Infrequent p53 gene mutations in medulloblastomas.

Cytogenetic and molecular studies of medulloblastomas have demonstrated frequent loss of sequences from the short arm of chromosome 17, possibly implicating loss or inactivation of the p53 tumor suppressor gene. We amplified exons 5 through 8 of the p53 gene by the polymerase chain reaction technique. These segments, which encompass the regions usually mutated in human tumors, were sequenced to search for p53 mutations in 12 medulloblastoma tumors, 8 xenografts, and 3 permanent cell lines. Mutation of the p53 gene was found in only 1 of 3 cell lines tested and in none of the xenografts or primary tumors studied. Our results suggest that p53 is mutated in an unusual way or that a second tumor suppressor gene on the short arm of chromosome 17 is involved in the pathogenesis of medulloblastoma.

Oncogene amplification in pediatric brain tumors.

Despite a considerable amount of information concerning chromosomal and molecular abnormalities found in gliomas in adults, relatively little is known regarding these abnormalities in pediatric brain tumors. We have analyzed DNA from 37 primary brain tumors and 4 tumor-derived cell lines for oncogene amplification. Probes utilized represent 11 known oncogenes (erbB1, gli, neu, myc, L-myc, N-myc, H-ras, K-ras, N-ras, sis, and src). Of 20 primary medulloblastomas studied, only one tumor was found to have erbB1 amplification. In contrast, of the 4 medulloblastoma cell lines studied, 1 had c-myc amplification, 1 had erbB1 amplification, and 1 had amplification of N-myc. Twelve glial brain tumors were analyzed, and only 1 case with amplification of the erbB1 oncogene was found. Other tumors studied include 1 meningioma, 2 ependymomas, 1 anaplastic ependymoma, and 1 cerebral primitive neuroectodermal tumor, none of which had oncogene amplification. These results suggest that oncogene amplification is relatively uncommon in primary medulloblastomas, but the frequency and diversity of oncogene amplification is greater in tumors that can be established as cell lines. The lower frequency of erbB1 amplification in glial brain tumors in children compared to adults is consistent with the generally lower grade of glial tumor histology seen in pediatric patients. However, the case with amplification of the erbB1 oncogene represented 1 of 2 cases of glioblastoma multiforme we studied, which suggests that pediatric glioblastoma multiforme may have a similar frequency of erbB1 oncogene amplification to glioblastomas seen in adults. Our results suggest that oncogene amplification is a relatively uncommon mechanism of oncogene activation in pediatric brain tumors, and they provide molecular evidence for heterogeneity in tumors classified as medulloblastomas.

Secondary acute myelogenous leukemia following safe exposure to etoposide.

PURPOSE: To present two patients as illustrations of the risk of developing secondary acute myelogenous leukemia (sAML) when theoretically safe doses of etoposide (VP-16) are used. PATIENTS AND METHODS: Patient no. 1 was a 15-year-old white girl diagnosed with stage IIa Hodgkin's disease. She was treated with a combination of vincristine, doxorubicin, bleomycin, and VP-16 (2 g/m2 total) over 4 months, followed by 25.5 Gy of involved-field radiotherapy. Patient no. 2 was an 11-year-old white boy diagnosed with virus-associated hemophagocytic syndrome (VAHS). He was treated with VP-16 intravenously (IV) and orally (0.3 g and 2.8 g/m2, respectively). RESULTS: Patient no. 1 developed AML 16 months from the diagnosis of Hodgkin's disease. Patient no. 2 developed AML 26 months from diagnosis. Both bone marrows were consistent with French-American-British (FAB) M4 disease. Both patients had abnormalities of the long arm of chromosome 11. CONCLUSION: The use of low-dose or oral VP-16 can be associated with the development of sAML. Clinicians should be cautious in the use of VP-16 in low-risk diseases.

Cyclophosphamide plus topotecan in children with recurrent or refractory solid tumors: a Pediatric Oncology Group phase II study.

