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.

Developing Genetic Tools to Study Oral Bacterial Pathogenesis

Han, Yiping Weng,Suh, Wenyuan Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.1

Molecular biology has revolutionized the medical sciences, especially in the field of microbiology. However, so far it has only limited impact on dentistry even though bacteria are the main cause of the periodontal diseases and dental caries. With the increasing number of antibiotic-resistant oral bacteria and the increasing cost of oral surgery, it is becoming very important to understand the molecular mechanisms of the pathogenesis of the oral bacteria, so one can develop new drugs for the infections as well as new diagnostic methods for quick identification of the oral pathogens for proper treatment. In order to study the molecular biology of the oral bacteria, it is essential to develop key genetic tools. This paper reviews the recent progress in this field, including setting up genetic systems and using these tools to study the genes related to pathogenesis.

16S rRNA Genes PCR-RFLP Analysis for Rapid Identification of Oral Anaerobic Gram-Positive Bacilli

Sato, Takuichi,Matsuyama, Junko,Takahashi, Nobuhiro Korean Academy of Oral Biology and the UCLA Dental 2000 International Journal of Oral Biology Vol.25 No.3

Oral anaerobic gram-positive bacilli are frequently isolated from oral infectious sites including carious, endodontic and periodontal lesions. Identification of these bacteria by conventional methods are labor-intensive and time-consuming because of poor growth and non-reactivity in most of the biochemical tests. In this study, in order to develop the rapid identification method for these bacteria, restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA genes〔16S rRNA genes PCR-RFLP〕was applied to anaerobic gram-positive bacilli such as Eubacterium, Mogibacterium and Pseudoramibacter, and the obtained findings were compared with the results of biochemical identification methods. 16S rRNA gene sequences were amplified from isolated genomic DNA samples by the PCR with universal primers. The PCR products were then purified and characterized by single digestion with restriction endonuclease HpaII, and this allowed discrimination among the respective reference strains〔type strains of ATCC〕. In addition, 55 strains were assigned to one of the reference species on the basis of their restriction profiles by digestion with HpaII, and these results were corresponded to those of biochemical identification methods. Furthermore, the 16S rRNA gene sequences were accessed from the GenBank and the RDP data base, and the restriction profiles expected are in accordance with the obtained RFLP patterns in this study. therefore, 16S rRNA genes PCR-RFLP is a rapid and reliable identification method for oral gram-positive bacilli including Eubacterium, Mogibacterium and Pseudoramibacter.

Comparison between 16S rRNA Genes PCR-RFLP Analysis and Biochemical Tests for Identification of Actinomyces naeslundii

Matsuyama, Junko,Sato, Takuichi,Takahashi, Nobuhiro Korean Academy of Oral Biology and the UCLA Dental 2000 International Journal of Oral Biology Vol.25 No.3

Restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction〔PCR〕-amplified 16S ribosomal RNA genes〔16S rRNA genes PCR-RFLP〕using MnlI has been shown to be a useful tool in defferentiating oral Actinomyces species. In this study, the method was applied to Actinomyces isolates from human dental plaque〔n=77〕, and the findings were compared with those obtained by biochemical identification methods. As a result, 46 strains out of 55 A. naeslundii genospecies 1 strains were mis-identified as A. israelii〔n=35〕 and A. meyeri〔n=11〕; and 2 strains out of 12 A. naeslundii genospecies 2 strains were mis-identified as A. israelii by biochemical identification methods. Therefore, one should pay attention to the mis-identification of a. naeslundii species by conventional biochemical tests.

G_2/M phase Arrest and Apoptosis Induction by DW2282 in Human Oral Carcinoma (KB) Cells

Piao, Wenhua,Kwon, Hee-Young,Ha, Jung-Sun,Kim, Yeo-Gab,Kim, Jeong Hee Korean Academy of Oral Biology and the UCLA Dental 2002 International Journal of Oral Biology Vol.27 No.3

We studied the effect of DW2282 on cell proliferation, cell proliferation, cell cycle progression and induction of apoptosis in human oral carcinoma (KB) cells. IC_50 value was determined to be. 0.04㎍/ml. The presence of mRNA synthesis or protein biosynthesis inhibitors did not significantly change IC_50 value of DW2282. Flow-cytometric analysis showed G_2/M phase accumulation then apperarance of subpG_1, apoptotic population. Apoptosis induction was confirmed by fragmentation of DNA in DW2282-treated cells. DW2282 induced-cell cycle arrest was accompanied by decrease in cdc2 and cyclin B1 that have critical roles in progression through the G_2/M phase. We obsered a decreased level of the anti-apoptotic protein Bcl-2, activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. Taken together, our data demonstrate that DW2282 suppresses cell growth by inducing apoptosis after G_2/M phase arrest in KB cells.

