Combination gene therapy for salivary gland cancer.

An established combination gene therapy strategy involving adenovirus vector delivery of the herpes thymidine kinase (tk) and murine interleukin-2 genes was adapted to treat salivary gland cancer in a murine model. Salivary tumors were generated by transcutaneous injection of 5 x 10(5) murine squamous carcinoma cells into the submandibular gland of syngeneic C3H/HeJ mice. After one week, established submandibular gland tumors were injected with a recombinant adenovirus containing therapeutic and control genes.

Some Properties and Uses of Torsional Overlap Integrals.

The first diagonalization step in a rho-axis-method treatment of methyl-top internal rotation problems involves finding eigenvalues and eigenvectors of a torsional Hamiltonian, which depends on the rotational projection quantum number K as a parameter. Traditionally the torsional quantum number vt = 0, 1, 2···is assigned to eigenfunctions of given K in order of increasing energy. In this paper we propose an alternative labeling scheme, using the torsional quantum number vT, which is based on properties of the K-dependent torsional overlap integrals .

Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells.

Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.

The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer.

Interleukin-2 (IL-2) gene therapy alone and in combination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ mice were directly injected with recombinant adenoviral vectors carrying both therapeutic and control genes. One week after adenoviral gene transfer, only the animals treated with combination IL-2+tk or tk alone demonstrated significant tumor regression.

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.

Quantification of diazeniumdiolate mutagenicity in four different in vitro assays.

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER/NO ([H2N(CH2)3NH(CH2)4N[N(O)NO-](CH2)3 NH3+]), which generate NO spontaneously with half-lives at 37 degrees C and pH 7.4 of 2 and 39 min, respectively.

DNA methylation directs a time-dependent repression of transcription initiation.

Background: The regulation of DNA methylation is required for differential expression of imprinted genes during vertebrate development. Earlier studies that monitored the activity of the Herpes simplex virus (HSV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-dependent repression of gene activity. Here, we examine the mechanism of methylation-dependent HSV tk gene regulation by direct determination of nucleoprotein organization during the establishment of a transcriptionally silenced state after microinjection of templates with defined methylation states into Xenopus oocyte nuclei.

Loss of retinoic acid receptors in mouse skin and skin tumors is associated with activation of the ras(Ha) oncogene and high risk for premalignant progression.

Retinoic acid receptor transcripts (RARalpha and RARgamma) are decreased in benign mouse epidermal tumors relative to normal skin and are almost absent in carcinomas. In this report, the expression of RARalpha and RARgamma proteins was analyzed by immunoblotting in benign skin tumors induced by two different promotion protocols designed to yield tumors at low or high risk for malignant conversion. RARalpha was slightly reduced in papillomas promoted with 12-O-tetradecanoylphorbol-13-acetate (low risk) and markedly decreased or absent in papillomas promoted by mezerein (high risk).

Early signaling events by endotoxin in PC12 cells: involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels.

We studied the effects of endotoxin from Escherichia coli (E. coli) on Ca2+ channel activity in PC12 cells using the cell-attached patch clamp technique. Endotoxin (1-100 ng/ml) decreased channel availability (n x Po) to about one third of control values, an effect that required 3.

Increased sensitivity to cisplatin by nm23-transfected tumor cell lines.

We report a functional link between expression of the metastasis suppressor gene nm23 and cancer cell sensitivity to the alkylating agent cisplatin. Cisplatin was 2-15-fold more inhibitory to the growth in vitro of nm23 transfectants of the K-1735 TK murine melanoma, MDA-MB-435 human breast carcinoma, and OVCAR-3 human ovarian carcinoma cell lines as compared to matched control transfectants. Administration of a single dose of cisplatin i.

Combination gene therapy for oral cancer in a murine model.

Combination therapy involving adenovirus-mediated transfer of the genes for herpes thymidine kinase (tk) and murine interleukin 2 (mIL-2) was used to treat head and neck cancer in C3H/HeJ mice. Tumors were generated by transcutaneous injection of 5 X 10(5) murine squamous carcinoma cells into the floor of the mouth of these syngeneic mice. After 1 week, recombinant adenoviral vectors containing both therapeutic and control genes in various combinations were injected directly into the established tumors, and subsequently all mice were administered ganciclovir twice daily (25 mg/kg) for 6 days.

Characterization of a chicken monoclonal antibody that recognizes the apical complex of Eimeria acervulina sporozoites and partially inhibits sporozoite invasion of CD8+ T lymphocytes in vitro.

A stable chicken hybridoma secreting a monoclonal antibody (mAb) that detects the apical complex of Eimeria acervulina sporozoites has been developed by fusing a thymidine kinase (TK)-deficient chicken myeloma with spleen cells from chickens immunized with sporozoite antigen. The mAb, designated as 6D-12-G10, recognized the apical complex of a sporozoite of 20-21 kDa molecular mass on western blots. Immunoelectron microscopic examination revealed that mAb 6D-12-G10 stained the conoid antigen.