PURPOSE: To determine the response rate of the combination of cyclophosphamide and topotecan in pediatric patients with recurrent or refractory malignant solid tumors. PATIENTS AND METHODS: A total of 91 pediatric patients, 83 of whom were fully assessable for response and toxicity, received cyclophosphamide (250 mg/m2/dose) followed by topotecan (0.75 mg/m2/dose), each given as a 30-minute infusion daily for 5 days. All patients received filgrastim (5 mcg/kg) daily until the absolute neutrophil count (ANC) was > or = 1,500 microL after the time of the expected ANC nadir. RESULTS: A total of 307 treatment courses were given to the 83 fully assessable patients. Responses (complete response plus partial response) were seen in rhabdomyosarcoma (10 of 15 patients), Ewing's sarcoma (six of 17 patients), and neuroblastoma (six of 13 patients). Partial responses were seen in two of 18 patients with osteosarcoma and in one patient with a Sertoli-Leydig cell tumor. Twenty-three patients had either minor responses (n = 6) or stable disease (n = 17); the median number of courses administered to patients with partial or complete response was six (range, two to 13 courses), and the median administered to those with stable disease was three (range, one to 11 courses). The toxicity of the combination was limited principally to the hematopoietic system. Of 307 courses, 163 (53%) were associated with grade 3 or 4 neutropenia, 84 (27%) with grade 3 or 4 anemia, and 136 (44%) with grade 3 or 4 thrombocytopenia. Despite the severe myelosuppression, only 34 (11%) of 307 courses were associated with grade 3 or 4 infection. Nonhematopoietic toxicity of grades > or = 3 was rare and consisted of nausea and vomiting (two courses), perirectal mucositis (one course), transaminase elevation (one course), and hematuria (two courses). CONCLUSION: The combination of cyclophosphamide and topotecan is active in rhabdomyosarcoma, neuroblastoma, and Ewing's sarcoma. Stabilization of disease was seen in osteosarcoma, although objective responses were rare in this disease. The therapy can be given with acceptable hematopoietic toxicity with the use of filgrastim support.

Phase I study of topotecan in combination with cyclophosphamide in pediatric patients with malignant solid tumors: a Pediatric Oncology Group Study.

PURPOSE: To determine the maximum-tolerated dose (MTD) and dose-limiting toxicity of topotecan when combined with cyclophosphamide in pediatric patients with recurrent or refractory malignant solid tumors. PATIENTS AND METHODS: A total of 33 patients received cyclophosphamide (250 mg/m2/dose) followed by topotecan in escalating doses (0.6 to 0.75 mg/m2/dose), each given as a 30-minute infusion daily for 5 days. A total of 154 fully assessable treatment courses were given to these patients. RESULTS: Neutropenia was the dose-limiting toxicity of the therapy at both topotecan dose levels. The addition of filgrastim allowed escalation of the topotecan dose to the 0.75-mg/m2 level with acceptable neutropenia. Other significant toxicities were anemia and thrombocytopenia. Nonhematopoietic toxicity of grades > or = 3 was not observed. Responses were reported in patients with Wilms' tumor (one complete response [CR], one partial response [PR]), neuroblastoma (one CR, one PR), rhabdomyosarcoma (one PR), and osteosarcoma (one PR). Pharmacokinetic studies indicate that cyclophosphamide administered on the schedule used in this study did not alter topotecan disposition on day 5. As with previous studies, a pharmacodynamic relation between systemic exposure and myelosuppression was noted. CONCLUSION: The combination of topotecan and cyclophosphamide shows activity in a wide variety of pediatric solid tumors and can be given with acceptable hematopoietic toxicity with the use of filgrastim support. We recommend that pediatric phase II trials use cyclophosphamide 250 mg/m2 followed by topotecan 0.75 mg/m2 daily for 5 days with filgrastim for amelioration of neutropenia.

Lack of bystander killing in herpes simplex virus thymidine kinase-transduced colon cell lines due to deficient connexin43 gap junction formation.

The efficacy of herpes simplex virus thymidine kinase (HSV-TK) gene therapy for colorectal carcinoma has been investigated in an in vitro system. The magnitude and the mechanism of the HSV-TK bystander effect was determined in three human colon tumor cell lines: HCT-116, HCT-8, and HT-29. Each HSV-TK(+) cell line was generated by stable transduction with a bicistronic retroviral vector containing the HSV-TK and neomycin resistance (neo) genes; each exhibited an IC50 for GCV of < or =4 microM. When GCV was added to HSV-TK(+) cells mixed with parental cells or known bystander-positive cell lines, no bystander killing was evident in the HT-29 or HCT-8 cells. Western blots detected the expression of the gap junction protein connexin43 (Cx43) in HCT-8 and HT-29 cells; however, immunolocalization studies indicated predominantly cytoplasmic staining of Cx43 and no cell surface staining in these cell lines. Stable transfection of HCT-8 and HT-29 cells with Cx43 resulted in increased levels of Cx43 expression with the same subcellular distribution as before, yet there was again no apparent bystander killing. In contrast, Cx43 expression was localized to the cell surface in the bystander-positive colon tumor cell line HCT-116. These results demonstrate that expression and proper surface localization of Cx43 gap junctions are necessary components of the bystander effect in human colon tumor cells. They also indicate that future combination gene therapy approaches using coexpression of HSV-TK and Cx43 genes may not be applicable to all tumor systems.

