Anti-invasive and Anti-metastatic Effects of Arg-asp (Rd) on Human Salivary Adenoid Cystic Carcinoma

Li, Fenghe,Yu, Guangyan,Li, Shenglin,Peng, Shiqi,Fu, Jia,Wu, Dengcheng Korean Academy of Oral Biology and the UCLA Dental 2001 International Journal of Oral Biology Vol.26 No.3

The objective of the present study is to test the anti-invasive and anti-metastatic effect of Arg-Asp (RD), a newly developed peptide with the same effect as that of RGD, on SACC-LM (a highly pulmonary metastatic salivary adenoid cystic carcinoma cell line) cells. The ability of SACC-LM cells to invade in a modified Boyden chamber was inhibited by 56%, 64% and 65% when the final concentration of RD was 1, 5, and 25㎍/ml, respectively. The anti-metastatic effect of orally administered RD on experimental metastatic SACC-LM was observed by the survival rate and the number of pulmonary metastatic foci/mouse. When RD (7.5, 30, and 120mg/kg) was administered orally the survival rate increased in the treated mice 66.14(14.47), 64.75(8.43), 69.86(12.77) days, respectively, compared with 43.56 (4.95) days in the control mice (p<0.05). The number of pulmonary metastatic foci decreased significantly (6.5 Vs 20.29, p<0.05) for 120mg/kg RD, whereas the incidence of pulmonary metastasis was not significantly decreased at lower doses of RD. The results of this study suggest that RD has low toxicity and possesses an anti-invasive and anti-metastatic effect on SACC-LM and that RD may be administered orally.

Genotoxic Stress-Induced Nucleotide Excision Repair Activity of Human Oral Keratiocytes is Abrogated by "High Risk" Human Papillomavirus: Role of p53

Gujuluva, Chandrasekhar N.,Park, No-Hee Korean Academy of Oral Biology and the UCLA Dental 1999 International Journal of Oral Biology Vol.24 No.4

Human oral keratinocytes immortalized with cloned "high risk" human papillomavirus DNA can readily convert to tumorigenic cells when exposed to chemical carcinogens. Tumorigenic conversion of immortalized cells may, in part, be due to their lack of DNA repair responses to genotoxic stress. To study this possibility, we determined the nucleotide excision repair (NER) capacities and the level of proteins associated with DNA damage/DNA repair (p53, p21^WAF1/CIP1, gadd45 and PCNA)from primary normal human oral keratinocytes (NHOK), normal human skin epidermal keratinocytes (NHEK), HPV-immortalized human oral keratinocytes, and several human oral cancer cell lines before and after genotoxic exposure. Genotoxic exposure significantly increased the levels of cellular p53, p21^WAF1/CIP1, and gadd45 proteins and the NER activity in NHOK and NHEK, whereas it did not alter the protein levels and the NER activities of eht HPV-immortalized oral keratinocytes and oral cancer cells. These data indicate that normal epithelial cells lining the human oral cavity can increase the DNS repair function when challenged by genotoxic stress, and in doing so may protect themselves from transformation or cell death. Infection of these cells with "high risk" human papillomaviruses seems to abrogate this protection mechanism.

PROCEEDINGS OF THE 21th ANNUAL SCIENTIFIC MEETING OF THE KJOREAN ACADEMY OF ORAL BIOLOGY : The meeting was held on 8~9 November 1996 at the College of Dentistry, Seoul National University, Seoul, Korea

Ryoo, Hyun-Mo Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.2

Effects of IL-1β Applied lontophoretically to Tooth Pulp Neurons in the Medullary Dorsal Horn of Cats

Choi, Hyo Sun,Kim, Hae Dong,Ahn, Dong Kuk Korean Academy of Oral Biology and the UCLA Dental 2003 International Journal of Oral Biology Vol.28 No.1

