Targeting adenoviral vectors for enhanced gene therapy of uterine leiomyomas.

STUDY QUESTION: Is targeted adenovirus vector, Ad-SSTR-RGD-TK (Adenovirus -human somatostatin receptor subtype 2- arginine, glycine and aspartate-thymidine kinase), given in combination with ganciclovir (GCV) against immortalized human leiomyoma cells (HuLM) a potential therapy for uterine fibroids? SUMMARY ANSWER: Ad-SSTR-RGD-TK/GCV, a targeted adenovirus, effectively reduces cell growth in HuLM cells and to a significantly greater extent than in human uterine smooth muscle cells (UtSM). WHAT IS KNOWN ALREADY: Uterine fibroids (leiomyomas), a major cause of morbidity and the most common indication for hysterectomy in premenopausal women, are well-defined tumors, making gene therapy a suitable and potentially effective non-surgical approach for treatment. Transduction of uterine fibroid cells with adenoviral vectors such as Ad-TK/GCV (herpes simplex virus thymidine kinase gene) decreases cell proliferation. STUDY DESIGN, SIZE, DURATION: An in vitro cell culture method was set up to compare and test the efficacy of a modified adenovirus vector with different multiplicities of infection in two human immortalized cell lines for 5 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immortalized human leiomyoma cells and human uterine smooth muscle cells were infected with different multiplicities of infection (MOI) (5-100 plaque-forming units (pfu)/cell) of a modified Ad-SSTR-RGD-TK vector and subsequently treated with GCV. For comparison, HuLM and UtSM cells were transfected with Ad-TK/GCV and Ad-LacZ/GCV. Cell proliferation was measured using the CyQuant assay in both cell types. Additionally, western blotting was used to assess the expression of proteins responsible for regulating proliferation and apoptosis in the cells. MAIN RESULTS AND THE ROLE OF CHANCE: Transduction of HuLM cells with Ad-SSTR-RGD-TK/GCV at 5, 10, 50 and 100 pfu/cell decreased cell proliferation by 28, 33, 45, and 84%, respectively (P < 0.05) compared with untransfected cells, whereas cell proliferation in UtSM cells transfected with the same four MOIs of Ad-SSTR-RGD-TK/GCV compared with that of untransfected cells was decreased only by 8, 23, 25, and 28%, respectively (P < 0.01). Western blot analysis showed that, in comparison with the untargeted vector Ad-TK, Ad-SSTR-RGD-TK/GCV more effectively reduced expression of proteins that regulate the cell cycle (Cyclin D1) and proliferation (PCNA, Proliferating Cell Nuclear Antigen), and it induced expression of the apoptotic protein BAX, in HuLM cells. LIMITATIONS, REASONS FOR CAUTION: Results from this study need to be replicated in an appropriate animal model before testing this adenoviral vector in a human trial. WIDER IMPLICATIONS OF THE FINDINGS: Effective targeting of gene therapy to leiomyoma cells enhances its potential as a non-invasive treatment of uterine fibroids.

Chronic estrogen deficiency causes gastroparesis by altering neuronal nitric oxide synthase function.