Two-drug combinations that increase apoptosis and modulate bak and bcl-X(L) expression in human colon tumor cell lines transduced with herpes simplex virus thymidine kinase.

Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5- to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X(L), was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X(L). In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X(L) protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.

KRN5500 induces apoptosis (PCD) of myeloid leukemia cell lines and patient blasts.

The purpose of this study was to determine if KRN5500, a spicamycin derivative with a unique acyl tail, would induce programmed cell death (PCD) of myeloid leukemia cell lines and cryopreserved leukemic blasts from newly diagnosed children with acute leukemia (AL). Cells were incubated with varying concentrations (0-5 ng/ml) of KRN5500 and the percent PCD determined using a modified in situ end labeling (ISEL) technique with Klenow fragment. The percent PCD was calculated using the formula: Percent PCD (% PCD)=[number of apoptotic cells/(viable cells+apoptotic cells)]x100. DMSO (0.30% w/v) was added to the cells in culture as the positive control for PCD; the negative control was media or albumin. KRN5500 increased the amount of PCD significantly in all five of the tested cell lines; U937 41+/-1.8%, KG1a 40+/-0.3%, HEL 14+/-2.2%, HL-60 41+/-0. 9%, K562 36+/-2% (mean PCD+/-SD). Patient blasts exposed to KRN5500 had an increase in PCD when exposed to 2 ng/ml of agent from 2 to 8 h; acute myeloid leukemia patients 7.5+/-0.5% at 2 h to 43.5+/-1.6% at 8 h, and acute lymphocytic leukemia patients rose from 12.4+/-3.8% at 2 h to 29.9+/-11.6% after 8 h (mean+/-SE). Overall the PCD for the patient samples was 3.7 versus 28+/-4% at 2 and 8 h, respectively. PCD was proportional to the dose of KRN5500 and incubation time. Further pre-clinical and clinical studies are required.

Metabolism and activities of 3'-azido-2',3'-dideoxythymidine and 2',3'-didehydro-2',3'-dideoxythymidine in herpesvirus thymidine kinase transduced T-lymphocytes.

T-lymphocytes transduced with the conditionally toxic herpesvirus thymidine kinase gene (HSV-1 TK) are increasingly becoming important tools in genetic therapy approaches for treating viral infections and cancers. Therefore, the effects of different antiviral nucleoside drugs on the growth inhibition of parental and HSV-1 TK-transduced human T-lymphocyte cell lines (H9 and CEM TK-) were examined. As expected, both transduced cell lines were most sensitive to growth inhibition by ganciclovir (GCV). While the presence of HSV-1 TK did not potentiate 3'-azido-2',3'-dideoxythymidine (AZT) growth inhibition of H9 cells containing cellular TK; transduction of HSV-1 TK into the cellular TK-deficient CEM cells (CEM TK-) restored sensitivity to AZT. In both transduced cell lines, an HSV-1 TK-dependent growth inhibition with 2',3'-didehydro-2',3'-dideoxythymidine (d4T) was observed and a Km of 143 microM for d4T and HSV-1 TK was determined. Metabolic labeling analysis showed that drug metabolism correlated with the observed effects on cell growth. The effects of HIV-1 replication in the CEM TK- cell lines in the presence of AZT or d4T was evaluated. CEM TK- cells are largely resistant to AZT or d4T inhibition of HIV-1 replication, however, transduction of HSV-1 TK into the CEM TK- cells completely restored AZT and d4T inhibition of HIV-1 replication. These studies confirm the requirement for a thymidine kinase activity for the anti-HIV activities of d4T and suggest that AZT, but not d4T, could be potentially administered to patients receiving HSV-1 TK-transduced lymphocytes.

Neuroblastoma, retinoblastoma, and brain tumors in children.

