Local immunomodulation with CD4 and CD8 antibodies, but not cyclosporine A, improves osteogenesis induced by ADhBMP9 gene therapy.

This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (P < 0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (P < 0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.29 +/- 0.01 cm3 in the viral control group, 0.17 +/- 0.03 cm3 in the Cs. A-ADhBMPs group, and 0.59 +/- 0.07 cm3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.

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 x 10(5) PFU/well of BMP viral vector was 4890 x 10(-12) U/well for ADCMVBMP-9, 302 x 10(-12) U/well for ADCMVBMP-4, 220 x 10(-12) U/well for ADCMVBMP-6, 45 x 10(-12) U/well for ADCMVBMP-2, and 0.43 x 10(-12) U/well for ADCMVBMP-7. The average volume of new bone induced by 10(7) PFU of BMP vector in athymic nude rats was 0.37+/-0.03 cm(3) for ADCMVBMP-2, 0.89+/-0.07 cm(3) for ADCMVBMP-4, 1.02+/-0.07 cm(3) for ADCMVBMP-6, 0.24+/-0.05 cm(3) for ADCMVBMP-7, and 0.63+/-0.07 cm(3) 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 10(8) PFU induced 0.10+/-0.03 cm(3) of new bone, whereas ADCMVBMP-9 at a lower viral dose of 10(7) PFU induced more bone, with an average volume of 0.29+/-0.01 cm(3).

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.

Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy.

OBJECT: Gene therapy has many potential applications in neurosurgery. One application involves bone morphogenetic protein-2 (BMP-2), a low-molecular-weight glycoprotein that induces bone formation in vivo. Numerous studies have demonstrated that the BMP-2 protein can enhance spinal fusion. This study was undertaken to determine whether direct injection of an adenoviral construct containing the BMP-2 gene can be used for spinal fusion. METHODS: Twelve athymic nude rats were used in this study. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-2 gene (Ad-BMP-2) was used. A second adenovirus constructed with the CMV promoter and beta-galactosidase (beta-gal) gene (Ad-beta-gal) was used as a control. In three groups (four rats each) 7.5 microl of virus (5x10(8) particles/microl) was injected percutaneously and paraspinally at the lumbosacral junction: Group 1 received Ad-BMP-2 bilaterally; Group 2 received Ad-BMP-2 on the right, Ad-beta-gal on the left; and Group 3 received Ad-beta-gal bilaterally. Computerized tomography (CT) scans of the lumbosacral spine were obtained at 3, 5, 8, and 12 weeks. At 12 weeks, the animals were killed and underwent histological inspection. Ectopic bone formation was observed both on three-dimensionally reconstructed CT scans and histological examination in all rats at sites treated with Ad-BMP-2. Histological analysis demonstrated bone at different stages of maturity adjacent to the spinous processes, laminae, and transverse processes. CONCLUSIONS: Results of this study clearly demonstrated that it is possible to produce in vivo endochondral bone formation by using direct adenoviral construct injection into the paraspinal musculature, which suggests that gene therapy may be useful for spinal fusion in the future.

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.

Mechanisms of increased NGF production in vascular smooth muscle of the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension. In vivo and in vitro evidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta 1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 +/- 18.0 min in SHR VSMCs, compared to only 36.5 +/- 11.6 min in WKY VSMCs. Sequence analysis of the 3' untranslated region (UTR) revealed no strain differences in cis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3'UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.

Platinum coil-mediated implantation of growth factor-secreting endovascular tissue grafts: an in vivo study.

PURPOSE: To demonstrate in vivo that platinum embolic coils can be used to deliver genetically modified, growth factor-secreting fibroblast grafts into the endovascular space with the long-term goal of improving fibrosis within coil-embolized cerebral aneurysms. MATERIALS AND METHODS: Murine fibroblasts that contained multiple inserts of the DNA for human basic fibroblast growth factor were grown in culture onto 10-mm-long segments of Guglielmi detachable coils. Control (n = 4) and fibroblast-bearing (n = 4) coils were implanted into the common carotid artery in nude rats. The arterial segments that contained the coil were harvested after 14 or 35 days. Cellular content and collagen formation in the treated vessels were assessed histologically. RESULTS: At both 14 and 35 days, samples with control coils showed primarily involuting blood elements with minimal fibroblast proliferation or collagen formation. At 14 days, samples with fibroblast-bearing coils showed extensive fibroblast proliferation. At 35 days, samples with fibroblast-bearing coils showed marked interval fibroblast proliferation and collagen formation. CONCLUSION: Platinum coils can be used as a cell delivery device. Direct intravascular implantation of growth factor-secreting fibroblast grafts leads to improved intravascular scar formation, therefore theoretically reducing the potential for aneurysm regrowth or coil compaction.

In vitro proliferation and adhesion of basic fibroblast growth factor-producing fibroblasts on platinum coils.

