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W F Anderson

Publications and source records attributed to W F Anderson.

At least 73 records · Page 4Linked to original sources
PubMed1998

Characterization of the proline-rich region of murine leukemia virus envelope protein.

Mammalian type C retroviral envelope proteins contain a variable proline-rich region (PRR), located between the N-terminal receptor-binding domain and the more highly conserved C-terminal portion of the surface (SU) subunit. We have investigated the role of the PRR in the function of murine leukemia virus (MuLV) envelope protein. In the MuLVs, the PRR contains a highly conserved N-terminal sequence and a hypervariable C-terminal sequence. Despite this variability, the amphotropic PRR could functionally substitute for the ecotropic PRR. The hypervariable region of the PRR was not absolutely required for envelope protein function. However, truncations in this region resulted in decreased levels of both the SU and TM proteins in viral particles and increased amounts of the uncleaved precursor protein, Pr85. In contrast, the N-terminal conserved region was essential for viral infectivity. Deletion of this region prevented the stable incorporation of envelope proteins into viral particles in spite of normal envelope protein processing, wild-type levels of cell surface expression, and a wild-type ability to induce syncytia in an XC cell cocultivation assay. However, higher levels of the SU protein were shed into the supernatant, suggesting a defect in SU-TM interactions. Our data are most consistent with a role for the PRR in stabilizing the overall structure of the protein, thereby affecting the proper processing of Pr85, SU-TM interactions, and the stable incorporation of envelope proteins into viral particles. In addition, we have demonstrated that the PRR can tolerate the insertion of a peptide-binding domain, making this a potentially useful site for constructing targetable retroviral vectors.

3T3 Cells↗
PubMed1998

Functional domains in the retroviral transmembrane protein.

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967-6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.

3T3 Cells↗
PubMed1998

Mechanisms for ganciclovir resistance in gastrointestinal tumor cells transduced with a retroviral vector containing the herpes simplex virus thymidine kinase gene.

Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tumor cells confers sensitivity to the cells to the viral drug ganciclovir (GCV). Although the efficacy of the HSV-TK/GCV approach is well studied, the mechanisms for the resistance of HSV-TK-transduced tumor cells to GCV are poorly understood. Here, we examined the mechanisms for GCV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines. Our results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines. GCV-resistant colonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-resistant colonies demonstrated that the HSV-TK gene was either partially or completely deleted from the resistant HSV-TK-transduced cells. In the HT-29 RM and MIAPACA-2 RM cells, a 220-bp deletion of the gene was found, whereas in the HT-29 R1-R5-resistant cells, the whole TK gene was found to be absent. Immunocytochemical studies using a polyclonal antibody to the TK protein demonstrated that the HSV-TK protein was absent in the GCV-resistant, HSV-TK-transduced cells. Transfection of the resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV. The presence of GCV-resistant cells was only demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect. Our results suggest that GCV resistance found in tumor cells transduced with a retroviral HSV-TK gene is due to the lack of a functional TK protein in the tumor cells rather than any intrinsic resistance of the cells to GCV. In tumor cells with a good bystander effect, the small percentage of TK-transduced cells that do not express the TK protein are probably killed by the bystander effect because GCV-resistant tumor cells were not found in these cell lines. GCV-resistant tumor cells were found only in tumor cell lines with a poor bystander effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.

Antiviral Agents↗
PubMed1998

Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene.

BACKGROUND: Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS: Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS: Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% +/- 0.7% vs 17.5% +/- 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer.

Animals↗
PubMed1998

Retrovirus-mediated gene transfer to photocoagulation-induced choroidal neovascular membranes.

PURPOSE: To determine the feasibility of experimental gene transfer to laser-induced choroidal neovascular membrane (CNVM) in rats, with a retroviral vector containing the reporter construct beta-galactosidase (beta-gal). METHODS: Laser photocoagulation was used to induce CNVM in rats. To ascertain the duration of beta-gal expression in the CNVM, 23 rats received 10 burns (75 microm, 100 mW, 0.1 seconds) in their right eyes, and beta-gal expression was examined from day 3 to 4 months. In addition, 14 pigmented rats were treated with 3 photocoagulation burns in their right eyes. beta-gal vector was injected into the vitreous or subretinal space 2 days later. On day 14, fluorescein angiography was performed to detect choroidal neovascularization. Then, beta-gal expression in each photocoagulation-induced CNVM was examined by observing the exposed fundus of the eyes stained with the beta-gal substrate X-Gal. RESULTS: beta-gal expression was identified in the CNVM induced by photocoagulation from day 5 (16.2% +/- 6.8% of the lesions) to 4 months (3.7% +/- 2.4%). Histopathologic examination revealed beta-gal-transduced macrophages and spindle-shaped cells, which amounted to 1.12% +/- 0.58% (at 2 weeks) of the total cells in the CNVM. beta-gal expression was restricted to the CNVM, and there was no beta-gal transduction in surrounding normal retinochoroidal tissue. There was no correlation between choroidal neovascularization formation and beta-gal expression. CONCLUSIONS: The feasibility of gene transduction targeted to the photocoagulation-induced CNVM was demonstrated using retroviral vectors. By transducing functional genes, this model could be useful for investigating the possibility of gene therapy to inhibit formation of the CNVM in age-related macular degeneration.

