제16형 HPV E6/E7 유전자의 변형 감염으로 배양된 SGT 세포주에서 유도된 편평세포 분화

이종현,박경주,이종헌 대한구강악안면병리학회 2010 대한구강악안면병리학회지 Vol.34 No.3

The origin of squamous cell components in salivary gland tumor has been not yet clarified in detail. The squamous cell differentiation from adenocarcinoma has been reported in various carcinoma by HPV transfection in vitro. The adenocarcinoma cells adjacent to the squamous cell carcinoma components were positive for HPV. This is thought to indicate that after adenocarcinoma cells are transfected with HPV, they undergo morphological changes, and that squamous cell differentiation follows. The purpose of this study were to examine the effects of HPV-16 E6/E7 gene transfection into SGT cell line from human salivary gland adenocarcinoma, and to study the relation between the E6/E7 gene and squamous differentiation. Plasmid pBR322 containing HPV-16 was transfected into cultured SGT cell line using lipofectin method. Hygromycin was used as a selection marker. The presence of HPV E6/E7, transglutaminase 1, and involucrin mRNAs and protein in E6/E7 gene transfected cells was investigated by RT-PCR and immunoslot blot method. The apoptosis index was analysed by flow cytometry. The growth rate of E6/E7 gene transfected cells was reduced. E6/E7 transfected SGT cells increased apoptosis index. Involucrin and TGase I mRNAs by the squamous cell differentiation was most conspicuous in the E6/E7 gene transfected cell compared with non transfected cells. Squamous cell differentiation demonstrated in the transfectedSGT cell line, which expressed E6/E7 fusion gene mRNA.E6/E7 gene transfected cells showed squamous cell differentiation, expressing involucrin and TGase 1 protein by immunoslot blotting. The transfected SGT cell which expressed E6/E7 gene mRNA showed the squamous cell differentiation particularly clearly, and apoptosis was also demonstrated. It suggested that E6/E7 gene transfection into human salivary gland adenocarcinoma cells might induce clear squamous cell differentiation and contribute to study the pathogenesis of human salivary gland adenocarcinoma.

Squamous Differentiation Induced by Transfection of Human Papillomavirus 16 E6/E7 Gene into Cultured SGT Cell Line

이종헌,박경주,이종현 대한구강악안면병리학회 2010 대한구강악안면병리학회지 Vol.34 No.3

The origin of squamous cell components in salivary gland tumor has been not yet clarified in detail. The squamous cell differentiation from adenocarcinoma has been reported in various carcinoma by HPV transfection in vitro. The adenocarcinoma cells adjacent to the squamous cell carcinoma components were positive for HPV. This is thought to indicate that after adenocarcinoma cells are transfected with HPV, they undergo morphological changes,and that squamous cell differentiation follows. The purpose of this study were to examine the effects of HPV-16E6/E7 gene transfection into SGT cell line from human salivary gland adenocarcinoma, and to study the relation between the E6/E7 gene and squamous differentiation. Plasmid pBR322 containing HPV-16 was transfected into cultured SGT cell line using lipofectin method. Hygromycin was used as a selection marker. The presence of HPV E6/E7, transglutaminase 1, and involucrin mRNAs and protein in E6/E7 gene transfected cells was investigated by RT-PCR and immunoslot blot method. The apoptosis index was analysed by flow cytometry. The growth rate of E6/E7 gene transfected cells was reduced. E6/E7 transfected SGT cells increased apoptosis index. Involucrin and TGase I mRNAs by the squamous cell differentiation was most conspicuous in the E6/E7 gene transfected cell compared with non transfected cells. Squamous cell differentiation demonstrated in the transfectedSGT cell line, which expressed E6/E7 fusion gene mRNA.E6/E7 gene transfected cells showed squamous cell differentiation, expressing involucrin and TGase 1 protein by immunoslot blotting. The transfected SGT cell which expressed E6/E7 gene mRNA showed the squamous cell differentiation particularly clearly, and apoptosis was also demonstrated. It suggested that E6/E7 gene transfection into human salivary gland adenocarcinoma cells might induce clear squamous cell differentiation and contribute to study the pathogenesis of human salivary gland adenocarcinoma.

