: Ad-BMP-9 on the left and Ad-beta-gal (control) on the right. Computed tomography scans and histological analysis were obtained 7, 14, 28, 42, 56, and 84 days postinjection. In vitro, human mesenchymal stem cells treated with Ad-BMP-9 (50 PFU/cell) showed signs of differentiation, whereas hMSCs treated with 250 and 1250 PFU/cell showed cytotoxicity. In vivo, computed tomography and histological analysis clearly demonstrated ectopic bone at hMSC/Ad-BMP-9 treatment sites, whereas the hMSC/Ad-beta-gal treatment sites showed no evidence of osteogenesis. None of the animals showed clinical evidence of toxicity. Ex vivo gene therapy with hMSC/BMP-9 may be efficacious for promoting bone formation for a variety of bone pathologies and certainly warrants further investigations.

Osteogenesis in rats induced by a novel recombinant helper-dependent bone morphogenetic protein-9 (BMP-9) adenovirus.

Although recombinant first‐generation BMP adenoviruses can induce ectopic bone formation in immune deficient animals, the osteoinductive activity of these BMP vectors is reduced in immune competent animals. Helper‐dependent adenoviral vectors have been developed to decrease the immune response and, therefore, increase gene expression in immune competent animals compared with first‐generation vectors. In the present study, the osteoinductive activity of a helper‐dependent GFP and BMP‐9 adenoviral vector (ADGBMP9) was evaluated in vitro and in vivo.

Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections

Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence of Pun1 were found associated with capsaicin in plants from both seasons. Our results support that CCR is an important control point for the flux of p-coumaric acid to specific biosynthesis pathways. KAS was found to regulate the major precursors for acyl moieties of capsaicinoids and may play a key role in capsaicinoid production. Candidate gene association mapping of Pun1 suggested that the accumulation of capsaicinoids depends on the expression of Pun1, as revealed by the most important associated SNPs found in the promoter region of Pun1.

Identification of the deleted in liver cancer 1 gene, DLC1, as a candidate meningioma tumor suppressor.

OBJECTIVEMeningiomas are the second most common primary tumors of the central nervous system. Meningiomas at the cranial base pose technical challenges and result in increased morbidity. To investigate the molecular mechanisms of meningioma formation, the expression profiles of 12 000 genes from meningiomas and dural specimens were compared. METHODSRibonucleic acid from 6 meningiomas (World Health Organization Grade I) and 4 dural specimens was profiled using U95A GeneChips (Affymetrix, Inc., Santa Clara, CA). Expression profiles of the 2 groups were compared using dChip and Data Mining Tool software packages (Affymetrix, Inc.) to identify differentially expressed genes. Down-regulation of a differentially expressed tumor suppressor gene, deleted in liver cancer 1 (DLC1), was verified by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Function and methylation of DLC1 were assessed by ectopic expression in 5 primary cultures, demethylation assay using 5-aza-2′-deoxycytidine, and methylation-specific polymerase chain reaction in 4 meningioma samples. RESULTSGene expression profiling revealed up-regulation of 5 genes (fibroblast growth factor 9, gibbon leukemia virus receptor 2, cyclin D1, eukaryotic translation initiation factor 5A, and 28S ribosomal ribonucleic acid) and down-regulation of 35 genes, including DLC1, in meningiomas. The down-regulation of DLC1 in meningiomas was confirmed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Transfection of DLC1 complementary deoxyribonucleic acid into primary cultures of 5 meningiomas resulted in decreased replication. Although demethylation decreased meningioma cell growth rates in vitro, methylation-specific polymerase chain reaction did not detect DLC1 promoter methylation. CONCLUSIONThe results suggest that DLC1 may function as a tumor suppressor gene in meningiomas. Furthermore, DLC1 promoter methylation does not appear to be responsible for the decreased DLC1 expression in these tumors.

Long-term tracing of adenoviral expression in rat and rabbit using luciferase imaging.

Luciferase optical imaging provides a novel method to monitor transgene expression in small living animals. As the genetic and immunological heritages of particular animals significantly affect the expression of adenovirus‐delivered transgenes, it is essential to know the expression patterns specific to athymic nude and Sprague‐Dawley rats, two strains commonly used in rodent models. In this study we set out to determine these patterns. At the same time, we tested luciferase optical imaging in a larger animal, the rabbit.