An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo.

Brain tumors have been treated clinically by intratumoral injection of cells that produce retroviral vectors encoding the herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic administration of the antiviral drug ganciclovir. In vitro and in vivo comparisons of two different HSV-TK vector producer clones, which were made using standard transfection and transinfection techniques, were conducted. The two clones, PA317/G1TkSvNa.

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid.

A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion.

Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J.

In vivo role of tumor necrosis-like factor in Eimeria tenella infection.

The effect of tumor necrosis-like factor (TNLF) on the pathogenesis of coccidiosis was investigated. Injection of crude chicken TNLF enhanced the weight loss caused by Eimeria tenella infection. Rabbit polyclonal antibody against recombinant human tumor necrosis factor-alpha (rhTNF) partially restored E.

Rapid turnover and impaired cell-surface expression of the human folate receptor in mouse L(tk-) fibroblasts, a cell line defective in glycosylphosphatidylinositol tail synthesis.

The human folate receptor (hFR) is a plasma membrane protein that is anchored to the membrane via a glycosylphosphatidylinositol (GPI) tail in some cell types. The KB hFR cDNA sequence predicts a hydrophobic, alpha-helical 31-residue carboxyl terminus that is thought to be the signal for cleavage and attachment of the GPI tail. Alternatively, this region may serve as a transmembrane domain if GPI attachment is not efficient.

Selective humoral immune response of Balb/C mice to Brucella abortus proteins expressed by vaccinia virus recombinants.

Genes encoding Brucella abortus Cu/Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54. Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the beta-gal reporter gene but no B. abortus DNA were also developed.

Eimeria acervulina infection: evidence for the involvement of CD8+ T lymphocytes in sporozoite transport and host protection.

Host-parasite interactions during coccidial infection are complex, and host responses leading to parasite elimination are not well understood. Interactions between host cells and parasites were investigated using two-color immunofluorescent staining of duodenal tissue infected with Eimeria acervulina. Tissues were removed from TK strain chickens 24, 48, and 72 h after primary infection with 2.

Characterization of the human glucocorticoid receptor promoter.

To elucidate the functional elements that are involved in the regulation of the human glucocorticoid receptor (hGR) gene, transient expression, DNase I footprinting, and gel mobility shift analyses were conducted. We found that the hGR promoter region between -700 and +38 bp contained 11 footprinted sites. Deletion of the -374 to -183 bp region, which is highly conserved between human and mouse (93%), induced a 5-24-fold reduction in promoter activity in HeLa, NIH3T3, CV1, and HepG2 cells.

Increased thymidylate synthase protein levels are principally associated with proliferation but not cell cycle phase in asynchronous human cancer cells.

We have analysed cell cycle variations in thymidylate synthase (TS) protein in asynchronously growing NCl H630 and HT 29 colon cancer and MCF-7 breast cancer cell lines. Western immunoblot analysis using the TS 106 monoclonal antibody revealed a 14- to 24-fold variation in TS levels between the peak exponential and confluent growth phase in the three cell lines. Similar variations in TS levels and TS activity were detected using the 5-fluorodeoxyuridine monophosphate and deoxyuridine monophosphate biochemical assays.

Thyroid hormone enhancement of estradiol stimulation of breast carcinoma proliferation.

Thyroid hormone (T3) and estradiol (Est) modulate biological processes by binding to nuclear receptor proteins that, through interactions with specific response elements in the regulatory regions of genes, modulate gene transcription. Est stimulation of estrogen receptor (ER)-positive breast carcinoma cell growth occurs through its ability to bind to the ER and activate gene transcription. We now report that physiological concentrations of T3 significantly enhance Est stimulation of growth of a number of human breast carcinoma cell lines.

Coexpression of a multidrug-resistance gene (MDR1) and herpes simplex virus thymidine kinase gene as part of a bicistronic messenger RNA in a retrovirus vector allows selective killing of MDR1-transduced cells.

A new retroviral vector, pSXLC/pHa, was constructed to coexpress drug-selectable markers with a second gene of interest as a part of a bicistronic mRNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system was used to develop a new retroviral vector pHa-MDR-IRES-TK which expresses a single mRNA from which translation of the MDR1 gene is cap dependent and translation of the herpes simplex virus thymidine kinase gene is IRES dependent. The pHa-MDR-IRES-TK transfectants showed high levels of P-glycoprotein expression and multidrug resistance.

Development and characterization of chicken-chicken B cell hybridomas secreting monoclonal antibodies that detect sporozoite and merozoite antigens of Eimeria.

Chicken-chicken B cell hybridomas that secrete monoclonal antibodies (mAb) detecting antigens located at the anterior tip of the sporozoites of Eimeria acervulina have been produced. Peripheral blood lymphocytes (PBL) obtained from chickens infected with E. acervulina were fused with a thymidine-kinase deficient (TK-) chicken B cell line.