구강 편평세포암종에서 Taxol과 Cyclosporin A의 세포사멸 상승 작용 효과

서민정,한세진,이재훈,Suh, Min-Jung,Han, Se-Jin,Lee, Jae-Hoon 대한악안면성형재건외과학회 2007 Maxillofacial Plastic Reconstructive Surgery Vol.29 No.5

Oral squamous cell carcinoma is the most prevalent oral cancer, which is characterized by its high metastasis and recurrent rates and poor prognosis. Taxol is an anticancer agent which is microbial products extracted from jew tree. It combines with the tubulin and induces apoptosis by inhibiting mitosis of cell with microtubule stabilization. Recently, it was reported to be effective in various solid tumors, but only very slight effect has been seen in oral squamous cell carcinomas due to its cell-specific potencies. Cyclosporin A is used as immune suppressant and is being applied in anticancer therapy as its mechanism of induction of change of apoptotic process in various cells have been known. In this study, oral squamous cell carcinoma HN22 cell line was used for in vitro experiment and as for the experimental group taxol and cyclosporin A were applied alone and to observe the synergistic effect of apoptosis, Taxol and cyclosporin A were coadministered with different concentration of taxol for comparison. The results were obtained as follow: 1. There was no difference in Bcl-2, Bax, caspase 3, 8, 9 mRNA expression when cyclosprin A or taxol was applied alone to HN 22 cell line. 2. Caspase 3, 9 mRNA expression was prominently increased when cyclosprin A and taxol were applied together to cancer cell. 3. No significant difference was observed when cyclosporin A and taxol($1{\mu}g/ml$ and $3{\mu}g/ml$) were applied together to cancer cell line. 4. No significant difference was seen in Bcl-2, Bax, and caspase 8 mRNA expression in all the groups of in vitro experiments. 5. When cyclosporin A was applied alone in vivo study on the nude mice, histopathologi cal findings was similar to those of the control group. Oral squamous cell carcinoma induced by inoculation of HN 22 cell line was not reduced after treatment of cyclosporin A. 6. When taxol was applied alone, the islands of squamous cell carcinoma still remained, which meant insignificant healing effect. There was a lesser volume increase compared with the cyclosporin A alone. 7. When taxol and cyclosporin A were applied together, the connective tissue and calcification were seen in the histopathologic findings. Oral squamous cell carcinoma was decreased and cancer cell was disappeared. In observing the tumor mass change with time, there was a gradual decreased size and healing features. As the results of the in vitro experiment, it could conclud that only when the two agents are applied together, mitochondria-mediated apoptosis occurred by considerable increase of caspase 3, 9 mRNA expression, irrespectable of the concentration of taxol. In vivo experiment, there was a discrete synergistic effect when the two agents were applied together. But single use of cyclosporin A was not effective in this study. Based on the results of this experiment, if further clinical studies are done, taxol and cyclosporin A could be effectively used in treatment of oral squamous cell carcinomas.

분화도 좋은 구강 편평상피세포암종에서 Dominant Negative p63 isoform의 발현

김인수(In-Su Kim),김철환(Chul-Hwan Kim),김경욱(Kyung-Wook Kim) 대한구강악안면외과학회 2007 대한구강악안면외과학회지 Vol.33 No.3