PURPOSE: To evaluate the growth and adhesion characteristics in vitro of genetically modified, basic fibroblast growth factor-producing fibroblasts on platinum detachable coils. MATERIALS AND METHODS: Coils of two sizes were coated with laminin, poly-L-lysine, fibronectin, and type I and type IV collagen and were cultured with basic fibroblast growth factor-secreting fibroblasts. Type I collagen strands were inserted in the lumen of some coils. Cellular proliferation and adherence during passage of coils through microcatheters were studied with both light and scanning electron microscopy. Growth factor concentration in the culture medium was measured. RESULTS: Rapid cellular proliferation was noted on all coated coils except those coated with type IV collagen. Proliferation on uncoated coils was slightly slower than on most coated coils, although confluent cell layers were present on uncoated larger-diameter coils within 48 hours. Cells had a marked propensity to grow between the primary coil windings into the coil lumen, except in coils that contained collagen filaments. Passage through microcatheters caused widespread stripping of cells from the outer surface of coils, especially the uncoated samples. Viable cells remained in the coil lumen. Supernatant contained high concentrations of growth factor. CONCLUSION: Platinum embolic coils are a promising mechanism of cell delivery for stimulation of scar formation or other desirable biologic effects.

Bone morphogenetic protein gene therapy for the induction of spinal arthrodesis.

Gene therapy has many potential applications in neurosurgery. One application involves bone morphogenetic protein-2 (BMP-2), a low-molecular-weight glycoprotein that induces bone formation in vivo. Numerous studies have demonstrated that the BMP-2 protein can enhance spinal fusion. This study was undertaken to determine whether direct injection of an adenoviral construct containing the BMP-2 gene can be used for spinal fusion. Twelve athymic nude rats were used in this study. Recombinant, replication-defective type-5 adenovirus with a universal promoter and BMP-2 gene (Ad-BMP-2) was used. A second adenovirus constructed with a universal promoter and beta-galactosidase (beta-gal) gene (Ad-beta-gal) was used as a control. Seven and one-half microliters of virus was injected percutaneously and paraspinally at the lumbosacral junction in three groups (four animals each): 1) Ad-BMP-2 bilaterally, 2) Ad-BMP-2 on the right, Ad-beta-gal on the left, and 3) Ad-beta-gal bilaterally. Computerized tomography (CT) scans of the lumbosacral spine were obtained at 3, 5, and 12 weeks. At 12 weeks, the animals were killed for histological inspection. Ectopic bone formation was seen both on three-dimensional CT reconstruction and histologically in all rats at sites treated with Ad-BMP-2. Histological analysis revealed bone at different stages of maturity adjacent to the spinous processes, laminae, and transverse processes. This study clearly demonstrated that it is possible to produce in vivo endochondral bone formation by using direct adenoviral construct injection into the paraspinal musculature, which suggests that gene therapy may be useful for spinal fusion in the future.

Transforming growth factor-Beta binds reversibly in vitro to guglielmi detachable coils.

SUMMARY: We determined the propensity for and reversibility of transforming growth factor-beta(TGFbeta) binding to uncoated Guglielmi Detachable Coils (GDC) and to GDC coated with extracellular matrix (ECM) proteins. Three 1.0 centimetre samples each of uncoated GDC-18 and of GDC-18 coated with either poly-L-lysine, laminin, type I collagen, type IV collagen, fibronectin, or poly-L-lysine and laminin were prepared. These samples were immersed briefly in a solution containing I(125)-labelled TGFbeta at a concentration of 0.225 mug/ml with initial specific activity of 123.3 mCi/mg (DuPont-NEN, Billerica, MA), and were counted using a scintillation counter. Each sample was then placed in a vial containing saline, shaken for 60 seconds, and counted again. Selected samples were immersed for varying periods within the TGFbeta solution and counted before and after saline rinse. Samples were rinsed one week after initial rinsing and counted again. The amount of binding between coil types was compared using the Student t test. For all samples initial binding of TGFbeta was in the order of 60-120 pg/cm. For the pre-rinse data there were no statistically significant differences between the amount bound to any single coil coating type relative to other coatings. Compared to the initial accumulations, the amount remaining after rinsing ranged from 40% (poly-L-lysine) to 63% (poly-L-lysine with laminin), with a mean of 55% among the seven coil types. After rinsing there was more growth factor remaining on uncoated coils than on poly-L-lysine-coated coils (p=0.05), fibronectin-coated coils (p=0.01), and type IV collagen-coated coils (p=0.04). There was a trend toward greater residual growth factor on coils coated with poly-L-lysine and laminin compared to coils coated with poly-L-lysine alone (p=0.10). Delayed, second rinsing of the samples one week after initial testing demonstrated only minor incremental loss of TGFbeta from the coil surfaces. After five minutes of immersion, accumulation was approximately 200% greater than that noted with brief submersion, but immersions lasting over five minutes did not yield increasing levels of TGFbeta binding. TGFbeta binds to GDC coils. Binding is not improved with ECM protein-coated coils compared to uncoated coils. The absolute amount of TGFbeta bound to the coil will likely result in local concentrations of growth factor in the order of those required for biological activity in vivo.

