Search PubMed⌕ Search

Biomedical subjects

E J Shillitoe

Publications and source records attributed to E J Shillitoe.

At least 19 recordsLinked to original sources
PubMed2006

Papillomaviruses as targets for cancer gene therapy.

The human papillomaviruses (HPVs) are a diverse group of infectious agents, some of which are a causative agent of human cancers. Cervical cancer and oral cancer are closely associated with specific types of HPV, and the tumors grow only if there is continual expression of the viral E6 and E7 genes. Evidence from in vitro studies shows that when expression of these genes is inhibited by gene therapy approaches such as antisense RNA, ribozymes, or siRNA, the transformed phenotype of the cells is lost. Although it seems possible that clinical applications of this approach could help in the management of cervical and oral cancers there have been no clinical trials of gene therapy for HPV-associated cancers. Since the basic information is now available, a shift to translational research would be greatly welcomed.

Cell Transformation, Neoplastic↗
PubMed2003

Gene discovery in oral squamous cell carcinoma through the Head and Neck Cancer Genome Anatomy Project: confirmation by microarray analysis.

The near completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells, including those derived from squamous carcinomas of the head and neck (HNSCC). As part of these efforts, representative cDNA libraries from patient sets, comprising of normal and malignant squamous epithelium, were generated and contributed to the Head and Neck Cancer Genome Anatomy Project (HN-CGAP). Initial analysis of the sequence information indicated the existence of many novel genes in these libraries [Oral Oncol 36 (2000) 474]. In this study, we surveyed the available sequence information using bioinformatic tools and identified a number of known genes that were differentially expressed in normal and malignant epithelium. Furthermore, this effort resulted in the identification of 168 novel genes. Comparison of these clones to the human genome identified clusters in loci that were not previously recognized as being altered in HNSCC. To begin addressing which of these novel genes are frequently expressed in HNSCC, their DNA was used to construct an oral-cancer-specific microarray, which was used to hybridize alpha-(33)P dCTP labeled cDNA derived from five HNSCC patient sets. Initial assessment demonstrated 10 clones to be highly expressed (>2-fold) in the normal squamous epithelium, while 14 were highly represented in the malignant counterpart, in three of the five patient sets, thus suggesting that a subset of these newly discovered transcripts might be highly expressed in this tumor type. These efforts, together with other multi-institutional genomic and proteomic initiatives are expected to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention in this disease.

Aged↗
PubMed2003

Clinical and pathological features of the murine AT-84 orthotopic model of oral cancer.

OBJECTIVE: The murine AT-84 orthotopic model of oral cancer was assessed to find how similar it is to human oral cancer. This was done because testing of new treatments for oral cancer requires the use of a realistic animal model. MATERIALS AND METHODS: Tumors were induced at orthotopic (oral) or heterotopic (flank) sites and their features were compared. The therapeutic effects of surgery, 5-fluorouracil and cisplatin were measured on the orthotopic tumors. RESULTS: Tumors had the histological appearance of a sarcomatoid carcinoma, invading locally and causing weight loss and death. The oral tumors metastasized to the lungs frequently. Tumors could be treated with some success by surgery or chemotherapy, but generally recurred. CONCLUSIONS: The similarities to human oral cancer suggest that the model will be very useful in the evaluation of experimental therapies for oral cancer.

Animals↗
PubMed2002

Effect of herpes simplex virus type-1 on growth of oral cancer in an immunocompetent, orthotopic mouse model.