The p53 which is well known as tumor suppressor gene is located at 17p13. p53 is a sequence-specific DNA binding transcription factor that responds to certain cytotoxic stresses, such as DNA damage, by enhancing the transcription of genes that regulate cell-cycle progression as well as programmed cell death. The p63 gene that is located at 3q27-29, is recognized members of the p53 family, and responsible for the transcription of 6 isoforms. Three isoforms (TAp63α, TAp63β, TAp63γ) contain an N-terminal transactivation (TA) domain and can induce apoptosis. The other 3 isoforms(ΔNp63α, ΔNp63β, ΔNp63γ) lack the TA domain and may function in a dominant-negative fashion by inhibiting the transactivation functions of p53 and TAp63 proteins, and thus act as oncoproteins. A number of studies have investigated the role of p63 in human squamous cell carcinomas from different organs. Only a few studies have examined ΔNp63 isoform in oral squamous cell carcinoma including normal epithelium. This study aimed to evaluate expression of ΔNp63 isoform in human oral squamous cell carcinoma tissue and normal mucosa. The 3 cases of well differenciated oral squamous cell carcinoma specimen including adjacent normal mucosa were examined, and immunohistochemical study with monoclonal antibody(4A4) and tumor cell apoptosis analysis with Transmission Electon Microscopy were studied. And, RT-PCR analysis was done for expression of ΔNp63 isoform. The results were as followed. 1. Normal gingiva showed the restricted p63 expression in basal cell layer. 2. Well differentiated squamous cell carcinoma showed mainly p63 expression in overall area of malignancy, especially in basal cell layer to adjacent stromal tissue. 3. Tumor cells around keratinized area with no p63 expression disclosed less micro-organelle in decreased size cytoplasm and severe chromatin margination with nuclear destruction that means apoptosis. 4. Comparison of mRNA expression of ΔNp63 isoform by RT-PCR showed variable expression of ΔNp63 isoform, but ΔNp63αwas most highly expressed in all 3 tumor specimen. From theses results, it should be suggested that ΔNp63 isoform expression in well differentiated squamous cell carcinoma was closely related to tumor oncogenesis, expecially overexpression of ΔNp63αis a most important factor in tumor genesis of oral squamous cell carcinoma.

원발성 및 전이성 구강편평세포암종 세포주에서 p21 및 p73 mRNA발현에 관한 연구

강정훈,김경욱,이재훈,Kang, Jeong-Hoon,Kim, Kyung-Wook,Lee, Jae-Hoon 대한구강악안면외과학회 2001 대한구강악안면외과학회지 Vol.27 No.6

There were many controversies in the cause and progress of tumorigenesis. Recently, studies on the mutation of genes related to the tumor have extensively been performed due to development of molecular biology. Structural and morphological changes of chromosomes, which are related to the abnormal activation of oncogenes or inactivation of tumor suppression genes, transform the normal cells into the tumor cells. p53 and Rb are well known tumor suppressor genes, while oncogenes include c-myc, bcl-2 and ras, etc. When exposed to cell damaging agents, p53 inhibits cell growth by inducing transcription of p21. Especially p73, which is homo-logy of p53, frequently deleted in melanoma, neuroblastoma, colon cancer, and breast cancer, when over produced, p73 activates the transcription of p21, bax-1 and inhibits cell growth by inducing apoptosis. For study on mRNA expression of p21 and p73, normal oral keratinocytes, and cell lines of primary and metastatic oral squamous cell carcinoma were cultured and then electrophoresis and RT-PCR(reverse transcription-polymerase chain reaction) were performed. 1. The mRNA of p21 and p73 in normal oral keratinocyte expressed lower than that of primary squamous cell carcinoma. 2. The mRNA of p21 in metastatic oral squamous carcinoma cell lines was expressed as various patterns compared with that of normal oral keratinocyte. 3. In the metastatic oral squamous cell lines, the mRNA of HN8 expressed higher than that of HN12 or HN19. 4. The mRNA of p73 in primary oral squamous cell lines expressed 4-5 times higher than that of normal keratinocyte. 5. In metastatic oral squamous cell lines, there was no significant expression of p73 mRNA compared with that of normal oral keratinocyte. From the results obtained in this study, mRNA expression of p73 in primary oral squamous cell lines was remarkable, while mRNA expression of p21 and p73 in metastatic oral squamous cell lines were statistically insignificant.

