No-Hee Park - US grants

Recently some studies have shown an association of herpes simplex virus (HSV) in human oral malignancies, and implied a link between chemical carcinogens found in tobacco and smoked tar and HSV in cancer induction. Furthermore many in vitro investigations have demonstrated the oncogenic capacity of HSV to be increased by chemical carcinogens. However, no study to date has examined the combined action of a chemical carcinogen and HSV on the oral carcinogenesis of laboratory animals. The specific aim of this proposal will be to investigate the combined action of a chemical carcinogen, 9,10-dimethyl-1,2-benzathracene (DMBA) and HSV on the development of oral cancer in hamsters. Hamster buccal pouches will be inoculated with HSV-1 or HSV-2, and a repeated topical application of DMBA on the inoculation site will be made for 15 weeks. HSV inoculation will be repeated during this period. At the end of the period, the pouches will be analyzed for the presence of infectious HSV and histopathologic changes. In order to study the effect of DMBA on the reactivation of latent HSV, a latent HSV infection will be established in the trigeminal ganglia of hamsters by primary HSV infection in buccal pouches. DMBA will then be repeatedly applied to the primary inoculation site. The presence of reactivated HSV will be determined and quantitated by swabbing of oral mucosa and homogenization of the sensory ganglia. Furthermore, we will examine the effect of DMBA on the development of oral cancer in hamsters with latent HSV infection in the trigeminal ganglia. Finally we will determine the presence of viral DNA and RNA in the developed tumor by in situ hybridization techniques. We expect to obtain significant information on the combined role of HSV and a chemical carcinogen on the development of oral cancer. Our long-term objective is to understand the mechanism of cocarcinogenic effect of HSV and to prevent it.

Recent animal studies have suggested a possible link between cellular prostaglandins (PGs) and the reactivation of latent herpes simplex virus (HSV). However, no study to date has examined the direct action of PGs on latent HSV. The specific aim of this proposal will be to investigate the effects of PGs, cyclooxygenase inhibitors, and a lipoxygenase inhibitor on the in vitro and in vivo reactivations of latent HSV in the sensory ganglia of laboratory animals. We will inoculate the orofacial area of mice with HSV to establish a latent infection in the trigeminal ganglia. During latent period (4-8 weeks after viral inoculation), the ganglia will be excised and cultured in the presence of PGE2, PGF2, PGD2, specific cyclooxygenase inhibitors, e.g. indomethacin, flurbiprofen or ibuprofen, or a lipoxygenase inhibitor, norhydroguaiaretic acid (NDGA). The effect of PGs and enzyme inhibitors on the reactivation of latent HSV in vitro will be monitored by quantitating the amount of reactivated virus in ganglia. In order to study the action of PGs synthesis inhibitors and NDGA on the reactivation of latent HSV in vivo, rabbit corneas will be inoculated with HSV and latent viral infection will be established in the trigeminal ganglia. Following the establishment of latency, the latent virus will be manipulated to be reactivated by iontophoresis of epinephrine into the rabbit cornea. The effect of systemic PGs synthesis inhibitors and NDGA on the reactivation of latent virus will be studied by monitoring the viral shedding in tears and the determination of virus in ganglia. Through the above study we expect to obtain significant information on factors affecting HSV latency and reactivation.