The present study was to characterize the extracellular response properties of trigeminal neurons that responded to electrical or/and heat stimulation of dental pulp. We also investigated the effects of IL-1β applied iontophoretically to tooth pulp neurons evoked electrical stimulation. Experiments were carried out on 32 adult cats weighing 1.5-3kg. Assessment of sensitivity to quantitative heat stimuli was carried out with a custom designed, computer-controlled contact thermode thet fitted around the clinical crown. Extracellular recordings were made with glass electrodes with resistance of 2-10㏁ The activities of 18 single neurons excited by noxious hear stimulation of canine were studied. All tooth pulp neurons were found in both superficial and deep nuclear regions in subnucleus caudalis (Vc) and at the interface between the nucleus caudalis and nucleus interpolaris (Vc/Vi). Although many neurons were excited by electrical stimulation of the canines, only 10% of the units responding to electrical stimulation responded to noxious heat stimualtion of the dental pulp. Eighteen of the tooth pulp neurons responding to cutaneous stimulation were classified as either NS (5) or WDR (13) neurons. Twenty-nine of tooth pulp were examined after pressure injection of IL-1β. Pressure injection of IL-1β (0.25 or 2.5 ng/μl) inhibited the neuronal resposes evoked by cyclic application of NMDA in 17 of 29 examined neurons. In 5 of 29 examined neurons, pressure injection of IL-1β (0.25 or 2.5 ng/μl) facilitated the neuronal responses evoked by cyclic application of NMDA. The present results provide evidence that Vc and Vc/Vi contain neurons that respond to natural noxious stimulation of the dental pulp. Central administration of IL-1β may modulate signal transmission from tooth pulp.

Understanding Dental Caries : An Infectious Disease, Not a Lesion

Tanzer, Jason M. Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.4

Caries is an infectious disease. Cavities are its resultant lesions. The lesions should not be confused with the disease, because it leads to misunderstandings of preventive and therapeutic possibilities. This paper reviews the fundamental evidence which establishes that dental caries is an infection caused primarily by the mutans group of streptococci. The infection is powerfully influenced by the diet, especially the consumption pattern of sucrose. While other bacteria probably can play some causative role, the evidence which implicates them is limited, and suggests these other bacteria to be relatively weakly cariogenic. Substantial information clarifies the dental plaque ecology, metabolism, transmission, and conditions which determine expression of virulence of mutans streptococci. These phenomena are strongly correlated with their metabolism of sucrose. Mutans streptococci are also associated with the inception of secondary carous lesions at the enamel/plaque interface. The initial lesions of tooth decay thus reflect a microbiologically specific dental plaque infection, influenced by diet. Much less is understood about either the microbiology of progression of lesions through dentin or secondary lesions of the deep structures of the teeth. Root surface caries, like coronal caries, is closely associated with mutans streptococcal colonization; however, Actinomyces viscosus may also be a significant cariogen in this setting. The incipient lesions of tooth decay are widely thought to reflect a complex series of demineralization and remineralization events occurring under the mutans-rich plaque in the surfaces of teeth. These events ate driven by the pattern and content of dietary carbohydrate consumption which, in turn, induces fermentative activity of the plaque, decrease and eventual increase of plaque pH, and solubilization and reprecipitation of the inorganic phase of enamel and salivary ions. The net flux of mineral components out of and into the tooth determines whether teeth ostensibly remain mineralized, demineralize, or become remineralized after development of initial lesions. Salivary flow, composition, and fluoride strongly influence this complex of fluxes, as does the consumption and fermentation of carbohydrate-rich foods, especially but not exclusively those rich in sucrose.

Actinomycin D-Mediated Sensitization of Malignant Oral Cell Lines to Fas-Mediated Cytotoxicity : Implication of p53 and FAP-1

Itakura, Masayuki,Mori, Shunsuke,Park, No-Hee,Bonavida, Benjamin Korean Academy of Oral Biology and the UCLA Dental 1999 International Journal of Oral Biology Vol.24 No.3