BACKGROUND: Gastroparesis affects predominantly females; however, the biological basis for this gender bias is completely unknown. Several lines of evidence suggest that nitrergic dependent stomach motility function is reduced in diabetic gastroparesis and that nNOS is estrogen-regulated. AIMS: The purpose of this study was to investigate whether reduced levels of estradiol-17ß (E2) down-regulates tetrahydrobiopterin (BH4, a cofactor for nNOS dimerization and enzyme activity) biosynthesis and therefore nNOS mediated gastric motility would be impaired in a mouse model of chronic estrogen deficiency, follicle stimulating hormone receptor knock-out female mice (FORKO). METHODS: In-bred 12-week-old female FORKO mice were obtained from our FORKO breeding colony. Gastric emptying was measured in overnight fasting mice. Nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric antrum strips prepared from WT and FORKO mice. Protein expression for nNOSα, BH4 biosynthesis enzymes (GCH-1, DHFR) and estrogen receptors (α, ß) were measured in gastric antrum by western blotting. Levels of BH4 and oxidized BH2, B biopterin levels were determined by HPLC. RESULTS: In FORKO, compared to wild type (WT) stomachs we indentified (1) reduced (%) gastric emptying (64 ± 2.5 vs. 77.6 ± 0.88), (2) greater reduction in nitregic relaxation (-0.13 ± 0.012 vs. -0.28 ± 0.012), (3) increased nNOS dimerization (0.48 ± 0.02 vs. 0.34 ± 0.05), (4) decreased NO release whether measured at 24 h (0.6 ± 0.04 vs. 1.7 ± 0.22, p < 0.05) or at 48 h (3.4 ± 0.26 vs. 5.0 ± 0.15, p < 0.05) of incubation, (5) decreased GCH-1 (1.9 ± 0.06 vs. 2.3 ± 0.04), DHFR (1.8 ± 0.14 vs. 2.4 ± 0.07) and ERα (2.7 ± 0.4 vs. 3.9 ± 0.4) and (6) increased oxidized biopterin levels and decreased ratio of BH4 versus BH2 + B. CONCLUSION: We conclude that chronic estrogen deficiency negatively modifies the function of both BH4 and nNOS thereby contributing to the development of gastroparesis in a FORKO mouse model.

Towards gene therapy of postoperative adhesions: fiber and transcriptional modifications enhance adenovirus targeting towards human adhesion cells.

Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.

Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of uterine fibroids.

PURPOSE: Fibroids are the most common smooth muscle overgrowth in women. This study determined the expression and the effect of hypoxia on two potent antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) on human fibroid cells. METHODS: Immortalized human leiomyoma (fibroid) and myometrial cells were subjected to hypoxia (2 % O2, 24 h). Total RNA and cell homogenate were obtained from control and treated cells; CAT and SOD mRNA and activity levels were determined by real-time RT-PCR and ELISA, respectively. RESULTS: Fibroid cells have significantly lower antioxidant enzymes, SOD and CAT mRNA and activity levels than normal myometrial cells (p < 0.05). Hypoxia treatment significantly increased SOD activity in myometrial cells while significantly decreasing CAT activity in fibroid cells (p < 0.05). There was no significant difference in CAT mRNA levels or activity in response to hypoxia in myometrial cells. Also, there was no significant difference in SOD mRNA levels in response to hypoxia in myometrial cells. CONCLUSION: This is the first report to show that uterine fibroids are characterized by an impaired antioxidant cellular enzymatic system. More importantly, our results indicate a role for hypoxia in the modulation of the balance of those enzymes in fibroid and myometrial cells. Collectively, these results shed light on the pathophysiology of fibroids thereby providing potential targets for novel fibroid treatment.

Towards non-surgical therapy for uterine fibroids: catechol-O-methyl transferase inhibitor shrinks uterine fibroid lesions in the Eker rat model.

BACKGROUND: Uterine leiomyomas (fibroids) are the most common pelvic tumors in women. We assessed the potential therapeutic utility of Ro 41-0960, a synthetic catechol-O-methyl transferase inhibitor (COMTI), in the Eker rat. METHODS: We randomized uterine fibroid-bearing Eker rats for treatment with Ro 41-0960 (150 mg/kg/12 h) versus vehicle for 2 and 4 weeks. The fibroids were measured by caliper and subjected to histological evaluation. Urinary levels of 2-hydroxy estrogen (E(2)), 16-hydroxy E2 and DPD (osteoporosis marker) and serum liver enzymes were evaluated. Expressions of Cyclin D1, proliferating cell nuclear antigen (PCNA), Poly [ADP-ribose] polymerase1 (PARP1), tumor suppressor gene (P53) and transforming growth factor (TGFß3) were assessed in fibroids using immunohistochemical analysis or RT-PCR. Apoptosis was confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL). RESULTS: Ro 41-0960-treated rats exhibited fibroid volumes of 86 ± 7% and 105 ± 12% of initial burden, at 2 and 4 weeks post-treatment, respectively, significantly lower than control group (240 ± 15% and 300 ± 18%; P< 0.01). Ro 41-0960 increased the urinary 2-hydroxy E2/16-hydroxy E(2) ratio, level of p53 mRNA and TUNEL positivity (P< 0.05) and decreased PARP1, PCNA and cyclin D1 proteins and TGFß3 mRNA (P< 0.05). Ro 41-0960 did not change normal tissue histology, liver functions or urinary DPD level. CONCLUSIONS: Ro 41-0960 (COMTI) arrested growth/shrunk uterine fibroids in Eker rats. This result may be related to modulation of estrogen-dependent genes involved in apoptosis, proliferation and extracellular matrix deposition via accumulation of 2-hydroxy estrogen. The efficacy and safety of Ro 41-0960 in rats suggest its candidacy for treatment of uterine fibroids.

Toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human FSH receptor restores folliculogenesis in FSHR(-/-) FORKO mice.

A homozygous missense mutation, C566T, in the follicle stimulation hormone receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called follitropin receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intra-ovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6-10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26 +/- 4 follicles/ovary in treated group with 8 +/- 2 follicles at the antral stage compared with only 5 +/- 2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2-3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring.

Burden of Uterine Fibroids: An African Perspective, A Call for Action and Opportunity for Intervention.

Black women carry the burden of uterine fibroids, (AKA uterine leiomyomas), at a much higher rate than their racial counterparts. Thus, increasing awareness and discovering a solution to an endemic problem that plagues Sub-Saharan Africa is of critical importance, not only for the region itself, but also for the medical community globally. A collaborative, patient oriented, cost effective, and culturally sensitive approach must be at the forefront of this endeavor. While the exact pathogenesis of uterine fibroid development remains elusive, the racial disparity is well documented. Moreover, in the developed world, women are able to seek treatment through surgical and non-surgical means; however, sub-Saharan regions face their own challenges that, if not addressed, can ultimately extinguish the lives of many suffering women. Unfortunately, the literature is scarce on how to prevent fibroid development, which may be critical for women who do not have access to effective interventions. Recent research from our group and others has shown that vitamin D deficiency plays an important role in fibroid development and may be a preventable risk factor. Daily vitamin D supplementation is a low cost, effective intervention that could be implemented throughout the Sub-Saharan region. Similarly, education and increased awareness as to the nature and symptoms of uterine fibroids could improve the quality of life, remove negative social stigma, and reduce morbidity and mortality rates in women who seek medical care with advanced uterine fibroids.

Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation.

Resistance ovarian syndrome is a heterogeneous disorder inherited as a Mendelian recessive trait and characterized by infertility, primary amenorrhea, normal karyotype and elevated serum FSH and LH levels. An inactivating mutation, C566T, in FSH receptor gene (FSHR) has been identified initially in Finland. We investigated if an adenovirus expressing a normal copy of human FSHR (Ad-hFSHR) has the ability to: (i) transfect granulosa cell lines, (ii) render the transfected cell lines responsive to FSH stimulation and (iii) transcomplement the malfunctioning form of human FSHR gene with C566T mutation. COS-7, JC-410, JC-410-P450-scc-luc and JC-410-StAR-luc cell lines were infected by Ad-hFSHR followed by treatment with FSH. Functional activity of the Ad-hFSHR was tested by measuring cyclic adenosine monophosphate (cAMP) or luciferase activity in response to FSH stimulation, and showed 2-4.6-fold increases in Ad-hFSHR transfected cells compared with untransfected or Ad-LacZ transfected cells, indicating that Ad-hFSHR is functionally active and expressing hFSHR. Generation of cAMP in cells expressing only mutated hFSHR-T566 showed minimal increase after FSH stimulation. Co-transfection of Ad-hFSHR in these cells carrying the malfunction form of human FSHR caused significant increases of 2.2-7.4-fold in FSH dependent cAMP generation (P = 0.0007). We concluded that adenovirus expressing a normal human FSHR can compensate the inactivating human FSHR-C566T mutation and restore FSH responsiveness.

Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells.

BACKGROUND: To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial. METHODS: We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content. RESULTS: Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels. CONCLUSIONS: Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.

Use of uterine artery embolization to prevent peripartum hemorrhage of placental abruption with fetal demise & severe DIC.