Herpes simplex virus type-1 has been proposed as an agent for the treatment of oral cancer. Experiments were designed to test its effectiveness in an animal model that had a high level of similarity to the human disease. The mouse oral cancer cell line, AT-84, was implanted at an orthotopic site--the base of the tongue--into syngeneic, immunocompetent C3H mice. As expected, tumors invaded the musculature of the tongue, eroded the mandible, and metastasized to the lungs. To obtain a suitable strain of HSV-1 for therapy we screened 17 fresh clinical isolates and selected one that grew to a high titer in vitro. The mouse tumors were then treated by injection of HSV-1 at a titer of 10(9) plaque-forming units/milliliter. To prolong the anti-tumor effect some mice were also given cyclophosphamide, hydrocortisone, or a second injection of virus. To find the importance of bystander killing of tumor cells, some mice were given virus with ganciclovir. A reduction in tumor volume for a limited period was seen after treatment by HSV-1, and was increased by a second injection of virus or by the administration of cyclophosphamide. Ganciclovir negated the anti-tumor effect. Virus was detectable in the tumors for up to 7 days, and loss of virus coincided with the time at which growth of tumors resumed. The mortality of the mice varied up to around 50%. It appears that (1) a non-attenuated strain of HSV-1 can inhibit the growth of an aggressive malignant oral tumor, but only to a limited extent and (2) inhibition depends on the ability of the virus to replicate in the tumor. It is suggested that mutations in the virus will be necessary to prevent mortality, but must be designed carefully so as not to reduce the virulence of the virus.

Animals↗
PubMed2000

Strength and specificity of different gene promoters in oral cancer cells.

Gene therapy of oral cancer will require expression of genes by promoters that are both powerful and relatively tumor specific. We compared the level of expression of a reporter gene from promoters of human cytomegalovirus (CMV), SV40 virus, mouse mammary tumor virus (MMTV), human papillomaviruses (HPV) types 16 and 18, and the human multi-drug-resistance gene (mdr1), in several lines of oral cancer cells. In the oral cancer cell line 686LN the rank order of expression levels was: CMV > SV40 > HPV > mdr1 > MMTV. Unlike in previous reports the mdr1 promoter was no more active in two cancer cell lines with mutations in the p53 gene than in two other lines with wild-type p53, and its expression level could not be increased by either doxorubicin or taxol. On the other hand, expression from the MMTV promoter was increased over 10-fold by the presence of 1 microM dexamethasone. Thus, by an appropriate choice of promoter and inducer a wide variety of expression levels, over a 3-log range, could be attained in 686LN cells. The oral cancer-specificity of each promoter was judged by comparing expression in the neuroblastoma line IMR32. The most specific promoters were those from papillomaviruses, which were up to 45 times more active in the oral cancer cells, and the least specific was the CMV promoter. In order to find if an HPV-derived promoter was sufficient to produce expression of a suicide phenotype the 686 promoter was cloned adjacent to the thymidine kinase gene of herpes simplex and the construct was expressed from an adenovirus vector. The vector reduced the growth of 686LN cells over a 5-day period by up to 32% when optimal concentrations of virus and ganciclovir were used. These data will be valuable in the design of new constructs for gene therapy of oral cancer.

Gene Expression↗
PubMed2000

Establishment of a human polyclonal oral epithelial cell line.

OBJECTIVE: To develop a human oral epithelial cell line to constitute a continuous source of cells readily available for human oral epithelial cell research. STUDY DESIGN: Oral epithelial cells from a 30-week gestational, stillborn male fetus were grown in serum-free medium and transfected by lipid-mediation with the shuttle vector plasmid, pZ189, containing the T-antigen coding region and replication origin from the SV40 virus. RESULTS: Resulting cultures produced foci of rapidly multiplying cells that failed to senesce, in contrast to controls. The transformed culture, designated GMSM-K, was polyclonal. The original culture possessed a normal human male karyotype, and the transformed line was largely hypotetraploid. Multiple clones, isolated from soft agar studies and low density plating, showed decreased doubling times. Electron microscopy and immunohistochemistry confirmed an epithelial phenotype. Cells did not generate tumors in nude mice. CONCLUSION: Few human epithelial cell lines are available to investigators and most are tumor-derived. The nontumor-derived GMSM-K line has value as a resource for human oral epithelial cell research.

Animals↗
PubMed2000

Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector.