Dysregulation of the normal apoptotic mechanism in normal cells can directly contribute to the pathogenesis of cancer and the development of resistance of tumor cells to cytotoxic drugs and lymphocytes. We have investigated the development of resistance to immune Fas-mediated apoptotic signals in oral cell lines as a function of progression from normal towards malignancy and the sensitizing effect of actinomycin D(ActD). Several lines of oral tissues that represent different stages of progression from the normal phenotype to the malignant phenotype were examined. Normal human oral keratinocytes (NHOK)were immortalized by transfection with the recombinant human papilloma virus ((HPV)-16 DNA (HoK-16B)). These cells were subsequently exposed to benzo(a)pyrene for 7days (HOK-16B-BaP) and remained non-tumorigenic in nude mice. HOK-16B-BaP exposed for longer periods (6 months) to benzo(a)pyrene (HOK-16B-BaP-T) developed tumors in nude mice when injected subcutaneously and a cell line was developed from the tumor (HOK-16B-BaP-T1). NHOK and HOK-16B were relatively more sensitive to anti-Fas antibody (CH-11) and Fas-ligand-mediated cytotoxicity than the more advanced lines. Western blot analysis and flow cytometry showed (a) decreaded surface espression of Fas, (b) decreased expression Of P53, (c) increased expression of Bcl-2 and decreased expression of Bax, as a function of tumor cell development from the normal to the malignant phenotype. Treatment with ActD augmented both anti-Fas antibody mediated and Fas ligand mediated cytotoxicity, and significantly up-regulated the p53 protein levels without changing the Bcl-2 and Bax protein levels. Fas-associated phosphatase-1 (FAP-1) is an anti-apoptotic molecule reported to interact with Fas and can block transduction of the apoptotic signal. Western blot analysis revealed that FAP-1 protein levels were increased with progression towards malignancy and was down-regulated by treatmint with ActD. These findings demonstrate the implication of several potential mechanisms by which oral cell lines become resistant to Fas-mediated apoptosis with the development of malignancy in vitro. These include decreased expression of pro-apoptotic proteins such as surface Fas, p53, and Bax, and increased expression of anti-apoptotic proteins such as Bcl-2 and FAP-1. Sensitization to Fas-apoptosis by ActD resulted in the upregulation of p53 and down-regulation of FAP-1 protein levels.

Development of an In Vitro Candida albicans Oral Infection Model

Lund, Trace,Kleinegger, Cynthia L.,Wertz, Philip W.,Drake, David R. Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.4

Objectives: The purpose of the present study was to develop an in vitro model for studies of Candida albicans oral infections. Design : Sheets of porcine buccal epithelium were cut at a thickness of 300 μm and glued with cyanoacrylate to a plastic backing. Circular 1-cm-diameter disks of the plastic-backed epithelium were cut, and incubated with Candida albicans. At various incubation times, numbers and appearance of Candida albicans on the tissue disks were assessed. Materials : Porcine tissue was obtained at a slaughterhouse. Candida selective Chromagar plates were purchased from Hardy Diagnostics of Santa Maria, CA. Other materials were from Sigma Chemical Company, St. Louis, Mo. Methods : Candida albicans was cultured in Sabouraud Dextrose Broth at 37℃, overnight. Tissue disks were incubated with standardized suspensions. At time of harvest, some disks were fixed for examination by scanning electron microscopy, while others were homogenized and spiral-plated onto Candida-selective agar. Results : At three hours, there were 2.1×10 exp (5) adherent yeast per disk. By 24 hours of incubation, there was noticeable hyphae formation, and by 48 hours hyphae predominated and tissue invasion was evident. Conclusions : The porcine buccal epithelial disk provides an in vitro model for oral Candida albicans infections in which adherence, growth,, and the bud to hyphae transition and tissue invasion process may be investigated.

Genetic Instability and the Development of Human Oral Cancer

Park, No-Hee,Joseph McQuirer,Frederick Rutherford,Shin, Ki-Hyuk,Liu, Xuan,Guo, Wentong,Bertolami, Charles N. Korean Academy of Oral Biology and the UCLA Dental 1998 International Journal of Oral Biology Vol.23 No.1