Uterine artery embolization (UAE) is typically not indicated in the pre-operative management of pregnancies with a live fetus, because risk of fetal death from reduced uteroplacental blood flow. However, pre-operative UAE in pregnancies with a fetal demise poses no fetal risk, and may offer maternal benefits. Patients with placental abruption resulting in fetal demise are at high-risk for developing disseminated intravascular coagulation (DIC), which could have devastating complications such as peri-operative hemorrhage and death. This case report describes the first successful execution of a pre-operative UAE that effectively prevented antepartum and postpartum hemorrhage in a patient with DIC secondary to a placental abruption and recent fetal demise.

Growth retardation--an unexpected outcome from growth hormone gene therapy in normal mice with microencapsulated myoblasts.

Recently, we have succeeded in using nonautologous myoblasts engineered to secrete mouse growth hormone (GH) to correct partially the growth retardation of the Snell dwarf mice, which suffer from pituitary GH deficiency. The allogeneic myoblasts were protected from immune rejection by enclosure in permselective microcapsules fabricated from alginate, thus validating the clinical efficacy of using universal nonautologous cells for somatic gene therapy. Because GH therapy is considered also for treating patients with normal pituitary function, we now apply this protocol to treat normal mice to evaluate the potential consequences of using GH gene therapy in subjects with no demonstrated GH deficiency. When microencapsulated allogeneic myoblasts engineered to secrete mouse GH were implanted into normal male and female mice, contrary to expectation, the treated animals became significantly shorter and lost weight; their internal organs became smaller and their tibial growth plates were less differentiated, indicating reduced skeletal growth. Females were more severely affected than males and 2 animals died by day 13 of unknown cause. By day 70, most of the abnormalities were restored to normal except for body weights, which remained below normal. In conclusion, although somatic gene therapy for GH delivery is beneficial for pituitary dwarfism, it may have adverse metabolic consequences in those with normal hypothalamic-pituitary functions, and the female mice were more severely affected than males.

Correction of the growth defect in dwarf mice with nonautologous microencapsulated myoblasts--an alternate approach to somatic gene therapy.

Most of the currently approved human gene therapy protocols depend on genetic modification of autologous cells. We propose an alternate and potentially more cost-effective approach by implanting genetically modified "universal" cell lines to deliver desired gene products to nonautologous recipients. The recombinant allogeneic cells are protected from rejection after implantation by enclosure within immuno-protective alginate-poly-L-lysine-alginate microcapsules. The clinical efficacy of this strategy is now demonstrated by implanting microencapsulated allogeneic myoblasts engineered to secrete mouse growth hormone into the growth hormone-deficient Snell dwarf mice. The treated mutants attained increases in linear growth, body weights, peripheral organ weights, and tibial growth plate thickness significantly greater than those of the untreated controls. Secondary response to the exogenous growth hormone stimulation also resulted in increased fatty acid metabolism during the first month post-implantation. The microcapsules retrieved after about 6 months of implantation appeared intact. The encapsulated myoblasts retained a viability of > 60% and continued to secrete mouse growth hormone. Thus, implantation of nonautologous recombinant cells corrected partially the pleiomorphic effects of a transcription factor mutation in the Snell dwarf mice and the encapsulated cells remained functional for at least 6 months. This simple method of delivery recombinant gene products in vivo is a benign procedure, obviates the need for patient-specific genetic modification, and is amenable to industrial-scale quality control. It should have wide applications in therapies requiring a systemic continuous supply of recombinant gene products.

Gene therapy of uterine leiomyoma: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells in vitro and in a nude mouse model.