Human oral cancer cells may have any of several genetic changes, but the role of the bcl-2 oncogene is relatively unexplored. To find out if this gene plays a significant role and whether it could act as a target for gene therapy of oral cancer, we have examined the effects of an anti-bcl-2 ribozyme on the phenotype of oral cancer cells. A hammer-head ribozyme was designed to cleave the bcl-2 transcript after nucleotide 279 and was confirmed to be effective against a synthetic bcl-2 transcript. A gene encoding the ribozyme was cloned into an adenovirus vector and transferred to the human oral cancer cell lines 686LN, 1483, and Tu183. Over a 6-day period, the growth of each cancer cell line was reduced, whereas growth of the fibroblast cell line FS7 was less inhibited. Inhibition of the oral cancer cells could be attributed to apoptosis, as indicated by the detection of histone-associated DNA fragments in an immunoassay. Northern blots showed no detectable reduction in the level of bcl-2 mRNA of Tu183 cells, but Western blots showed a reduction of Bcl-2 protein at 24 h after infection with the ribozyme-expressing adenovirus vector. The results imply that (a) expression of the bcl-2 oncogene is necessary for the survival of oral cancer cells, (b) the bcl-2 gene transcript presents a target for gene therapy by ribozymes, and (c) an adenovirus vector is a suitable method for transfection of the ribozyme-expressing gene.

Adenoviridae↗
PubMed2000

Genome-wide analysis of oral cancer--early results from the Cancer Genome Anatomy Project.

The Cancer Genome Anatomy Project (CGAP) is a large cooperative effort sponsored by the US National Institutes of Health designed to find, catalog and annotate genes that are expressed during cancer development. In the past 2 years, the CGAP has sequenced over 700,000 clones from approximately 140 cDNA libraries, resulting in the identification of over 30,000 new human genes. As a first step in applying this project to oral cancer we entered four cell lines--two from oral cancer, one from primary oral keratinocytes, and one from oral keratinocytes which had been immortalized by human papillomavirus. Libraries of cDNA were made and sequenced and the data were deposited in GenBank. The expressed genes were then identified where possible. The cell lines, and the total number of expressed genes that were cloned from each were: HN3 (oral cancer), 263 genes; HN4 (oral cancer), 550 genes; HN5 (primary keratinocytes), 237 genes; HN6 (immortalized keratinocytes), 408 genes. The total number of different genes that were found was 1160. A total of 38 new genes, of unknown function, were discovered. The data presented here represent a beginning of the application of the CGAP technology to oral cancer. Even though the data are still quite incomplete, they already represent a large quantity of new information and clones of potential utility to the oral cancer community, and provide a glimpse of the data sets to be forthcoming from the Project. It must therefore be expected that there will soon be a large expansion in the volume of data regarding the genetics of oral cancer. Those who study this disease must be prepared to develop new methods of analysis and storage for handling the oncoming volumes of information.

Clone Cells↗
PubMed1999

Effects of herpes simplex virus on human oral cancer cells, and potential use of mutant viruses in therapy of oral cancer.

The herpes simplex virus type-1 (HSV-1) might be useful in treatment of oral cancer because it is strongly cytolytic, and its natural target tissue is the source of oral squamous cell carcinomas. Use of a wild-type virus would be limited by its spread and neurotoxicity, but it might be possible to develop mutants whose range could be restricted to oral cancers. Thus we have investigated the effects of HSV-1 on human oral cancer cells and have used both wild-type virus and a mutant that lacks UL42--an essential gene of the virus. Growth of the oral cancer cell line 686LN was readily inhibited by wild-type HSV-1, with only 10(2) plaque forming units (pfu) per milliliter required for 50% inhibition. In contrast, the mutant HSV-1 required a titer of 10(6) pfu/ml for 50% inhibition of growth. The mutant virus did, however, inhibit cell growth through the activation of ganciclovir and thus might be able to amplify its cytotoxicity through a bystander effect. When wild-type HSV-1 was injected into 686LN cells which were growing as tumors in nude mice, the virus spread through the tumor. Treated tumors were smaller, of lower weight, and significantly more necrotic than either untreated tumors or tumors which had been treated with the mutant virus. The wild-type virus spread to the skin and nervous system of most animals causing zosteriform skin rash, neurological symptoms and death, while the mutant virus produced none of these side-effects. These results show that HSV-1 might be used to treat oral cancer if its replication could be limited to the tumor cells, and that controlled expression of the UL42 gene would be one way to obtain that limitation.

