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J A Roth

Publications and source records attributed to J A Roth.

623 records · Page 35Linked to original sources
PubMedDate not supplied

p53 involvement in activation of the cytokeratin 8 gene in tumor cell lines.

We show that the expression of the human cytokeratin 8 (CK8) gene is regulated by wild-type p53. DNA sequence data indicate that the 5' untranslated region of the CK8 gene contains a putative p53-like binding site. In this study we focused on the effect of the p53 protein on the regulation and expression of the CK8 gene. Cotransfection of the H358 p53-negative human lung cancer cell line with a CK8 promoter CAT expression vector and a plasmid expressing the wildtype p53 indicated that p53 induces CK8 expression. A transient assay in which a p53-negative cell line was cotransfected with a CK8 promoter CAT expression vector and a plasmid expressing wildtype or mutant p53 indicated that only wildtype p53 induces the CK8 promoter. Deletion of the putative p53-binding site from the CK8 promoter or introduction of mutations in the p53-binding sequences abolished the wild-type p53-mediated transactivation of CK8. A gel-retardation assay was used to measure DNA binding by the wild-type p53 protein. A 24-bp oligonucleotide corresponding to the putative p53 binding site was used for this assay. The wild-type p53 protein bound weakly to this DNA sequence but much more strongly when three tandem repeat of the binding sequences was used. These studies suggest that the CK8 gene is a downstream target whose expression is regulated by wild-type p53.

Base Sequence↗
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Functional inactivation of p53 by antisense RNA induces invasive ability of lung carcinoma cells and downregulates cytokeratin synthesis.

Previously we had identified a p53 DNA-binding motif in the 5' region of the CK8 gene This finding led us to study the role of p53 protein in the regulation of CK8 gene expression and its role in tumorigenesis. Human lung cancer cell lines stably transfected with antisense p53 cDNA that expressed little p53 protein were analyzed. CK8 mRNA and the protein expression in these p53 antisense clones were very low as revealed by northern and western blot analysis. Immunofluorescence studies indicated that the cytokeratin networks around the nuclei of these cells collapsed; although some staining was observed around the nuclei. Antisense clones were highly invasive on in vitro matrigel assay. Scanning electron microscopy of the surface topography of these antisense clones revealed a large number of microvilli on their surfaces, a phenotype characteristic of tumor cell invasion. These findings suggest that functional inactivation p53 protein could be an important step in tumor progression.

Blotting, Northern↗
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Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor.

We have developed a targeted DNA delivery system for the treatment of lung cancer by gene therapy. This system uses epidermal growth factor (EGF), which has receptors that are overexpressed on lung cancer cells, and couples receptor-mediated endocytosis with an endosomal lysis agent for targeted gene delivery. Recombinant human EGF was chemically modified to allow for its attachment to DNA through the use of the poly-cation poly-L-lysine (PLL). The EGF/PLL conjugate was able to bind DNA and when incubated with several different lung cancer cell lines, high levels of gene expression resulted when uptake was performed in the presence of replication-defective adenovirus, which was used only as an endosomal lysis agent. When the adenovirus was coupled directly to the EGF/DNA complex, the levels of gene expression were up to fourfold higher. Depending on the lung cancer cell line and the type of complex used, approximately 14% to 99% of the cells showed DNA uptake, whereas a colon adenocarcinoma cell line that does not express the EGF receptor at the cell surface showed little or no DNA uptake. As a result, the EGF/DNA complex can deliver therapeutic genes to lung cancer cells by targeting to specific receptors.

Adenoviridae↗
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High levels of gene transduction in human lung tumors following intralesional injection of recombinant adenovirus.

To effect gene transfer into large solid malignancies for the purpose of clinical application, new treatment strategies using intralesional administration of adenovirus were studied. Replication-deficient adenovirus Ad5LacZ, containing the Escherichia coli beta-galactosidase (beta-gal) gene (LacZ), was injected directly into 1-cm x 1-cm subcutaneous xenograph tumors of human large cell lung cancers (H460 and H1299). Each tumor received a single injection or three injections of purified virus, diluted in 200 microL of phosphate-buffered saline. The tumors were harvested 3 days after the last injection, serially sectioned, and stained with X-gal. The cells expressing beta-gal were counted by using digital image analysis and the percentage of tumor cells transduced was calculated. After a single viral injection of 1 x 10(9) PFU, 5 x 10(9) PFU, or 1 x 10(10) PFU solid tumor transduction increased significantly with dose escalation. At a dose of 1 x 10(10) PFU, transduction of the H1299 and H460 tumors was 80.2% and 46.7%, respectively. Dividing the viral dose into three injections given on alternating days had no significant effect on viral transduction. These data demonstrate that a large portion of an established human lung cancer cell line tumor undergoes gene transduction after a single intralesional injection of recombinant adenovirus.

Adenoviridae↗
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Growth inhibitory effect of anti-K-ras adenovirus on lung cancer cells.

An adenoviral vector carrying a 2-Kb fragment of the K-ras proto-oncogene inserted in antisense orientation with respect to the cytomegalovirus promoter was constructed and used to infect H460a lung cancer cells (codon 61 K-ras mutation). The gene was efficiently transferred, and a high level of expression of antisense K-ras was achieved. At a multiplicity of infection to achieve 65% transduction of cells, the expression of K-ras protein was reduced by 70% in the lung cancer cell line H460a as compared with cells infected with control vectors or noninfected cells. This reduction produced a 47% inhibition of monolayer growth and a 90% inhibition of colony formation. At a similar level of transduction in the cell line H358 (codon 12 K-ras mutation), a 59% inhibition of monolayer growth compared with control vectors occurred; however the inhibition of H322 cells (wild-type k-ras) growth was no different than control vector infected cells. These data suggest that the adenoviral K-ras H322a antisense vector may have therapeutic potential in tumors in which K-ras is mutated.