M6P/IGF2 receptor: a candidate breast tumor suppressor gene.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) functions in the activation of TGFbeta, a potent growth inhibitor for most cell types, the degradation of the mitogen, IGF2, and the intracellular trafficking of lysosomal enzymes. We have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs) and recently reported loss of heterozygosity (LOH) at this locus with mutations in the remaining allele in human liver tumors. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of M6P/IGF2r to screen breast tumors for LOH. Forty of 62 (65%) patients were informative (heterozygous) and 12/40 (30%) breast tumors had LOH; 5/19 (26%) carcinomas in situ (CIS) and 7/21 (33%) invasive carcinomas. To investigate the early molecular genetic events in breast carcinogenesis, we screened the CIS with LOH for mutations. In 2/5 (40%) of these tumors, missense mutations were found in the remaining allele that gave rise to significant amino acid substitutions. These findings provide evidence that M6P/IGF2r allelic loss is an early event in the etiology of breast cancer, that this gene functions as a tumor suppressor gene in the breast.

Phenobarbital selectively promotes initiated cells with reduced TGF beta receptor levels.

Phenobarbital (PB) is a potent tumor promoter in rodent liver. In this study we investigated whether PB selectively promotes a population of initiated cells with reduced levels of transforming growth factor-beta (TGF beta) receptors types I, II and III. Liver tumors were induced in male Fischer F344 rats by diethylnitrosamine (DEN). Following induction the animals were divided into PB-treated (DEN/PB) and untreated groups (DEN). After 3 months of treatment half of the PB-treated rats were removed from PB for the final month (DEN/PB/OFF). At 4 months, the livers from rats in the three treatment groups were removed, tumors excised and frozen with matched surrounding normal liver tissue. The mRNA levels for the TGF beta receptors types I-III were significantly decreased in tumor tissue from DEN/PB rats when compared with surrounding normal liver tissue or tumors from age-matched untreated controls. In tumors from DEN/PB/OFF rats the TGF beta receptor types I-III were also significantly reduced compared with controls and not different to tumors from DEN/PB rats. There was no difference in the mRNA levels for the TGF beta receptors in tumors from rats exposed to DEN alone, when compared with the surrounding normal tissue. These results demonstrate that PB selectively promotes initiated cells with reduced levels of TGF beta types I-III receptors and suggests a mechanistic role for TGF beta in PB-induced liver tumor promotion.

M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity.

The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) functions in the intracellular trafficking of lysosomal enzymes, the activation of the potent growth inhibitor, transforming growth factor beta 2, and the degradation of IGF2 (ref. 1), a mitogen often overproduced in tumours. We have recently shown that 70% of human hepatocellular tumours have loss of heterozygosity (LOH) at the M6P/IGF2R locus which maps to chromosome 6q26-q27 (ref. 8). Using a coarse screen, we have now identified point mutations in the remaining allele of 25% of human hepatocellular carcinomas (HCCs) with LOH. These mutations give rise to truncated receptor protein and significant amino acid substitutions, and provide evidence that the M6P/IGF2R gene functions as a tumour suppressor in human liver carcinogenesis.

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Frequent loss of heterozygosity on 6q at the mannose 6-phosphate/insulin-like growth factor II receptor locus in human hepatocellular tumors.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the activation of transforming growth factor beta, and previously we have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs). Therefore, we have postulated that loss of the M6P/IGFIIr gene may be mechanistically involved in liver carcinogenesis. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of the M6P/IGFIIr gene to screen non-cirrhotic, hepatitis virus negative patients with hepatocellular tumors for LOH. Twenty-two of 36 (61%) patients were informative (heterozygous), and 14/22 (64%) liver tumors had LOH; 11/16 (69%) carcinomas, 1/3 (33%) fibrolamellar tumors and 2/3 (67%) adenomas. This is the first report of LOH at the M6P/IGFIIr locus in human hepatocellular tumors, and the presence of LOH in adenomas suggests that allelic loss may be an early event in the etiology of HCCs. These results support the hypothesis that the M6P/IGFIIr gene may function as a tumor suppressor gene in the liver.

Regulation of mannose 6-phosphate/insulin-like growth factor-II receptors and transforming growth factor beta during liver tumor promotion with phenobarbital.

Chronic exposure of rats to the liver tumor promoter phenobarbital (PB) significantly reduces the ability of normal hepatocytes, but not of initiated hepatocytes, to respond to mitogenic stimuli. This reduced proliferative ability of normal hepatocytes was correlated with a marked elevation in hepatic concentration of the potent mito-inhibitory factor, transforming growth factor-beta 1 (TGF-beta 1). PB also increased the mannose 6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor concentration in hepatocytes, with a concomitant up-regulation in gene expression. Since the M6P/IGF-II receptor facilitates the proteolytic activation of TGF-beta 1, this suggests that PB increases the capacity of normal hepatocytes to activate TGF-beta 1. In contrast, a subset of preneoplastic lesions induced with N-nitrosodiethylamine did not demonstrate elevated levels of the M6P/IGF-II receptor or TGF-beta 1 in response to PB. These findings emphasize the potential importance of TGF-beta 1 during liver tumor promotion with PB and suggest that reduction of M6P/IGF-II receptor levels in liver tumors may provide the tumor cells with an important selective growth advantage.