Animals↗
PubMed1999

Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo.

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAAr5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker gene and the HSV oris sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAAr5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G. C-to-T. A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.

Animals↗
PubMed1997

Mutations in the long control region of human papillomavirus DNA in oral cancer cells, and their functional consequences.

Oral cancers frequently contain DNA of human papillomavirus (HPV) type 16 or 18, but the functional significance of this is unclear. A role for HPV in the progression of oral cancer would be more plausible if the viral transforming genes were likely to be overexpressed in oral cancer cells. We therefore isolated and sequenced the long control region (LCR) of HPV-16 or HPV-18 from three oral cancer cell lines and two lines of HPV-16-immortalized oral keratinocytes, using PCR. The functional activity of each LCR was measured by cloning it into a luciferase expression vector, followed by transfection into both normal oral keratinocytes and oral cancer cells. For comparison, the LCRs of the wild-type HPV-16 and HPV-18 were studied. Several mutations were found in the LCRs isolated from oral cancer cells and HPV-immortalized oral epithelial cells. The promoter activity of the mutated LCRs was significantly higher than that of the equivalent wild-type LCRs in oral cancer cells that contained the same HPV type. These results imply that mutations in the LCR of HPVs in oral cancer could lead to increased expression of HPV-transforming proteins, which might contribute to the carcinogenic process.

Base Sequence↗
PubMed1997

Ribozymes. Their functions and strategies for their use.

The ability to alter genes in order to regulate their expression has become an undeniable reality. This can be performed in vitro and in cells, and the possibility of treating diseases and even preventing them now exists through such gene manipulation. A particularly intriguing form of manipulation that has been investigated for just over a decade is one that involves the use of ribozymes. These are short segments of RNA that form complementary base-pairing with mRNA. However, it is their enzymatic properties that set them apart from other antisense RNA molecules and allow them to cleave and destroy mRNA in a very specific manner. The ribozyme then dissociates from the cleaved substrate RNa, and repeatedly hybridizes to and cleaves additional substrate RNA molecules. Problems being addressed as this technology evolves involve optimization of ribozyme:substrate binding efficiencies and their effective transmission into cells. This article points out the origin of ribozymes, analyzes and summarizes the current strategies for designing ribozymes, and outlines a basic procedure for ribozyme development.

Animals↗
PubMed1997

The high risk human papillomaviruses and oral cancer: evidence for and against a causal relationship.

Oncogenic human papillomaviruses (HPVs) have been detected in oral squamous cell carcinoma (SCC). HPV16 is the most frequently detected HPV type in oral SCC and is present in up to 22% of cases, either alone or in combination with other HPV types. HPV18 is present in up to 14% of cases. HPV16 and HPV18 are present together in approximately 6% of cases. However, HPV16 and 18 are also detected in normal oral mucosae (10% and 11% of subjects, respectively). These data suggest that high risk HPV infection may be a co-factor in oral carcinogenesis and that latent HPV infection of the oral mucosa is common. A role for HPV infection in oral carcinogenesis is supported by the ability of high risk HPVs to immortalize oral keratinocytes in vitro. Immortalization may involve (i) deactivation of pre-formed tumor-suppressor proteins by viral oncoproteins, (ii) blocking of tumor-suppressor gene transcription as a result of HPV oncogene insertion or (iii) stimulation of cellular oncogene transcription by the upstream insertion of HPV-derived transcription activating sequences. Hence, infection of oral keratinocytes with high risk HPV may be involved in the pathogenesis of some oral SCCs although the evidence implicating HPV in oral carcinogenesis is, at present, mainly circumstantial.