Animals↗
PubMed1997

Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein.

Targeted gene delivery to vascular lesions is a major challenge in the development of gene therapy protocols for cardiovascular diseases. One approach would be to enable retroviral vectors to accumulate at sites of vascular injury and enhance local vector concentration. An early step in wound repair is the adhesion of platelets to collagen exposed from damaged vasculature. Hence, the Moloney murine leukemia virus (MoMLV) envelope (env) protein was engineered to incorporate a high-affinity collagen-binding domain derived from von Willebrand clotting factor, and expressed in Escherichia coli and in mammalian cells. The chimeric env protein bound tightly to collagen, and virions bearing this collagen-binding env protein exhibited viral titers approaching those of virions expressing wild-type (WT) env protein. The chimeric virions were concentrated on collagen matrices, and they retained their infectivity under conditions in which virions bearing WT env protein were washed away. Targeted delivery of the chimeric env protein to injured mouse aorta and selective binding of the collagen-targeted virions to injured rabbit artery were observed. In comparative studies, vascular smooth muscle cell transduction was demonstrated in catheter-injured carotid arteries following infusion of collagen-targeted virions but not of virions bearing WT env protein. Taken together, these observations demonstrate the ability of collagen-targeted virions to localize gene delivery to sites of vascular injury.

3T3 Cells↗
PubMed1997

Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice.

Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.

Animals↗
PubMed1997

Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-beta1-von Willebrand's factor fusion protein for retrovirus-mediated delivery of coagulation factor IX.

Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes, including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells, which are capable of self-renewal and undergo expansion in the presence of transforming growth factor-beta1 (TGF-beta1). The present study was designed to test the concept that mesenchymal progenitor cells could be selected and expanded by virtue of their intrinsic physiologic responses to TGF-beta1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-beta1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-beta1-vWF). Histologic examination of TGF-beta1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. The TGF-beta-responsive cells were expanded to form stromal/fibroblastic colonies by serum reconstitution, and further to form osteogenic colonies upon supplementation with osteoinductive factors. In comparative studies, both marrow-derived progenitor cells and mature stromal cells were transduced with a retroviral vector bearing a human factor IX construct. Both the transduced progenitor cells and mature stromal cells expressed the factor IX transgene at levels comparable to those reported for human fibroblasts. Transplantation of murine progenitor cells bearing the human factor IX vector into syngeneic B6CBA mice resulted in detectable circulating levels of the human factor IX antigen. Taken together, these data demonstrate a novel physiologic approach for the selection of mesenchymal precursor cells followed by mitotic expansion, transduction, and transplantation of these progenitor cells with retroviral vectors bearing therapeutic genes.

Animals↗
PubMed1997

Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation.

BACKGROUND: The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation. METHODS AND RESULTS: In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with down-regulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector-transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation. CONCLUSIONS: These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.

Animals↗
PubMed1997

Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay.

A single chain antibody (scFv) was constructed from a hybridoma expressing the anti-CD34 monoclonal antibody My10. The scFv was expressed in the mouse fibroblast cell line NIH 3T3, and purified from culture supernatant via an epitope tag fused to the C-terminus of the protein. The scFv equilibrium dissociation constant (KD) was determined to be 2.4 X 10(-7) M using a fluorescence based flow cytometry assay involving recognition of the epitope tag, and bound approximately 24-fold less avidly to CD34 expressing KG-1a cells than the native antibody My10. This novel and previously unreported method for determining antibody binding affinity offers several advantages over alternative methods. It is rapid and simple, and unlike methods that directly label the antibody, it involves no covalent modifications of antibody variable domain residues that could potentially interfere with antigen binding. The KD for the anti-CD33 antibody HuG1 (Caron et al. (1992) The biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res. 52, 6761-6767) was determined as well. The close agreement of this value and the previously reported value, determined by a radioligand competition method, validates the use of this assay for antibody affinity determination. We discuss various potential applications for this anti-CD34 scFv.

3T3 Cells↗
PubMed1997

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer.

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3') was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer.

Animals↗
PubMed1997

Retrovirus-mediated transfer of the suicide gene into retinal pigment epithelial cells in vitro.