Uterine leiomyomas (LM) affect a high percentage of reproductive-age women. They develop as discrete, well-defined tumors that are easily accessible with imaging techniques--making this disease ideal for localized gene therapy approaches. In this study, we determined the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) as a potential therapy for LM. Rat ELT-3 LM cells and human LM cells were transfected with different multiplicity of infections (10-100 plaque forming units [PFU]/cell) of Ad-TK and treated with GCV (5, 10, or 20 microg/ml) for 5 days. To test the bystander effect, Ad-TK-transfected ELT-3 cells (100 PFU/cell) or LM cells (10 PFU/cell) were cocultured with corresponding nontransfected cells at increasing percentages and treated with GCV followed by cell counting. In ELT-3 cells transfected with Ad-TK/GCV (10, 20, 50, or 100 PFU/cell), the cell count was reduced by 24, 42, 77, and 87%, respectively, compared with the control cells (transfected with Ad-Lac Z/GCV). Similarly, in LM cells transfected with Ad-TK/GCV (10, 50, or 100 PFU/cell), the cell count was reduced by 31, 62, and 82%, respectively, compared with the control. A strong bystander effect was noted in both ELT-3 and LM cells with significant killing (p = 0.001) at a ratio of infected:uninfected cells of only 1:99 and maximal killing at 1:4. This study demonstrates the potential efficacy of the Ad-TK/GCV gene therapy approach as a viable nonsurgical alternative treatment for uterine LM.

Gene therapy of epithelial ovarian cancer using adenoviral vectors.

Ovarian cancer is the fourth most common cause of death in women. Gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) treatment has been successfully applied in the treatment of different cancers in experimental animals and in humans. In a recent report, we have demonstrated that the HSV-tk/GCV system can be used efficiently to kill human epithelial ovarian cancer cells (Gynecol Obstet Invest 1997;43:268-75). In this work, we wanted to test the ability of the HSV-tk/GCV to treat ovarian cancer in an animal model.The immune-deficient nude mice model was employed, and mice were injected intraperitoneally with the human epithelial ovarian cancer cell line OVCAR3, 10(8) cell/mouse. The mice were divided into three different groups, groups 1 and 2 were treated by intraperitoneal injection of adenovirus carrying the HSV-tk gene (ad-tk) on day 3 after cell implantation. Group 1 received 2 x 10(8) pfu/mouse; group 2 received 20 x 10(8) pfu/mouse. Group 3 did not receive any viral injection and served as our negative control. All mice received GCV 10 mg/kg IP bid for 6 days. All mice were hosted in the same facilities and had access to food and water ad libitum. Mice in group 3 started to show clinical manifestations of disease by day 10, and all mice were dead by day 21 (16 +/- 1.5). At this point mice in groups 1 and 2 appeared perfectly healthy. Autopsy done on group 3 mice demonstrated multiple cancer implants in the abdominal cavity plus hemorrhagic ascitis. In contrast, autopsy on sample mice from groups 1 and 2 at the same time point failed to demonstrate any macroscopic or microscopic cancer.On further follow-up, mice in groups 1 and 2 started to show cancer-related signs, eg, weight loss, movement difficulty, poor reflex response, and finally death. Survival varied between 50 and 101 days with a mean of 66 +/- 17 days for group 1 and 74 +/- 13 days for group 2. Autopsy done on treated mice demonstrated multiple cancer implants and ascitis. In conclusion, a single injection of ad-tk/GCV was able to improve survival in an ovarian cancer mouse model from an average of 16 days to 74 days. Trials with multiple injection in a novel immune-competent mouse model of ovarian cancer are underway in our laboratory.

Applying the herpes simplex virus thymidine kinase/ganciclovir approach to ovarian cancer: an effective in vitro drug-sensitization system.

Ovarian cancer is a major clinical problem with no rewarding treatment protocol currently available. In other malignancies transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells using a viral vector followed by administration of ganciclovir (GCV) provides a potentially effective strategy for treatment. In this work human ovarian epithelial cancer cell lines were infected with a recombinant adenoviral vector expressing the HSV-tk (AdRSV-tk) and were rendered sensitive to doses of GCV that were 100-200 times less than for untransfected cells. A strong bystander effect was noted with significant killing at a ratio of infected:uninfected cells of only 1:20 and maximal killing at 1:3. Normal human ovarian surface epithelial cells were also highly sensitive to the AdRSV-tk/GCV system. This study demonstrates the potential efficacy of the HSV-tk/GCV approach in ovarian cancer gene therapy.

Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems.

The Yersinia enterocolitica O:3 lipopolysaccharide O-antigen is a homopolymer of 6-deoxy-L-altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementation experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for O-antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP-L-rhamnose biosynthesis. Rhamnose and 6-deoxy-L-altrose are C3-epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O-antigen exporters.