Carcinoma, Squamous Cell↗
PubMed1996

Evaluation of topical gene therapy for head and neck squamous cell carcinoma in an organotypic model.

The organotypic (raft) culture system has been shown to be a useful model for examining the effects of biochemical manipulations on various epithelial cell types, using in vitro conditions that simulate the in vivo environment of the tissue of origin. To investigate this method as a model for topical gene therapy, we cultured the oral head and neck squamous cell carcinoma cell line TR146 on fibroblast-containing collagen gels at the air-medium interface and assessed the efficiency of transduction of a topically applied adenoviral vector containing beta-galactosidase cDNA. Diffuse expression of -galactosidase activity in multiple cell layers demonstrated effective penetration of the vector. Transduction efficiency and therapeutic activity of a replication-defective recombinant adenovirus containing wild-type p53 cDNA linked to a FLAG marker (AdCMV-p53-FLAG) were then assessed in TR146 organotypic cultures transduced by topical application. Twenty-four, 48, and 72 h after transduction, the cultures were harvested, and residual cell number and FLAG peptide expression were determined. The number of cells in p53 transduced cultures was significantly reduced in comparison to controls at all three time points (P < 0.001), which resulted from the induction of apoptosis as determined by in situ DNA end labeling. In addition, the FLAG peptide was expressed diffusely in the residual cells, further confirming effective transduction and expression of the exogenous gene products throughout multiple layers. We conclude that the organotypic culture is an effective in vitro model for assessing the efficacy of topically applied gene therapy on head and neck squamous carcinomas and premalignancies.

Adenoviridae↗
PubMed1995

The effect on cell phenotype of the mutagenic peptide of herpes simplex virus type-1.

The transforming region of the genome of herpes simplex virus type-1 (HSV-1) encodes a peptide that raises the mutation frequency of cells. To find the effect of this peptide on cell phenotype, three types of cells were transfected with a shuttle vector plasmid that expressed the peptide. When immortalised rat fibroblasts were transfected they rapidly became anchorage-independent with high efficiency, but were not tumorigenic in nude mice. When monkey kidney cells were transfected, five clonal cell lines were isolated, of which one became anchorage-independent but was not tumorigenic in nude mice. When human oral keratinocytes were transfected they did not become immortalised. The peptide therefore induced some of the features of transformation in different cell types, but did not induce a malignant phenotype in any cell. This suggests that interaction with co-factors would be necessary for the peptide to contribute to the development of oral cancer.

Amino Acid Sequence↗
PubMed1995

The antiviral spectrum of Listerine antiseptic.

The mechanism of activity and the antiviral spectrum of Listerine antiseptic have not been examined thoroughly. We therefore tested its effect on laboratory strains of herpes simplex type 1 and type 2 (enveloped DNA viruses), influenza A virus (enveloped RNA virus), rotavirus (nonenveloped RNA virus), and adenovirus type 5 (nonenveloped DNA virus). Each virus was mixed with an equal volume of Listerine for 30 seconds to 5 minutes, and the residual infectivity of the virus was assessed. An antiviral effect was defined as greater than 95% reduction of infectivity. Exposure to Listerine for 30 seconds had an antiviral effect against herpes simplex type-1 and type-2 (96.3% and 100% reduction in infectious virus, respectively) and influenza A (100% reduction). In contrast, rotavirus-induced plaque formation was reduced by 12.2% after 30 seconds of exposure to Listerine, whereas 5 minutes of exposure to Listerine resulted in a 21.5% increase in plaque formation. Exposure of adenovirus to Listerine had a minimal effect on the cytopathocity of the virus, with a 33.4% reduction in virus levels after 5 minutes. The antiviral activity of Listerine is thus not related to the viral genome but is probably directed to the viral envelope.

Adenoviridae↗