Considerable progress has been made recently in the biologic understanding and the clinical management of pediatric tumors of the peripheral and central nervous system. Here we review important representative studies published primarily in the past 2 years regarding neuroblastoma, retinoblastoma, and brain tumors in children. Highlights include 1) the importance of tumor DNA content, N-myc amplification, and chromosome 1 deletion in predicting outcome of patients with neuroblastoma; 2) the impact of mass screening for neuroblastoma in Japan and elsewhere; 3) improvements in the clinical management of neuroblastoma, retinoblastoma, and brain tumors; and 4) neurologic sequelae of these tumors and their treatment.

Expression of human alpha 1 antitrypsin in murine hematopoietic cells in vivo after retrovirus-mediated gene transfer.

For patients with alpha1 antitrypsin (alpha 1AT) deficiency, the expression of alpha 1AT in hematopoietic cells may results in a number of benefits not provided by gene transfer strategies involving local modification of the respiratory epithelium or liver-directed gene transfer. We investigated the expression of alpha 1AT in murine hematopoietic cells after retrovirus-mediated gene transfer. For this purpose we constructed an LNL-6-derived recombinant retrovirus vector (L alpha 1ED) that expresses the alpha 1AT cDNA from the Moloney murine leukemia virus (MoMuLV) U3 promoter/enhancer and coexpresses the cDNA for a mutant form of the murine dihydrofolate reductase molecule (*DHFR) from the encephalomyocarditis virus (emc) internal ribosome entry site (IRES). All of the mice transplanted with bone marrow transduced with the L alpha 1ED vector expressed the alpha 1AT protein at the 3-week time point after transplantation. By the 6-week time point the alpha 1AT levels declined to a lower level, where they generally remained for the duration of the experiment. This study demonstrates the potential utility of hematopoietic cell gene transfer for gene therapy of alpha 1AT deficiency.

Hematopoietic cytokine-inducible gene expression from retroviral vectors.

Retroviral vectors capable of cytokine-inducible gene expression will be useful for a number of gene therapy applications. We explored one mechanism whereby cytokine inducibility may be imparted to the retroviral U3 promoter/enhancer by utilizing the JAK-STAT signal transduction pathway that is activated by a number of hematopoietic cytokines. We used PCR mutagenesis to insert a consensus binding site for the ubiquitous transcription factor Sp1 into the Moloney murine leukemia virus U3 followed by the insertion of multimers of a STAT-binding oligonucleotide with the core sequence 5'-TTCCCGGAA. After insertion of the modified U3s into a retroviral vector expressing the luciferase reporter gene and transduction of the HepG2 cell line, luciferase expression was induced with recombinant human IFN-gamma. The level of induction reached a maximum of 9.9-fold higher than the uninduced vector when the Sp1-U3 contained four STAT oligos. When this optimal vector was compared with the wild-type and Sp1 vectors, respective values of 17.9- and 16.7-fold higher expression were achieved with IFN-gamma treatment. Retroviral vectors incorporating these cytokine-inducible U3s will be useful for gene therapy in a number of situations involving gene transfer to hematopoietic, hepatic and other cytokine-responsive cell types.

Prenatal detection of neuroblastoma by fetal ultrasonography.

PURPOSE: We report three cases of neuroblastoma diagnosed by prenatal ultrasound examination and examine the biologic features of tumors diagnosed prenatally. PATIENTS AND METHODS: Neuroblastoma is the most common tumor detected in the newborn period. Thus, some of these tumors develop prenatally and should be detectable by maternal ultrasound. Here we report three cases in which a neuroblastoma was suspected on prenatal ultrasonography. In addition, we review selected features of 17 additional cases reported in the literature. RESULTS AND CONCLUSIONS: These data indicate that, although the majority of patients have favorable clinical and biological features and do well, some patients do not, and the DNA index may be the most important predictor of outcome.

Defective asialoglycoprotein receptor endocytosis mediated by tyrosine kinase inhibitors. Requirement for a tyrosine in the receptor internalization signal.