We established an in vitro multistep oral carcinogenesis model by sequential exposure of normal human oral keratinocytes (NHOK) to "high risk" human papillomavirus (HPV) and chemical carcinogens. First, we immortalized primary NHOK by transfecting the cells with either cloned HPV-16 or HPV-18 genome. The immortalized human oral keratinocytes (HOK) were further transformed into tumorigenic cells when they were exposed to chemical carcinogens. However, under the same chemical carcinogen exposure, NHOK did not transform. Although the reason for these differences in normal and HPV-immortalized HOK's response to chemical carcinogens remains unknown, we postulate that it may be due to the differences in the cells' ability to maintain genomic integrity. Genomic integrity is normally maintained by the cell's ability (1) to assess the status of the genome at a given time point, (2) to signal cell cycle progression or arrest, and (3) to repair damaged DNA. Disruptions in any of these pathways may ultimately result in the loss of genomic integrity, a hallmark of neoplastic cells. We demonstrated that, in HPV-immortalized HOK, the E6 and E7 oncoproteins of "high risk" HPV disrupt cellular signals and perturb cell cycle control and DNA repair, particularly when challenged by genotoxic agents. Finally, the effect of "high risk" HPV on the stability of cellular chromosomes was indirectly determined by checking the mutation frequency using a shuttle vector plasmid in NHOK and cells harboring "high risk" HPV genome. We found that infection with "high risk" HPV tremendously enhanced the mutation frequency of the shuttle vector. These data indicate that, in NHOK, genomic integrity is maintained through controlling cell cycle progression and DNA repair when challenged by genotoxic stresses, but "high risk" HPV infection, such as HPV-16 or HPV-18, jeopardizes such cell cycle controlling and DNA repair capacities and causes genetic instability.

Telomerase Expression in Precancerous Oral Verrucous Hyperplasia

Greer, Robert O.,George Hoernig,Kenneth Shroyer Korean Academy of Oral Biology and the UCLA Dental 1999 International Journal of Oral Biology Vol.24 No.2

Activation of telomerase and stabilization of telomeres are thought to be required for both cellular immortality and oncogenesis. Telomerase is an essential ribonucleoprotein that maintains telomere length and whose activity is associated with escape from cellular senescenec. Telomeres have been purported to function as mitotic clocks, shortening with age in a replication dependent manner. The role of telomerase in oral dysplastic lesions has not been well defined. The purpose of this study is to investigate the role of telomerase in the precancerous dysplastic lesion verrucous hyperplasia. Using a modifies PCR-based assay for telomerase activity, 23 cases of verrucous hyperplasia were examined using a telomertric repeat amplification protocol (TRAP assay). Telomerase positive controls included enzyme abstracts from HELA cells, a squamous cell carcinoma cell line. Negative controls included RNAse pretreatment of tissue extracts. Fresh tissue samples from 23 cases of verrucous hyperplasia were assayed. Telomerase activity was detectable in 14 of 23 cases. Negative controls which included cheek biting leukoplakias failed to demonstrated telomerase activity. This study suggests that telomerase activation may indeed be associated with a precancerous phase of oral squamous cell carcinoma known as verrucous leukoplakia and that telomerase may serve as a biomarker for cancer risk assessment in patients with certain oral precancers.

Loss of p16^INK4a Expression Is Associated with Hypermethylation of 5'-CpG Island of INK4A in Oral Cancer Cell Lines

Kang, Mo K.,Phillip Liu,Kim, Reuben H.,Lim, Jung,Park, No-Hee Korean Academy of Oral Biology and the UCLA Dental 1998 International Journal of Oral Biology Vol.23 No.4

Normal somatic cells undergo limited cell doublings until replicative senescence, at which cells completely lose their potential to divide and yet maintain their metabolic activity. The p16^INK4a, a cyclin-dependent kinase inhibitor, has been postulated as a putative cellular factor responsible for the onset of senescence in normal cells. Inactivation of INK4A, the gene encoding p16^INK4a, is frequently found in many types of human cancer cells that escaped from senescence. To correlate the status of p16^INK4a expression and oral cancer, we determined the expression level of p16^INK4a in seven human oral cancer cell lines and human oral keratinocyte (HOK) cell line immortalized with type 16 human papillomavirus (HPV-16) genome. p16^INK4a was undetected in all tested cancer cell lines except those cells harboring "high risk" HPV DNA, high expression of p16 was found. We also found that 5'-CpG island of INK4A was hypermethylated in the cell lines in which p16^INK4a expression was undetected. Exposure of cells to 5-aza-2deoxycytidine (5-azaCdR), a demethylating agent, significantly reduced the proliferation rate, and at least one cancer cell line was heavily stained with senescenec-associated β-galactosidase (SA β-gal) by 5-aza CdR. Taken together, the above findings suggest that cancer cells escape from senescence, in part, by inactivation of p16^INK4a due to hypermethylation of 5-CpG island of INK4a, and that exposure of cells to a demethylating agent may trigger the senescence program in oral cancer cells.