Rapid method for isolation and staining of bacterial lipopolysaccharide.

Classically bacterial lipopolysaccharide (LPS) purification and silver staining take several days. We designed a simple and fast method for LPS isolation which when combined with silver staining using Pharmacia PhastSystem both can be completed in few hours. The purity of LPS isolated by this simple method may not be comparable to that by the phenol-water method hence we recommend this rapid isolation and staining procedures for simple and fast study of LPS patterns in gels.

Expression cloning of Yersinia enterocolitica O:3 rfb gene cluster in Escherichia coli K12.

The genes of Yersinia enterocolitica serotype O:3 (YeO3) that determine the synthesis of the O-side-chain of the lipopolysaccharide, the rfb region, were cloned into plasmid pBR322. The O-side-chain of YeO3 was expressed by the clone both in Escherichia coli and Salmonella typhimurium indicating that the entire rfb region was included in the clone. It was shown by restriction mapping, deletion analysis and transposition mutagenesis that about 10.4 kilobase pairs of DNA was essential for the synthesis and expression of the O-side-chain. The correct assembly of the O-side-chain on the cell surface of the clone was confirmed by immunofluorescence microscopy and slide agglutination. Immunoblotting using monoclonal antibody specific for the O-side-chain of YeO3 revealed that the O-side-chain material synthesized by the clone in E. coli was similar to that of YeO3. The clone did not show the in vitro temperature variation in O-side-chain expression characteristic of YeO3. Instead analogous O-side-chain was produced both at 25 degrees C and at 37 degrees C. Using transposon Tn2507, which carries a promotorless chloramphenicol acetyltransferase (CAT) gene, transcriptional fusions with the target DNA were generated. When testing the ability of mutated clones to produce CAT, transcription was shown to occur in a uniform direction throughout the whole rfb region. In colony hybridizations, using the cloned insert as a probe, homologous DNA was detected only in pathogenic Y. enterocolitica serotypes.

The effect of growth temperature on the biosynthesis of Yersinia enterocolitica O:3 lipopolysaccharide: temperature regulates the transcription of the rfb but not of the rfa region.

The rfb region of Yersinia enterocolitica O:3 (YeO3) that determines the synthesis of the O-side chain of the lipopolysaccharide was cloned and expressed in Escherichia coli K12 previously. The clone did not show the in vitro temperature variation in O-side chain expression known for YeO3, instead analogous O-side-chain was produced at 25 degrees C and at 37 degrees C and both were similar to that produced by YeO3 at 25 degrees C. In Northern blot analysis marked reduction in the amount of rfb-specific mRNA was observed from YeO3 grown at 37 degrees C when compared with those grown at 25 degrees C. On the other hand, equal amounts of rfb-specific mRNA were detected in the E. coli clone at both growth temperatures. This indicates that the transcription of YeO3 rfb region is dramatically repressed at 37 degrees C. The repressor gene is located outside the rfb region with no analogous locus in E. coli chromosome. We cloned 18.2 kilobase pairs of YeO3 chromosomal DNA that rendered E. coli K12 reactive with 2B5, a YeO3 core-specific monoclonal antibody, in colony blotting, indirect immunofluorescence and immunoblotting. Hence this clone contains the YeO3 rfa region, encompassing the genes involved in the core oligosaccharide biosynthesis. No apparent difference, in the aforementioned tests, was noticed in the expression of the 2B5 epitope at different growth temperatures either in YeO3 or in E. coli. In Northern blot analysis comparable amounts of rfa-specific mRNA were detected at 25 degrees and 37 degrees C. This argues that in YeO3 the core oligosaccharide biosynthesis is not temperature-regulated.

Association between mutations of the follicle-stimulating-hormone receptor and repeated twinning.

Follicle-stimulating hormone (FSH) has a role in folliculogenesis and spontaneous twinning. Using the candidate gene approach, we searched for mutations in the gene encoding the FSH receptor in a woman who had given birth to two sets of dizygotic twins without fertility treatment. We identified two linked mutations (Thr307Ala and Asn680Ser) that were closely associated with this phenotype. We suggest that expression of both mutations increases the sensitivity of the receptor to FSH.