Regulated endocytosis by growth factor receptors requires intact receptor-associated tyrosine kinase activity. To determine whether a similar requirement exists for the asialoglycoprotein (ASGP) receptor which lacks intrinsic tyrosine kinase activity and participates in constitutive endocytosis, we examined the effect of three tyrosine kinase inhibitors, tyrphostin, genistein, and staurosporine, on receptor-mediated endocytosis in the human hepatoma line HepG2. These compounds inhibited early receptor internalization from the plasma membrane to internal protease-resistant sites in a concentration-dependent manner. This effect correlated with their inhibition of tyrosine phosphorylation of the ASGP receptor in vitro. Receptor trafficking subsequent to receptor internalization was unaffected. Endocytosis of another constitutively internalized protein, the transferrin receptor, was also inhibited by these compounds. In contrast, pinocytosis of the fluid-phase marker Lucifer yellow was not inhibited. The tyrosine kinase inhibitors also decreased the endocytic rate of transfected ASGP receptor H1 subunit in SK-Hep-1 cells. Therefore an intact ASGP receptor heterooligomeric complex is not required for this effect. Mutation of the single cytoplasmic tyrosine at position 5 of the H1 subunit to phenylalanine produced an ASGP receptor which was endocytosed regardless of treatment with the tyrosine kinase inhibitors. We conclude that tyrosine kinase activity modulates the rate of receptor endocytosis at a point early in the internalization process.

2-Chlorodeoxyadenosine (2-CDA) for the treatment of refractory or recurrent Langerhans cell histiocytosis (LCH) in pediatric patients.

BACKGROUND: Pediatric patients with Langerhans cell histiocytosis (LCH) may become refractory to conventional therapy or present with repeated recurrences over several years. Current therapeutic options such as prednisone, vinblastine, etoposide, and cyclosporine are associated with significant acute toxicities and late effects. Recent reports suggested that 2-chlorodeoxyadenosine (2-CDA) may be an effective agent in adults with LCH. The purpose of this study was to determine the safety and efficacy of 2-CDA in children with LCH. METHODS: This report presents the data collected from the first three patients that have completed this trial. Patients were enrolled in a prospective study after informed consent was obtained. Patients had a confirmed diagnosis of LCH that had recurred several times or not responded to standard therapy. Patients were given a starting dose of 5 mg/M2 of daily continuous infusion for three days duration. Two patients had their dose increased to 6.5 mg/M2/ day. A total of 4-6 courses were given, and courses were repeated every 3-4 weeks. Thirteen of fifteen courses were given as outpatients at home. RESULTS: Each patient completed therapy with myelosuppression the primary toxicity. Pt. 1 initially received a higher dose of 2-CDA and developed sepsis. The dose was reduced to current study levels and no other incidence of infection, fever, and neutropenia, or blood product transfusion was required. All three patients are free of active disease 10-18 months after completing 2-CDA. CONCLUSION: Three patients with LCH refractory to standard therapy had CR to 2-CDA, given at 5-6.5 mg/M2/day for 3 days, without significant toxicity.

Bone marrow stromal cells as a vehicle for gene transfer.

Adoptive transfer of genetically modified somatic cells is playing an increasingly important role in the management of a wide spectrum of human diseases. Hematopoietic stem cells and lymphocytes have been used to transfer a variety of genes, however, they have limitations. In this study, the feasibility of retroviral gene transduction of bone marrow stromal cells, and the engraftment characteristics of these cells following infusion, was investigated in a murine transplantation model. Stromal cells derived from Balb/c mouse bone marrow were transduced with a replication-defective retrovirus containing the LacZ gene. Following three rounds of transduction, between 5 and 40% of the cells were positive for the LacZ gene. A total of 2 x 106 cells were infused into the same mouse strain. After the infusion, the LacZ gene was detected by PCR in the bone marrow, spleen, liver, kidney and lung; however, only the spleen and bone marrow samples were strongly positive. Quantitative PCR demonstrated that between 3 and 5% of spleen and bone marrow cells, and 1% of liver cells contained the LacZ gene at 3 weeks after infusion; <0.2% transduced cells were found in other organs. No difference was noted in engraftment between mice with or without irradiation before transplantation, suggesting that engraftment occurred without myeloablation. The infused transduced cells persisted for up to 24 weeks. Self-renewal of transplanted stromal cells was demonstrated in secondary transplant studies. Ease of culture and gene transduction and tissue specificity to hematopoietic organs (bone marrow, spleen, liver) is demonstrated, indicating that stromal cells may be an ideal vehicle for gene transfer.

In vivo hepatocyte transduction with retrovirus during in-flow occlusion.