Adenoviridae↗
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Inhibition of esophageal cancer proliferation by adenovirally mediated delivery of p16INK4.

We have been investigating the molecular basis of esophageal carcinogenesis in an attempt to develop gene therapy of established esophageal cancers and premalignant esophageal lesions. In this report we demonstrate the efficacy of adenovirally mediated p16 gene replacement in esophageal squamous cell cancers. Adenoviral p16 efficiently transduced epidermoid cancer cells containing p16 mutations, and expression of exogenous p16 in these cells correlated with G1 cell cycle arrest and profound growth inhibition. No effects were observed in esophageal adenocarcinoma cells due to extremely poor transduction of these cells by adenovirus. These results may have implications regarding the design of clinical trials using p16 gene replacement strategies for intervention in esophageal cancers.

Adenocarcinoma↗
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In vivo adenovirus-mediated p53 tumor suppressor gene therapy for colorectal cancer.

BACKGROUND: The p53 tumor suppressor gene is altered in up to 70% of colorectal cancers. MATERIALS AND METHODS: We infected the colorectal cancer cell lines SW620 and KM12L4, in which p53 is mutated, with the replication-defective adenovirus Ad5/CMV/p53 to evaluate the effects of adenovirus-mediated wild-type p53 gene transfer. Gene transduction was measured by cytochemical staining of cells infected with the Ad5/CMV/beta-gal virus and expression of the wildtype p53 protein in these cells was demonstrated by immunoblotting. RESULTS: Significant suppression of in vitro cell proliferation and induction of apoptosis (as measured by TUNEL assay labeling) were observed following Ad5/CMV/p53 infection. More importantly, similar effects were observed in vivo in an established nude mouse subcutaneous tumor model; significant suppression of tumor growth (60%-70%) and induction of apoptosis were observed following intratumoral injections of Ad5/CMV/p53. CONCLUSION: This form of therapy may provide a novel approach to colorectal cancer.

Adenoviridae↗
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Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex.

Current viral delivery systems suffer from disadvantages that may limit the rate at which therapeutic gene expressing constructs can be tested both in vitro and in vivo. In this study, our focus was to develop a simple gene delivery system for the rapid and reproducible testing of therapeutic genes in cancer cells both in vitro and in vivo. We report here that a delivery system based on using a conjugated adenovirus in complex from with a DNA plasmid can be used for not only delivering genes in vitro but also for efficient and reproducible delivery in vivo. Replication defective adenoviral particles were chemically modified by covalent attachment of poly-L-lysine (PLL) to the viral capsid, allowing for direct interaction with DNA. The adenovirus/PLL conjugate (Adv/PLL) was used to deliver the plasmid pCMV/beta-gal to several different cancer cell lines (i.e., lung, cervical) in vitro and resulted in transduction efficiencies as high as 52% as determined by histochemical staining. On direct intralesional injection of the Adv/PLL/DNA complex into subcutaneous tumors, transduction efficiencies greater than 35% could also be achieved. As a result, this system provides a simple method for delivering and testing therapeutic genes in cells both in vitro and in vivo, prior to the further development of gene therapy vectors for both malignant and benign disease.

Adenoviridae↗
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Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex.

An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312, allowing for coupling with plasmid DNA by an ionic interaction. We have previously demonstrated that this adenovirus/DNA complex can efficiently transduce malignant cells with a plasmid expressing the beta-galactosidase gene both in vitro and in vivo. In this report, we show that this system can deliver a therapeutic gene that encodes for the tumor suppressor protein p53 to lung cancer cells, both in vitro and in vivo, leading to significant biological effects. Transfection of the p53-negative human lung cancer cell line H1299 with the adenovirus/DNA complex carrying a plasmid expressing the p53 gene resulted in high levels of p53 protein and induction of apoptosis. Injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment. Three and six injections of the complex carrying the p53 gene into H1299 subcutaneous tumor nodules led to significant dose-related tumor growth suppression 18 days after the first injection compared with control-treated tumors. This adenovirus/DNA complex, therefore, is capable of efficiently delivering the p53 gene into malignant cells in vitro and in vivo and now provides a general gene delivery vector that is simple to construct and capable of testing therapeutic genes in malignant cells.

Adenoviridae↗
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Restoration of tumour suppressor gene expression for cancer.

Gene therapy has the potential to provide cancer treatments based on novel mechanisms of action with potentially low toxicities. This therapy may provide more effective control of loco-regional recurrence in diseases such as non-small cell lung cancer (NSCLC), as well as systemic control of micrometastases. Despite current limitations, retroviral and adenoviral vectors can in certain circumstances provide an effective means of delivering therapeutic genes to tumour cells. Although multiple genes are involved in the process of carcinogenesis, mutations of the p53 gene are the most frequent abnormality identified in human tumours. Pre-clinical studies both in vitro and in vivo have shown that restoration of p53 function can induce apoptosis in cancer cells. Phase I clinical trials now show that p53 gene replacement therapy is feasible and safe using both retroviral and adenoviral vectors, and that it induces tumour regression in patients with advanced NSCLC and recurrent head and neck cancer. Other pre-clinical studies indicate that gene therapy may have useful synergy with cytotoxic and radiation therapy. This paper describes the different gene therapy strategies under investigation and the pre-clinical data that provides a rationale for the gene replacement approach, reviews clinical trial data and presents novel ideas for improving current vectors and gene delivery to tumours.

Adenoviridae↗