Effect of a caspase inhibitor, zVADfmk, on the inhibition of breast cancer cells by herpes simplex virus type 1.

The oncolytic effects of herpes simplex virus-1 (HSV-1) are limited, possibly because of premature death of infected cells by apoptosis, which limits the amount of progeny virus that is produced. It has been proposed that inhibition of apoptosis in infected tumor cells would allow increased viral persistence, replication and therapeutic effect. To test this hypothesis, we infected monocyte chemoattractant factor-7 (MCF-7) and MDA-MB-231 breast cancer cells with HSV-1 strain 17(+) and 17Δγ34.5 in the presence or absence of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk), a pan-caspase inhibitor. At low doses of HSV-1 strain 17(+) and 17Δγ34.5, the growth of MCF-7 cells was reduced to 37% or 42%, respectively, of uninfected cells. However, when cells were infected in the presence of zVADfmk, cell growth was further reduced to 24 and 33%. Similar results were seen in MDA-MB-231 cells. Cells treated with zVADfmk contained roughly 10 times more infectious viral particles than cells infected without zVADfmk, as shown by both plaque-forming and quantitative polymerase chain reaction assays. To model the situation within an infected tumor, supernatant fluids were collected from infected and non-infected cell cultures and then passed to non-infected cells. In the presence of zVADfmk, the cell growth inhibitory effect became stronger with repeated passages and was attributed to viral replication, because it could be prevented by anti-HSV antibody. These results suggest that caspases represent a novel target for drugs that increase the therapeutic efficacy of oncolytic herpes viruses against breast cancer.

Development of an oncolytic herpes simplex virus using a tumor-specific HIF-responsive promoter.

We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, infected cell polypeptide 4 (ICP4), was placed under the regulation of an HIF-responsive promoter and then introduced into the thymidine kinase locus (U(L)23) of HSV d120, which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV was isolated and their derivation from d120 was verified by expression of a truncated, non-functional form of ICP4 protein. Disruption of the U(L)23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported-reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.

An oncolytic mutant of herpes simplex virus type-1 in which replication is governed by a promoter/enhancer of human papillomavirus type-16.

Although herpes simplex virus type-1 (HSV-1) can be used as an oncolytic virus it has the undesirable side effect of neurotoxicity. To create a virus with improved specificity for oral cancer we used a fragment of human papillomavirus type-16, which is frequently found in oral and cervical cancers, but not elsewhere. The upstream regulatory region, URR16, was shown to have a high level of transcriptional activity in three of four oral cancer cell lines but low activity in three cell lines derived from brain cancers. URR16 was therefore placed in HSV-1, replacing the promoter of the essential gene ICP4, and the resulting virus was named HSPV-1. When cells were infected with HSPV-1, ICP4 was expressed at levels that were not associated with the level of activity of URR16. The virus replicated in each type of cell to a final titer that showed a correlation with the level of expression of ICP4, but with no correlation to either the tumor of origin or the presence of HPV sequences in the cells. To find if some function of HSV-1 was affecting the activity of URR16, oral cancer cells were transfected with a URR-reporter construct and were then infected with virus. This induced transcription, which was attributed to immediate-early viral genes other than ICP4. A promoter/enhancer from a papillomavirus therefore has the potential to regulate the functions of an oncolytic strain of HSV-1, and is affected by functions of both the host cell and of HSV-1 itself.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Transduction of normal and malignant oral epithelium by particle bombardment.

Although genetic approaches to the treatment and prevention of oral cancer are being developed, there are no suitable methods of transduction of the oral mucosa or early cancers. We therefore tested the technique of particle bombardment for its ability to transduce oral cancer cells in vitro and normal epithelium of the hamster cheek pouch in vivo. A gene gun was used to transfer a plasmid that encoded a marker/suicide fusion gene, beta-galactosidase-thymidine kinase (GAL-TEK), under control of a CMV promoter. For comparison we used the method of lipofection and an adenovirus vector. Particle bombardment transduced up to 13% of cells in culture, resulting in a 24.3% reduction in growth in the presence of ganciclovir. The efficiency of transduction was similar to that of lipofection but was much less than that of the adenovirus vector, which transduced 54% of cells and completely inhibited their growth in the presence of ganciclovir. Transduction of the hamster cheek pouch by particle bombardment produced expression of beta-galactosidase as judged by macroscopic staining, for up to 5 days. However, histological examination showed that the transduced cells were rare and superficial, and that administration of systemic ganciclovir did not lead to any changes in the tissue. Improvements in efficiency are necessary before the gene gun can be used in the management of oral cancer.

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.

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.

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.

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.