Edward J. Shillitoe

Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.

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.

Antisense-mediated inhibition of the bcl-2 gene induces apoptosis in human malignant glioma

BACKGROUND The bcl-2 protooncogene represses a number of cellular apoptotic pathways and is known to be expressed in increasing amounts in glial tumors of higher malignancy. We tested whether antisense oligonucleotides to the bcl-2 gene would affect glioma cell viability. METHODS Antisense oligonucleotides directed to the first six codons of the human bcl-2 gene, and nonsense oligonucleotides as a control, were transfected into malignant glioma cells. Two human Bcl-2 positive glioblastoma cell lines from our tumor bank (Jon52 and Roc) were both transfected in vitro with bcl-2 antisense (AS) and nonsense (NS) oligonucleotides at 1 microm and 5 microm concentrations for 5 and 24 hr. Cell viability was assessed at 2, 4, 5, and 7 days by using an MTT mitogenic assay and by cell counting via direct visualization using a hemocytometer. RESULTS There was up to a log-fold decrease in cell growth of the bcl-2 AS treated cells compared to the NS transfected cells for both Roc (p = 0.007 and p = 0.004) and Jon52 (p = 0.02 and p = 0.004) at 5 and 24 hr of transfection. There was as much as 50% cytotoxicity in both glioblastoma cell lines at 1 microm and 5 microm concentrations after 24 hr transfection with AS bcl-2 oligonucleotides (all p < 0.01). Western blot analysis demonstrated a decrease in the expression of the Bcl-2 protein in one cell line, whereas there was a statistically significant increase in the apoptotic index of both cell lines (p < 0.05 by chi square analysis). CONCLUSIONS Our results suggest that transfection of human glioma cells with antisense bcl-2 results in an increase in apoptotic death. This provides evidence that Bcl-2 plays a role in tumor progression of glioma by acting as an oncogene, and suggests that inhibition of the bcl-2 gene could have a therapeutic effect.

The role of viruses in squamous cell carcinoma of the oropharyngeal mucosa.

The development of squamous cell carcinomas of the oropharyngeal mucosa may involve many factors, including viruses. This review examines the evidence that viruses could be involved in the etiology of oral cancer, and shows that the evidence for a role of different viruses varies from very weak to very persuasive. Papillomaviruses are probably involved in the etiology of some carcinomas, particularly those of the oropharynx, and some herpes viruses may be involved as well. On the other hand some viruses can cause cancer in experimental situations but not in humans. Thus the importance of viruses in oral cancer is not always clear and must be evaluated with care. Those viruses that are associated with the disease provide targets for therapy and for diagnostic assays.

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.

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 (UL23) 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 UL23 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.

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.

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.

The mechanism of exogenous B7.1-enhanced IL-12-mediated complete regression of tumors by a single electroporation delivery

Electroporation‐based mono‐gene therapy has received great interest in recent years but coadministration of different therapeutic genes for treatment of tumors has not been well explored. We hypothesize that electroporation is capable of delivering multiple genes that induce an additive or synergistic antitumor effect. To test this hypothesis, we used mice that were bearing SCCVII or TRAMP tumors. Established tumors with a diameter of 4–5 mm were injected with control plasmid DNA or plasmid DNA encoding B7.1, IL‐12 or both via electroporation. Tumor regression, CTL activity and the level of B7.1, IL‐12 and Stat1 expression were determined in both wild‐type mice and in mice with a knock‐out of the Stat1 gene. Remarkably, a single coadministration of the plasmids that encoded IL‐12 and B7.1 eradicated tumors in 80% of mice. The therapeutic effect was associated with high levels of endogenous B7.1 expression, activity of cytotoxic lymphocytes, and activation of Stat1. Both exogenous B7.1 and IL‐12 were required for inducing a high level of Stat1 activation in tumors, which occurred through a mechanism that was independent of the host Stat1. Both stimulators were also required for inducing the strong cytotoxic lymphocyte activity and for increasing the level and extending the duration of endogenous B7.1 expression. We therefore propose a 2‐signal stimulation model to explain the synergistic effect of the coadministration of IL‐12 and B7.1 on the regression of the tumors.

The oral microflora in obesity and type-2 diabetes

Background : Type 2 diabetes mellitus (T2DM) is prevalent in people with obesity. It has been proposed that these conditions are related to specific features of the microflora of the mouth and lower gastrointestinal (GI) tract. Hyperglycemia often resolves quickly after Roux-en-Y gastric bypass (RYGB) but the role of the GI microflora cannot be examined easily because of reduced intestinal mobility. We propose that the study of microorganisms present in the mouth of patients undergoing RYGB will contribute to our understanding of the role of bacteria in the pathogenesis of T2DM. Objective : To conduct a feasibility study to examine differences in oral microbes in obese patients with and without T2DM and to determine whether it is feasible to measure changes after gastric bypass surgery. Methods : Individuals with morbid obesity (n=29), of whom 13 had T2DM, were studied. Oral rinses, stool samples, and blood samples were obtained before RYGB, and oral rinses and blood samples were obtained at 2 and 12 weeks postsurgery. Results : Prior to surgery, participants with T2DM had slightly higher total levels of oral bacteria than those without diabetes. Those with HbA1c > 6.5% had rather lower levels of Bifidobacteria in the mouth and stool. At 2 weeks post-RYGB, patients with T2DM were able to reduce or discontinue their hypoglycemic medications. Stool samples could not be obtained but oral rinses were readily available. The levels of oral Bifidobacteria had increased tenfold and levels of circulating endotoxin and tumor necrosis factor-alpha had decreased. Conclusions : The study of oral bacteria before and after RYGB is feasible and should be tested in larger patient populations to increase our understanding of the role of microorganisms in the pathogenesis of obesity and T2DM.