Gene therapy research would be facilitated by a technically simple procedure for transducing hepatocytes in vivo. Previously reported methods have employed partial hepatectomy followed 24 hr later by asanguineous perfusion of the regenerating liver with retrovirus. We have developed a simpler method of in vivo transduction in which we deliver an intraportal bolus of retrovirus to the regenerating rodent liver during a brief period of hepatic in-flow occlusion. On Day 0, adult male Sprague-Dawley rats (N = 19) underwent 70% hepatectomy to induce hepatocyte replication. On Day 1, retroviral supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegalovirus promoter driving expression of the Escherichia coli beta-galactosidase (beta gal) gene. Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 x 10(5) colony-forming units of retrovirus by intraportal injection during a 3-min occlusion of the hepatic artery and portal vein. Control rats (N = 2) received intraportal medium (without retrovirus), also during in-flow occlusion. The procedure required 20-25 min, and the survival rate was 84%. Cryostat sections were prepared from liver biopsies obtained on Post-transduction Days 8 and 15 and stained with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside to detect beta gal expression. Light microscopic examination of Day 8 sections from surviving experimental rats (N = 14) revealed 0.10-1.00% blue (i.e., transduced) hepatocytes per low power field, while sections from control rats (N = 2) exhibited no blue cells. Day 15 sections from experimental rats revealed a somewhat lower frequency of hepatocytes expressing beta gal.(ABSTRACT TRUNCATED AT 250 WORDS)

STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone.

Interleukin (IL)-6-type cytokines stimulate osteoclastogenesis by activating gp130 in stromal/osteoblastic cells and may mediate some of the osteoclastogenic effects of other cytokines and hormones. To determine whether STAT3 is a downstream effector of gp130 in the osteoclast support function of stromal/osteoblastic cells and whether the gp130/STAT3 pathway is utilized by other osteoclastogenic agents, we conditionally expressed dominant negative (dn)-STAT3 or dn-gp130 in a stromal/osteoblastic cell line (UAMS-32) that supports osteoclast formation. Expression of either dominant negative protein abolished osteoclast formation stimulated by IL-6 + soluble IL-6 receptor, oncostatin M, or IL-1 but not by parathyroid hormone or 1,25-dihydroxyvitamin D3. Because previous studies suggested that IL-6-type cytokines may stimulate osteoclastogenesis by inducing expression of the tumor necrosis factor-related protein, receptor activator of NF-kappaB ligand (RANKL), we conditionally expressed RANKL in UAMS-32 cells and found that this was sufficient to stimulate osteoclastogenesis. Moreover, dn-STAT3 blocked the ability of either IL-6 + soluble IL-6 receptor or oncostatin M to induce RANKL. These results establish that STAT3 is essential for gp130-mediated osteoclast formation and that the target of STAT3 during this process is induction of RANKL. In addition, this study demonstrates that activation of the gp130-STAT3 pathway in stromal/osteoblastic cells mediates the osteoclastogenic effects of IL-1, but not parathyroid hormone or 1, 25-dihydroxyvitamin D3.

Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction.

Liver-directed gene therapy will be applicable to many inherited diseases. Although various protocols have been devised for in vivo delivery of retrovirus, comparison of hepatocyte transduction frequencies has been difficult due to variations in retroviral titer and a paucity of DNA data. We have previously reported an in vivo rat hepatocyte transduction technique which involves 70% hepatectomy followed 24 hr later by portal vein injection of retrovirus during hepatic in-flow occlusion. In this study, we employed this method and concentrated retroviral preparations to achieve transduction of up to 15% of hepatocytes as determined by a quantitative PCR assay. As an initial step toward identifying promoters which lead to high-level long-term expression of retroviral transduced genes, we used our in vivo delivery system to compare the Moloney murine leukemia virus long terminal repeat (LTR) promoter with the promoter for the large subunit of murine RNA polymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as the reporter gene to facilitate long-term analysis of expression. Serum hAAT levels were higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50 ng/ml). This difference was consistent with the higher transduction frequency observed for the Pol-II-hAAT vector. Although serum hAAT expression was sustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and three of five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level and proviral DNA frequency, in vivo expression was significantly lower than in transduced tissue culture cells. We conclude that a high frequency of in vivo transduction can be achieved by using retroviral vectors and our rapid transduction protocol, but transduced gene expression remains a serious problem. The quantitative assays described herein will facilitate in vivo comparisons of gene regulatory elements.