Recently some studies have shown an association of herpes simplex virus (HSV) in human oral malignancies, and implied a link between chemical carcinogens found in tobacco and smoked tar and HSV in cancer induction. Furthermore many in vitro investigations have demonstrated the oncogenic capacity of HSV to be increased by chemical carcinogens. However, no study to date has examined the combined action of a chemical carcinogen and HSV on the oral carcinogenesis of laboratory animals. The specific aim of this proposal will be to investigate the combined action of a chemical carcinogen, 9,10-dimethyl-1,2-benzathracene (DMBA) and HSV on the development of oral cancer in hamsters. Hamster buccal pouches will be inoculated with HSV-1 or HSV-2, and a repeated topical application of DMBA on the inoculation site will be made for 15 weeks. HSV inoculation will be repeated during this period. At the end of the period, the pouches will be analyzed for the presence of infectious HSV and histopathologic changes. In order to study the effect of DMBA on the reactivation of latent HSV, a latent HSV infection will be established in the trigeminal ganglia of hamsters by primary HSV infection in buccal pouches. DMBA will then be repeatedly applied to the primary inoculation site. The presence of reactivated HSV will be determined and quantitated by swabbing of oral mucosa and homogenization of the sensory ganglia. Furthermore, we will examine the effect of DMBA on the development of oral cancer in hamsters with latent HSV infection in the trigeminal ganglia. Finally we will determine the presence of viral DNA and RNA in the developed tumor by in situ hybridization techniques. We expect to obtain significant information on the combined role of HSV and a chemical carcinogen on the development of oral cancer. Our long-term objective is to understand the mechanism of cocarcinogenic effect of HSV and to prevent it.

There is compelling evidence that carcinogenesis is a multistep process and multiple genetic lesions are necessary to develop cancer in human. Along the genetic lesions, the alteration of p53 protein (due to mutation of p53 gene or by infection of "high risk" human papillomaviruses [HPV], e.g., type 16 or 18 HPV) is the most frequently found genetic disorder in human cancers including oral cancer. A significant body of evidence supports p53 mutation as an early event in oral carcinogenesis. Our studies have also shown that human oral keratinocytes (HOK) containing negligible amount of wild-type (wt) p53 protein (because of HPV DNA integration) and HOK expressing mutant (mt) p53 protein are immortal, but not tumorigenic. These cells convert to tumorigenic cells when exposed to tobacco-carcinogens. whereas cells with a normal complement of p53 do not, indicating that p53 dysfunction appears to be an early event in oral carcinogenesis. Therefore, the dysfunction of p53 protein appears be an early event at least in oral carcinogenesis and also be necessary for subsequent genetic disorders of other genes to convert normal cells to tumor cells in the human oral cavity. Inasmuch as wt p53 protein plays a major role in the regulation of cell cycle arrest, we hypothesize that normal human oral keratinocytes containing wt p53 protein repair damaged DNA more efficiently than oral keratinocytes with defective p53 function. As demonstrated by many studies including our preliminary data, cells expressing wt p53 protein have the ability to establish transient delays in the progression of cell cycle when exposed to genotoxic agents, but cells with defective p53 function do not possess such ability. Since the transient arrest of the cell cycle progression is necessary to repair damaged DNA prior to replication of damaged DNA template and segregation of damaged chromosome, cells with defective p53 function may fail or have limited ability to repair the damaged DNA when exposed to genotoxic agents. In the proposed study, we will test the above hypothesis by determining the effect of major tobacco- carcinogens on (1) the progression of cell cycle, the expression of major growth arrest and DNA damage inducible genes (e.g., p53, WAF1/CIP1, and gadd45), and the activity of cyclin-dependent kinases (cdks); (2) the genotoxicity of host chromosomal DNA (DNA adducts and single strand DNA breaks); (3) the repair of damaged DNA; and (4) the mutation frequencies and spectrum in normal HOK expressing wt p53, HOK expressing HPV-16 or HPV-18 E6 protein, HOK expressing mutant p53 protein, and HPV-immortalized oral keratinocytes. These proposed studies should help us gain more insight into molecular mechanisms of tobacco-related oral carcinogenesis.