PURPOSE: Human retinal pigment epithelial (HRPE) cells are a major cell component in proliferative vitreoretinopathy (PVR) membranes. We investigated the feasibility of killing HRPE cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, also known as the suicide gene, into HRPE cells followed by ganciclovir treatment, and to study the so-called bystander effect. Such a treatment plan might serve as a possible therapy for PVR. METHODS: Transduction efficiency was determined using retroviral vectors encoding the beta-galactosidase reporter gene. To evaluate the efficacy of suicide gene therapy. HRPE cells were transduced with retroviral vectors encoding the HSV-tk gene (G1TkSvNa), with empty vectors or without vectors, and were treated with 5 micrograms/ml ganciclovir. Sensitivity of HSV-tk positive HRPE cells to various concentrations of ganciclovir was evaluated. To demonstrate the bystander effect, HSV-tk positive cells were cultured with HSV-tk negative cells at varying proportions. RESULTS: Transduction efficiency in vitro was 15.1 +/- 4.8%. Cell growth was significantly inhibited after transduction with G1TkSvNa followed by ganciclovir treatment (P < 0.01). Ganciclovir showed dose- and time-dependent cytotoxicity only on HSV-tk positive cells. The concentration that resulted in 50% inhibited was 0.1 micrograms/ml. In terms of the bystander effect, after ganciclovir treatment the viability of co-cultured cells decreased with increasing populations of HSV-tk positive cells. CONCLUSIONS: HRPE cells were successfully transduced with the HSV-tk gene via retroviral vectors and displayed a strong bystander effect after treatment with ganciclovir. These results suggest that retrovirus-mediated suicide gene therapy might be a feasible treatment strategy for PVR.

Antiviral Agents↗
PubMed1997

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus.

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.

3T3 Cells↗
PubMed1997

Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein.

The cytoplasmic tail of the immature Moloney murine leukemia virus (MoMuLV) envelope protein is approximately 32 amino acids long. During viral maturation, the viral protease cleaves this tail to release a 16-amino-acid R peptide, thereby rendering the envelope protein fusion competent. A series of truncations, deletions, and amino acid substitutions were constructed in this cytoplasmic tail to examine its role in fusion and viral transduction. Sequential truncation of the cytoplasmic tail revealed that removal of as few as 11 amino acids resulted in significant fusion when the envelope protein was expressed in NIH 3T3 cells, similar to that seen following expression of an R-less envelope (truncation of 16 amino acids). Further truncation of the cytoplasmic tail beyond the R-peptide cleavage site toward the membrane-spanning region had no additional effect on the level of fusion observed. In contrast, some deletions and nonconservative amino acid substitutions in the membrane-proximal region of the cytoplasmic tail (residues L602 to F605) reduced the amount of fusion observed in XC cell cocultivation assays, suggesting that this region influences the fusogenicity of full-length envelope protein. Expression of the mutant envelope proteins in a retroviral vector system revealed that decreased envelope-mediated cell-cell fusion correlated with a decrease in infectivity of the resulting virions. Additionally, some mutant envelope proteins which were capable of mediating cell-cell fusion were not efficiently incorporated into retroviral particles, resulting in defective virions. The cytoplasmic tail of MoMuLV envelope protein therefore influences both the fusogenicity of the envelope protein and its incorporation into virions.

3T3 Cells↗
PubMed1997

Functional interactions between monomers of the retroviral envelope protein complex.

Retroviral vectors have been widely used in human gene therapy protocols. Entry into target cells is directed by the retroviral envelope protein, with receptor binding and postbinding fusion functions contributed mainly by the SU and TM subunits, respectively. We have generated mutants of the Moloney murine leukemia virus (MoMuLV) envelope protein with mutations in both the receptor binding domain of SU and throughout the TM subunit that are functionally inactive when expressed individually. However, the coexpression of these two classes of mutants partially restores envelope protein function and allows transduction. Several lines of evidence indicate that this complementation occurs in trans within envelope protein heterooligomers. The finding that the binding and postbinding functions of a retroviral envelope protein can be contributed by two different monomers should assist in the engineering of envelope proteins for tissue-specific gene delivery.

3T3 Cells↗
PubMed1997

A conditional self-inactivating retrovirus vector that uses a tetracycline-responsive expression system.

We developed a novel conditional self-inactivating (C-SIN) vector, TL-SN, by replacement of the enhancer-promoter of the 3' long terminal repeat of Moloney murine leukemia virus with a synthetic tetracycline operator-cytomegalovirus promoter (tetP) from the tetracycline-responsive expression system (TRES). The other component of the TRES, a chimeric transactivator (tTA), was stably incorporated into PA317 amphotropic packaging cells, thus generating the packaging cell line PA317-tTA. C-SIN amphotropic G418-resistant virus particles were generated with a titer of 2 x 10(5) CFU/ml within 2 days of transinfection of PA317-tTA cells with TL-SN ecotropic virus particles. This titer was approximately 2 log units higher than that obtained by transinfection of parental PA317 cells and was due to the high level of viral transcripts originating from the tetP promoter at the 5' end of the transduced vector in the presence of tTA. Our C-SIN vector has the potential for use in human gene therapy since it incorporates the advantages of previous SIN vectors in having weak tetP promoter activity (in the absence of tTA in target cells) while at the same time achieving high viral titers with PA317-tTA packaging cells.

3T3 Cells↗