Effect of Tempol on the prevention of irradiation-induced mucositis in miniature pigs.

OBJECTIVE: The goals of this study were to (i) establish a useful miniature pig (minipig) model for irradiation-induced oral mucositis and (ii) evaluate the effect of Tempol to prevent its development. METHODS AND MATERIALS: Minipigs were irradiated with 6 Gy for five consecutive days targeting the entire oral cavity. To prevent radiation damage, minipigs were treated with 30 mg kg-1 Tempol 10 min before irradiation (n = 4), while the radiation-alone group was similarly injected with saline (n = 4). Lesions were graded using an oral mucositis score and visual inspection every 3 days, and biopsy of multiple sites was performed at day 18. Weight and chest and abdominal circumferences were measured every 3 days. RESULTS: Lesions began about 12 days after the first irradiation fraction and healed about 30 days after irradiation. Epithelial thickness was calculated on the lingual and buccal mucosa on the 18th day after the first irradiation fraction. Tempol provided modest protection from ulceration after irradiation using this treatment strategy. CONCLUSIONS: This study established a useful large animal model for irradiation-induced oral mucositis and showed modest beneficial effects of Tempol in limiting tissue damage. The latter finding may be potentially valuable in preventing oral mucositis in patients receiving irradiation for head and neck cancers.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.

Persistence of hAQP1 expression in human salivary gland cells following AdhAQP1 transduction is associated with a lack of methylation of hCMV promoter.

In 2012, we reported that 5 out of 11 subjects in a clinical trial (NCT00372320) administering AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first-generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). As subject peak responses were at times much longer (7-42 days) than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. This hypothesis was supported in human salivary gland primary cultures and human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. Conversely, when we transduced mouse and rat cell lines in vitro and submandibular glands in vivo with AdhAQP1, the hCMVp was gradually methylated over time and associated with decreased hAQP1 expression and function in vitro and decreased hAQP1 expression in vivo. These data suggest that the hCMVp in AdhAQP1was probably not methylated in transduced human salivary gland cells of responding subjects, resulting in an unexpectedly longer functional expression of hAQP1.

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone.

OBJECTIVES: Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. MATERIALS AND METHODS: We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. RESULTS: We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. CONCLUSION: This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.

AdLTR2EF1α-FGF2-mediated prevention of fractionated irradiation-induced salivary hypofunction in swine.

Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of fibroblast growth factor-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by 16 weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR2EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.

Transgenic α-1-antitrypsin secreted into the bloodstream from salivary glands is biologically active.

OBJECTIVES: Salivary glands are potentially a valuable target for gene therapeutics. Herein, we examined the expression and biochemical activity of human alpha-1-antitrypsin (hA1AT) produced in rodent submandibular glands after gene transfer. METHODS: A serotype 5 adenoviral vector (Ad.hA1AT) was constructed and first characterized by dose response and time course studies using SMIE cells in vitro. hA1AT expression was analysed by ELISA and the biologic activity determined by the inhibition of human neutrophil elastase (hNE) and formation of hA1AT-hNE complexes. Ad.hA1AT was administered to submandibular glands of rats and mice. The levels and activity of hA1AT were analysed in saliva, serum and gland extracts. Treatment with endoglycosidase H and Peptide N-Glycosidase F was used to assess N-linked glycosylation. RESULTS: Transgenic hA1AT, expressed in submandibular glands following Ad.hA1AT administration, was secreted into the bloodstream, N-glycosylated and biochemically active. CONCLUSION: After in vivo gene transfer, rodent salivary glands can produce a non-hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation-dependent biologic activity.

Convenient and reproducible in vivo gene transfer to mouse parotid glands.

OBJECTIVES: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. METHODS: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. RESULTS: The chosen volume for mouse parotid vector delivery was 20µL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. CONCLUSION: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

AAV2-mediated transfer of the human aquaporin-1 cDNA restores fluid secretion from irradiated miniature pig parotid glands.

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Transient TWEAK overexpression leads to a general salivary epithelial cell proliferation.

OBJECTIVES: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. METHODS: A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Wharton's duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. RESULTS: AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. CONCLUSION: Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

Differential sorting of human parathyroid hormone after transduction of mouse and rat salivary glands.

Gene transfer to salivary glands leads to abundant secretion of transgenic protein into either saliva or the bloodstream. This indicates significant clinical potential, depending on the route of sorting. The aim of this study was to probe the sorting characteristics of human parathyroid hormone (hPTH) in two animal models for salivary gland gene transfer. PTH is a key hormone regulating calcium levels in the blood. A recombinant serotype 5 adenoviral vector carrying the hPTH cDNA was administered to the submandibular glands of mice and rats. Two days after delivery, high levels of hPTH were found in the serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in the saliva. Two days after vector infusion into rats, a massive secretion of hPTH was measured in saliva, with little secretion into serum. Confocal microscopy showed hPTH in the glands, localized basolaterally in mice and apically in rats. Submandibular gland transduction was effective and the produced hPTH was biologically active in vivo. Whereas hPTH sorted toward the basolateral side in mice, in rats hPTH was secreted mainly at the apical side. These results indicate that the interaction between hPTH and the cell sorting machinery is different between mouse and rat salivary glands. Detailed studies in these two species should result in a better understanding of cellular control of transgenic secretory protein sorting in this tissue.

Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Toxicity and biodistribution of a first-generation recombinant adenoviral vector, in the presence of hydroxychloroquine, following retroductal delivery to a single rat submandibular gland.

OBJECTIVE: We examined the toxicity and biodistribution associated with a single administration of a first-generation, serotype 5, adenoviral vector encoding human growth hormone (hGH; AdCMVhGH) to a single rat submandibular gland in the presence of hydroxychloroquine (HCQ). Previously, we showed that hGH is primarily secreted into saliva (approximately ninefold serum level) when expressed as a transgene in salivary glands (e.g. Baum et al, 1999), but administration of HCQ substantially increases the hGH levels secreted into the bloodstream (Hoque et al, 2001). A potential application of this observation is for patients with adult hGH deficiency. METHODS: Six groups of male and female adult rats (n = 12 each) were studied, with zero to 1.5 x 10(11) particles of AdCMVhGH, +/-HCQ, administered retroductally. Multiple clinical and pathological parameters, as well as vector tissue distribution, were assessed. RESULTS: All animals survived until the scheduled day of sacrifice, and essentially no untoward events were observed clinically or at gross necropsy. We observed no vector-related effects on clinical hematology evaluations and a single, transient significant change on clinical chemistry evaluations (increased serum globulin levels). Three days after AdCMVhGH administration, the vector distributed to all tissues analyzed with the exception of gonads and heart. By day 29, most organs, other than the targeted and contralateral submandibular glands, were negative for the presence of vector. On day 3, none of the animals tested positive for the presence of replication competent adenovirus in either their blood or saliva. CONCLUSION: Salivary gland delivery of AdCMVhGH +/-HCQ appears associated with limited toxicity in rats.

Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands.

The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

A recombinant adenoviral vector encoding functional vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) is a small neuropeptide, which exerts pleiotropic functions. Based on its immunomodulatory, secretory, and possibly trophic effects, VIP is a valuable candidate molecule for the management of autoimmune disease. The purpose of this study was to develop a recombinant viral vector capable of directing the expression of functional VIP. The vector rAd5CMVhVIP was constructed and used to infect 293 cells. VIP expression was measured by an ELISA and function was evaluated by measurement of intracellular cAMP formation. rAd5CMVhVIP directed VIP expression and the transgenic VIP elicited a dose-dependent increase of intracellular cAMP, mediated through the VIP receptor VPAC(1). This is the first report showing the construction of a recombinant viral vector encoding biologically active VIP.

Using HSV-thymidine kinase for safety in an allogeneic salivary graft cell line.

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 microM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.

Cationic liposome-mediated gene transfer to rat salivary epithelial cells in vitro and in vivo.

BACKGROUND: Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo. METHODS: Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts. RESULTS: Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed. CONCLUSIONS: The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

Insulin-like growth factor-I (IGF-I) receptor activation rescues UV-damaged cells through a p38 signaling pathway. Potential role of the IGF-I receptor in DNA repair.

The activated insulin-like growth factor-I receptor (IGF-IR) is implicated in mitogenesis, transformation, and anti-apoptosis. To investigate the role of the IGF-IR in protection from UV-mimetic-induced DNA damage, 4-nitroquinoline N-oxide (4-NQO) was used. In this study we show that the activation of the IGF-IR is capable of rescuing NWTb3 cells overexpressing normal IGF-IRs from 4-NQO-induced DNA damage as demonstrated by cellular proliferation assays. This action was specific for the IGF-IR since cells expressing dominant negative IGF-IRs were not rescued from 4-NQO UV-mimetic treatment. DNA damage induced by 4-NQO in NWTb3 cells was significantly decreased after IGF-IR activation as measured by comet assay. IGF-I was also able to overcome the cell cycle arrest, observed after 4-NQO treatment, thereby enhancing the ability of NWTb3 cells to enter S phase. Interestingly, the p38 mitogen-activated protein kinase pathway was shown to represent the main signaling pathway involved in the IGF-IR-mediated rescue of UV-like damaged cells. The ability of the IGF-IR to induce DNA repair was also demonstrated by infecting NWTb3 cells with UV-irradiated adenovirus. Activation of the IGF-IR resulted in enhanced beta-galactosidase reporter gene activity demonstrating repair of the damaged DNA. This study indicates a direct role of the IGF system in the rescue of damaged cells via DNA repair.