There is compelling evidence that carcinogenesis is a multistep process and multiple genetic lesions are necessary to develop cancer in human. Among the genetic lesions, the dysfunction of p53 protein (because of mutation of p53 gene or infection by "high risk" human papillomaviruses [HPV], e.g., type 16 or 18 HPV) is the most frequently found genetic disorder in human cancers including oral cancer. In spite of such frequent p53 dysfunction in oral cancer cells, alter p53 function (by transfection with HPV DNA or mutant p53 cDNA) alone is not sufficient for neoplastic conversion of normal human oral keratinocytes in vitro. Therefore, the dysfunction of p53 protein may be an early event at least in oral carcinogenesis and also be necessary for subsequent genetic disorders of other genes to convert normal cells to tumor cells in the human oral cavity. In fact, our recent studies show that human oral keratinocytes containing negligible amount of wild-type (wt) p53 protein (because of HPV transfection) convert to tumorigenic cells when exposed to tobacco-carcinogens, but the normal counterpart does not. Inasmuch as wild-type p53 protein plays a major role in the regulation of cell cycle arrest, we hypothesize that normal human oral keratinocytes containing wt p53 protein repair damaged DNA more efficiently than oral keratinocytes with defective p53 function (by mutation of p53 gene or by infection with "high risk" HPV). As demonstrated by many studies, cells expressing wt p53 protein have the ability to establish transient delays in the progression of cell cycle when exposed to genotoxic agents. However, cells with defective p53 protein do not possess such ability. Since the arrest of the cell cycle progression is assumed to be necessary for cells to repair damaged DNA prior to replication of damaged DNA template and segregation of damaged chromosome, cells with defective p53 function may fail to repair the damaged DNA when exposed to DNA damaging agents. In the proposed study, we will test the above hypothesis by (1) investigating the carcinogen-induced mutation frequencies: (2) determining the repair of damaged DNA: and (3) determining the effect of chemical carcinogens, alone or in combination with ethanol, on the progression of cell cycle, the activity of cyclin-dependent kinases (cdks) and the expression of major growth arrest and DNA damage inducible genes (p53, WAF1/C1P1, gadd45, and gadd153) in normal human oral keratinocytes with normal p53 function, HOK expressing HPV-16 or HPV-18 E6 protein, HOK expressing mutant p53 protein, and HPV-immortalized oral keratinocytes. These proposed studies would help us gain more insight into molecular mechanisms of oral carcinogenesis.

The immense racial, ethnic, and cultural richness of the greater Los Angeles metropolitan area is unparalleled in the United States. Correspondingly, however, the social disparity between minority and majority populations is all too evident--leading, even, to overt civil unrest in recent years. It is within this context that the UCLA-King/Drew Regional Research Center for Minority Oral Health (RRCMOH), Phase I, was initiated on September 29, 1992. Its two-fold mission is to train minority researchers and to improve the oral health of racial and underrepresented minorities in the single largest population center in the Western United States. The present Phase II application seeks continuation funding to allow the Center to build upon the substantial accomplishments of the Phase I program and to achieve the Center's long- term objectives: (1) To study oral health problems uniquely relevant to the needs of minority populations. (2) To expand research and research training opportunities for minority scientists by encouraging their participation in oral health research. (3) To develop and strengthen the biomedical and behavioral oral health research capacity of the Charles R. Drew Medical University/Martin Luther King, Jr. Medical Center. The Phase II Center will continue to develop and conduct multidisciplinary research on three key aspects of oral health-related morbidity, disability, and mortality among minority populations. Each corresponds to a major subproject and each shares the common theme of orofacial injury. The Phase II Center is based on the unifying conception that minority populations in the United States suffer disproportionately the consequences of orofacial injury. They are the victims of violence and bear a disparate share of the short-term and the long-term consequences of acute and chronic orofacial injury. African-American and Hispanic minority populations, in particular, exhibit a familial predisposition toward abnormal soft tissue repair secondary to injury and they are disproportionately vulnerable to oral malignancy arising from long-term, low-grade traumatic injury to oral mucosa. The Phase II subprojects and pilot projects therefore address (a) the increased vulnerability of minority populations to serious orofacial trauma (Subproject 1 and Pilot Project 1), (b) the increased predisposition of such populations to abnormal healing after sustaining such injury (Subproject 2 and Pilot Project 2), and (c) the disproportionate vulnerability of such populations to overt malignant neoplastic disease consequent to chronic oral injury (Subproject 3 and Pilot Project 3). These are not classic dental diseases in the usual sense, but they are all identified, appropriately, in the NIH/NIDR Phase II Program Announcement as important areas relevant to minority oral health needs. More importantly, they reflect the pressing, practical, and real-life oral health research needs of minority populations in an urban setting.