구강편평세포암종과 법랑아세포종에서 c-fos, c-jun의 발현에 관한 면역조직화학적 연구

마경화,조재오 대한구강악안면병리학회 2005 대한구강악안면병리학회지 Vol.29 No.3

The purpose of this study was to evaluate the role of c-fos and c-jun expression in the oral squamous cell carcinoma and ameloblastomas. For this study, 12 subjects diagnosed as squamous cell carcinoma and 7 subjects of ameloblastomas referred to the Dept. of Oral Path. School of Dentistry, Kyung Hee University, 2 subjects of normal oral mucosa without any inflammatory changes were used as experimental, control groups respectively. All the tissues ; experimental and control group were fixed in neutral formalin solution and embedded in paraffin, serial tissue section were made 5㎛ in thickness and processed in the standard way for immunohistochemical method, using primary and secondary antibodies, for c-fos (Anti-c-fos protein, rabbit polyclonal kit at 1:100 dilution), c-jun( anti-c-jun protein, rabbit polyclonal at 1:100 dilution), all BioGenex U.S.A. made, followed by the Streptavidin - Horse Radish Peroxidase(InnoGenex, Human-Avidin kit) application, counter stained with Mayer's hematoxylin stain method, mounted. And examined under the biologic microscope, with the criteria-(no epithelial stain), +(weak or focal epithelial stain), ++(moderate or focal intensive epithelial stain), +++(intense generalized epithelial staining) for the epithelial, and connective tissue component in squamous cell carcinomas, ameloblastomas and normal mucosal epithelium on each. Attained results as follows ; 1. In oral mucosa, c-fos and c-jun intensely expressed on the all cell layers except on the basal layer. Intense reaction is noted in the c-jun than in the c-fos. and a few cells with positive cytoplasm, negative nuclear are scattered in all layer. 2. The response to c-fos in ameloblastomas, is various according to the histological type, but intense resposes are nodted in nuclear and cytoplasm on the tall columnar cells at the periphery of the follicles compare to that on the stellate cells. 3. The respone to c-fos in squamous cell carcinoma, intense reaction is noted in cytoplasm and nuclei of the tumor cells in well differentiated, poorly differentiated type. 4. The response to c-jun in ameloblastoma, it is noted that moderate respone in nuclear and cytoplasm, at the tall columnar cells at the periphery of follicular or plexiform type but intense respone was notes on the columnar cells, and stellate cell in cytoplasm and nuclear of acanthomatous type. 5. The respon to c-jun in squamous cell carcinoma, it is noted that intensive responses only in cytoplasm in well differentiated type, but intensive responses in nuclei and cytoplasm in the poorly differentiated type are revealed. Intensive responses on c-fos, c-jun were noted on the high atypical cells. This results suggest that c-fos and c-jun may be affected to the reactivation on growth and development of the squamous cell carcinoma.

Cyclosporin A가 구강편평상피세포암 세포주에 미치는 항암효과

임한욱(Han-Wook Lim),김경욱(Kyung-Wook Kim) 대한구강악안면외과학회 2004 대한구강악안면외과학회지 Vol.30 No.6

Squamous cell carcinoma is the most prevalent oral cancer, which is characterized by its low survival rate, high malignancy, mortality with facial defects, and poor prognosis. Exact cause and pathogenesis of the squamous cell carcinoma is still unknown. Various routes including smoking, radiation, and viral infections predispose its genesis, and recent studies revealed that genetic defects which fail to prevent cancer proliferation play a role. Generally, a cancer develops from the decreased rate of apoptosis which is an active and voluntary cell death, and from the altered cell cycles. Anticancer effect can be obtained by recovering the apoptotic process, and by suppressing the cell cycles. Among the apoptosis related factors, bcl-2, caspase-9, and VDAC (voltage-dependent anion channel)are produced in mitochondria of the cell. Cyclosporin-A is known to induce apoptosis through its activation with VDAC. This study was to reveal the anticancer effect of Cyclosporin A to the oral squamous cell carcinoma. The inverted microscope was used to find alterations in the tissue, and sensitivity test to the anticancer cells was performed with MTT (Tetrazolium-based colorimetric) assay. Following cell line culture of primary and metastastic oral squamous cell carcinoma, electrophoresis was performed with extracted total RNA. Finally, semi-quantitative study was carried out through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The results of this study are as follows: 1. The inverted microscopic observation revealed a poorly defined cytoplasm at 2000ng􀅭3000ng/ml, indistinct nucleus, and apoptosis. 2. The Growth of cancer cells was decreased at 1000ng/ml of cyclosporin-A. No cancer cell growth was observed at over 2000ng/ml concentration of cyclosporin-A, and at one week, growth of cancer cells was ceased. 3. The MTT assays were decreased as cyclosporin-A concentration was increased. This means that the activation of succinyl dehydrogenase in mitochondria was decreased following administration of cyclosporin A. 4. A result of RT-PCR showed that amount of mRNA of VDAC-2 was decreased half times at a cyclosporine-A concentration of 2000ng/ml. In bcl-2, amount of mRNA was significantly decreased 1/5 times at 2000ng/ml. caspase-9, however, showed slight increase compared to the control group. From the results obtained in this study, administration of cyclosporin-A to the cell lines of oral squamous cell carcinoma induced alterations in morphology and growth of the cells as its concentration increased. Since apoptosis related factors such as VDAS-2, bcl-2, and caspase-9 also showed distinct alterations on their mRNAs, further research on cyclosporin A as an anti-cancer agent will be feasible.