Effectiveness of Different Promoters in Adeno-associated Virus Vectors for Expression of Genes in Oral Cancer Cells

Shillitoe, E.J.,Hermonat, P.L.,Noonan, S.,Shi, B. Korean Academy of Oral Biology and the UCLA Dental 2001 International Journal of Oral Biology Vol.26 No.1

The p5 and p40 promoters of the human adeno-associated virus (AAV) were tested for ability to promote expression of either the lacZ marker gene or the thymidine kinase (TK) suicide gene in human oral cancer cells. For comparison, the early gene promoter of the SV40 virus and the immediate-early promoter of human cytomegalovirus (CMV) were used. In the oral cancer cell line Tu183 the highest levels of expression of the marker gene was obtained from the CMV promoter. Lower levels were seen from the SV40 promoter and lower still from the p5 and p40 promoters. Nonetheless, both p5 and p40 were sufficiently active to produce a toxic level of expression of the TK suicide gene, with the p5 promoter being extremely effective. Testing in neuroblastoma cells revealed that the p5 and p40 promoters were less cancer-specific than the other promoters. Neither adenovirus nor hydroxyurea increased the expression from any promoter. The results imply that 1) the promoters of AAV could be used in gene therapy of human squamous cell carcinomas for expression of toxic drugs or other purposes, 2) the presence of DNA-damaging agents should not be expected to enhance the effects of the promoters, and 3) no specificity for oral cancer cells may be expected.

The Role of the Tonsils in Oral Immune Surveillance

Chiappelli, Francesco,Romeo, Horacio Korean Academy of Oral Biology and the UCLA Dental 2001 International Journal of Oral Biology Vol.26 No.1

Basic science research has clearly demonstrated the teleological relevance of the tonsils by producing evidence in support of the critical role these structures play both as a key immune gate-keeper in the oral cavity, and in communicating to the central nervous system that a local immune response has been initiated and must be modulated by the brain. By contrast, clinical practice often recommends the surgical resection of the tonsils, suggesting tonsillectomy has no counter productive effect on the immune system, despite studies that may indicate the opposite. This paper reviews our fundamental and clinical knowledge of the tonsils.

Morphological Characterization of Normal Human Oral Keratinocytes during Serial Subculture

Bibb, Carol A.,Kang, Mo K.,Park, No-Hee Korean Academy of Oral Biology and the UCLA Dental 1999 International Journal of Oral Biology Vol.24 No.3

This study described the morphology of normal human oral keratinocytes (NHOK) at different phases of an in vitro model of cell aging using phase contrast and electron microscopy. During serial subculture in low clacium conditions, NHOK proliferated exponentially, then entered replicative senesence, producing a prolferation curve with three phases: exponential, senescing, and senescent. Under phase contrase microscopy exponential phase NHOK were small and polygonal in shape with large nuclei containing prominent nucleoli while senescent phase cells were larger overall and contained clear, round, vacuolated spaces in the perinuclear cytoplasm. A better understanding of the cellular morphology was obtained by comparing the ultrastructure of representative cells from the etponential, senescing, and senescent phase of culture. Exponential phase cells had a high nuclear to cytoplasmic ratio, typical housekeeping organelles, and a perinuclear arrangement of keratin bundles. In cells entering esnescence there was a dramatic increase in clear, membrane bound cytoplasmic vacuoles which apperared to coalesce and filled much of the cytoplasmic compartmint in senescent phase cells. The vacuoles, housekeeping organelles, and keratin bundles were in a perinuclear location, leaving a peripheral rim of cytoplasm devoid of structural elements. In cells from all phases, nuclei were intact with diffuse chromatin and prominent nucleoli, although in senescent cells they appeared compressed by the accumulation of vacuoles. Our findings provide morphological evidence that the majority of NHOK in this in vitro model remain viable from exponential growth into senesence. The most notable change in phenotype was an accumulation of cytoplasmic vacuoles in the senescent phase cells.

Ethanol Upregulates the Expression of p21^WAF1/CIP1, Prolongs G_1 Transition, and Prevents PGA_2 Induced Apoptosis in Human Epithelial Cells

Guo, Wentong,Park, No-Hee Korean Academy of Oral Biology and the UCLA Dental 1998 International Journal of Oral Biology Vol.23 No.1

Extensive consumption of alcoholic beverages is a risk factor associated with the development of various human epidermoid cancers, including oral and pharyngeal squamous cell carcinomas. However, the mechanisms of alcohol associated carcinogenesis are unknown. We report here that exposure of human epithelial cells to ethanol, at concentrations (100-200 mM) that do not cause cell death, upregulates the transcription of the WAF1/CIP1 gene and reduces the rate of cellular proliferation by inducing a delay in G_1 phase transition in human epithelial cells, including normal human oral keratinocytes (NHOK), human oral squamous cell carcinoma (SCC4, SCC9) and human colorectal carcinoma (RKO) cells. RKO cells undergo apoptosis when exposed to prostaglandin A_2 (PGA_2), but are protected by ectotypical overexpression of p21^(WAF1/CIP1) (Gorospe et al., 1996b). We examined the effect of ethanol on PGA_2-induced apoptosis which followed DNA fragmentation, accumulation of cellular p53 and G_2/M arrest. Ethanol alone enhanced the cellular levels of p21^(WAF1/CIP1) and p27, and decreased Cdk2, resulting in prolongation of the G_1 to S transition. When non-synchronized exponentially proliferating cells were pretreated with ethanol, for 8 hours to establish the maximum effect of ethanol and subsequently exposed to both PGA_2 and ethanol, the cells stopped proliferating but apoptosis was prevented. Ethanol prevented PGA_2-induced DNA fragmentation and apoptosis, but did not prevent PGA_2-induced G_2/M arrest.

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.