The long-term objective of this project is to develop innovative therapeutic modes for the treatment of advanced forms of human oral cancers. Oral cancer is a major health problem not only because of the significant mortality rates associated with the disease, but also because of the functional defects and disfigurement often associated with its treatment. Early stage tumors can usually be managed through surgery and radiotherapy, however, successful treatment is inversely proportional to the extent of the disease at the time of treatment. Unfortunately, diagnosis is often delayed until the tumor is in a later stage as early lesions frequently do not show symptoms. Of oral cancers diagnosed between 1986-1991, 52 percent were at an advanced stage and the five-year survival rate of these cases was 55 percent among whites and 33 percent among blacks. These bleak statistics warrant extensive experimentation for treating advanced head and neck tumors. A particularly promising approach involves stimulating the host's immune system to identify and destroy tumor cells. There is strong evidence that oral cancers can be detected by the immune system, as infiltrating lymphocytes have been reported in the majority of tumors examined to date. The presence of T-lymphocytes has been associated with a favorable prognosis in early studies of animal models and human biopsies. The possibility of treating tumors using immunostimulatory cytokine therapy is beginning to gain acceptance. IL-2 is one of the leading candidates for therapy of cancers as exogenously administered IL-2 stimulates the activation and proliferation of infiltrating T- lymphocytes. Administration of IFN-gamma, has resulted in the reduction of tumors as well as rendering tumors more susceptible to IL-2 immunotherapy, possibly by up regulating MHC I/II or co-stimulatory molecules (B7-1), resulting in effective tumor-antigen presentation to T lymphocytes. Toxic effects, however, are a major problem when high doses of cytokines are administered. Since oral cancers are easily accessible, we propose to deliver the genes for IL-2 and IFN-gamma into the tumor cells where they will be expressed using non-replicating adenovirus vectors. We hope to produce high concentrations of IL-2 and IFN-gamma within the micro-environment of the tumor and stimulate anti- tumor immune responses. Although levels of cytokines will be high in the micro-environment, the systemic levels will remain low to avoid the toxicity problems associated with high exogenous cytokine dosage. We will test the usefulness of cancer gene therapy using non-replicating adenovirus vectors expressing immunostimulatory cytokines in a mouse model of oral cancers.

The UCLA School of Dentistry requests funds for the development of the Jane and Jerry Weintraub Center for Reconstructive Biotechnology (WCRB), with the express purpose of addressing the substantial challenges of restoring the craniofacial complex facing both patients and health professionals. Specifically, we are requesting matching funds for the conversion of 5,386 gross square feet of newly-acquired space in the UCLA Center for Health Sciences into a research complex that houses the WCRB in one central facility. The WCRB has been formed out of recognition that defects in the head and neck secondary to tumor ablation, injury, or congenital malformation can result in severe functional impairment and facial disfigurement. The WCRB's primary research objective, i.e., restoration of the craniofacial complex, involves the study of cellular and molecular biology of wound healing, tissue remodeling and regeneration, biomaterials and bioengineering, a range of translational research, and clinical outcome studies. The existing programs at UCLA in implant and maxillofacial prosthodontics, plastic and reconstructive surgery, head and neck surgery, orthopedic surgery, radiation oncology and dermatology are the strength for our unique capabilities and offer outstanding potential for rapid continued growth. Building on the clinical and basic research in areas related to acquired defects of the head and neck by scholars at UCLA, the WCRB will become a regional and national resource for collaborative research, education, and patient care relevant to the treatment of these defects. In 1998, the School of Dentistry purchased and assumed ownership of 11,000 gross square feet of non-research space on two underground levels in the Center for Health Sciences complex. One of these levels is expressly dedicated as physical research space for the WCRB. This grant will provide matching funds for the construction of WCRB laboratories and core facilities and core facilities in support of research, as well as research training and education, within this dedicated space. The requested NCRR funding represents 35% of the total of the cost for the design, demolition, and construction of this facility, as well as for fixed core equipment necessary for the WCRB to attain its research, training and educational goals. The proposed laboratories will provide space for primary investigators, their research teams, and research collaborators, all of whom are conducting interdisciplinary investigation for craniofacial reconstruction.

DESCRIPTION: (Provided by Applicant) Frequent infection with human papillomavirus (HPV) in the oral cavity has been noted in HIV immunocompromised children and adults. HIV-infected individuals are more susceptible to infection with multiple HPV subtypes, including types 16 and 18. These "high risk" HPVs that are closely associated with development of malignant oral cancer: the viral DNA is frequently found in oral cancer cells and tissue. Moreover, transfection of normal human oral keratinocyte (NHOK) cells with cloned "high risk" HPV genome immortalizes these cells, which can convert to fully transformed cells when exposed to chemical carcinogens. Since (1) the same chemical carcinogens cannot transform NHOK cells and (2) the loss of genomic integrity is the hallmark of neoplastic cells, "high risk" HPV must play a critical role in the malignant transformation of NHOK cells by disrupting cells' ability to maintain genomic integrity. Genomic integrity is maintained by constant repair of DNA damage; thus, disturbance of DNA repair results in mutations, which ultimately induces malignant transformation of cells. The central hypothesis of the project is that infection of NHOK cells with "high risk" HPV oncogenes disrupts DNA repair; and that inhibition of HPV oncogene expression allows pre-neoplastic human oral epithelial cells expressing high risk" HPV oncogenes to regain their DNA repair activities and genomic integrity. To test this hypothesis, the applicants propose the following specific aims: (1) to determine the basal and genotoxic agent-induced DNA repair activities of NHOK cells, HOK cells transfected with the HPV-16 genome, and pre-neoplastic oral epithelial cells (derived from lesion biopsies) expressing "high risk" HPV; (2) to investigate the effects of HPV-16 oncogenes on basal and genotoxic agent-induced DNA repair activities of NHOK cells; and (3) to study the effect of "high risk" HPV ribozymes on DNA repair activities and mutation frequency (and rate) of hypoxanthine phosphoribosyl transferase (hprt) gene of pre-neoplastic and neoplastic human oral epithelial cells (expressing "high risk" HPV) derived from lesion biopsies. The applicants expect to answer the following questions: Does "high risk" HPV disrupt the repair of DNA damage in NHOK cells? If so, which DNA repair process is impaired? Are viral oncogenes responsible for such disruption? If so, is the inactivation of p53 or pRB by HPV oncogenes solely responsible for the disruption? Do pre-neoplastic or neoplastic cells (expressing "high risk" HPV) derived from human oral lesion biopsies have the same spectrum of DNA repair defects as NHOK cells transfected with "high risk" HPV genome? Does the disruption of viral oncogene transcripts restore the DNA repair activites and genomic integrity of HPV-immortalized HOK cells, pre-neoplastic and neoplastic human oral epithelial cells (from lesion biopsies) expressing "high risk" HPV oncogenes?

DESCRIPTION (provided by applicant): This grant application requests matching funds for the conversion of 6,660 square feet of a 37-year old existing research space in the UCLA Center for Health Sciences into a state of the art research complex "The UCLA Yip Center for Oral/Head &Neck Oncology Research (UYCOHNOR)" supporting basic and translational research in oral/head &neck oncology. The UYCOHNOR is formed out of the recognition of an urgent need for the consolidation and significant expansion of oral and head &neck cancer research at UCLA, in particular NIH-supported translational research. The UCLA School of Dentistry has secured over 20 million dollars of NIH funds towards oral/ head &neck oncology research and research training in the last two years. Significant amounts of pending NIH grants will significantly benefit from this proposed Center. The proposed UYCOHNOR, with the envisioned leadership and significant institutional support in place, will serve as a focal center for oral and head &neck oncology research at UCLA and regionally in Southern California to foster multidisciplinary collaborative research, particularly in genomics and proteomics. Short-term goals include the development of a research environment and infrastructure to foster genomics and proteomics research of oral/head &neck cancer with a translational research focus at the UCLA School of Dentistry. The long-term goal is to elevate and enhance the UCLA oral/head &neck oncology research program to be the premier research center and research training program in the country with significant level of NIH research support.