Epigallocatechin-3-gallate (EGCG) Induces Cell Death of Oral Squamous Carcinoma Cells

박형목,이은경,정진,박봉수,박혜련,유미현 대한구강악안면병리학회 2009 대한구강악안면병리학회지 Vol.33 No.4

Green tea, derived from the plant Camellia sinensis, is one of the most common beverages consumed worldwide. Epigallocatechin-3-gallate (EGCG) is the most abundant and bioactive polyphenolic constituent in green tea. Understanding how intracellular signaling pathways respond to EGCG may provide a clue to the difference of cell responses and basis for usefulness of EGCG as a chemopreventive and/or chemotherapeutic agent. In the present study, we tried to check whether EGCG could be a useful agent in chemotherapeutic treatment of oral squamous carcinoma. Furthermore, we investigated which signaling pathway is involved in biologic activities of EGCG. EGCG induced the cell death of oral squamous carcinoma cells. Furthermore, it increased phosphorylation of Akt in serum-strarved oral squamous carcinoma cells. But, initial increase of Akt activation did not affect cell survival. Activities of Raf-1 and Erk showed inconsistent response to EGCG treatment, but Erk phosphorylation is consistent with Raf-1 activity in YD 10B cells. These changes of Raf-1 and Erk activity in EGCG treated cells were different depending on cell line type. Supposedly, the difference of cell component may affect the Raf-1 and Erk reactivity to EGCG treatment. Akt activation by EGCG is independent on activities of PDK1 and PTEN, and expression of bax and bcl-2 proteins were not changed by EGCG treatment. Therefore, EGCG treatment did not induce the apoptosis of YD 10B cell. On the other hand, vascular adhesion molecule (VCAM) was decreased by EGCG treatment, so it is possible that decrease of VCAM can play certain role in survival and/or cell death in EGCG treated cells.

자궁경부 상피내병변과 침습성 편평세포 암종에서의 bcl-2 발현양상

성정희,오수섭,기근홍 조선대학교 부설 의학연구소 2002 The Medical Journal of Chosun University Vol.27 No.2

Background and Objectives: Squamous cell carcinomas (SCC) of the uterine cervix arise from the carcinoma in situ developed in a field of atypical squamous cell proliferation. So, clinical significance of the cervical intraepithelial neoplasm (CIN) are widely accepted. The purpose of this study is to investigate the oncoprotein changes in the sequence of low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), keratinizing type of squamous cell carcinoma (K-SCC) and nonkeratinizing type of squamous cell carcinoma (NK-SCC). Materials and Methods: Immunohistochemical stains for anti-bcl-2 oncoprotein to 120 conized cervical tiussues or resected uterus specimens diagnosed as squamous cell lseions including 30 cases of LSIL, 30 cases of HSIL, 30 cases of K-SCC, and 30 cases of NK-SCC were done. The evaluation of the bcl-2 immunostaining was based on the percentages of the positive neoplastic cells. And statistical analysis of data for the relation between each lesion was performed using the Student t-test. Results: The average positive cell rate was 2.0%, 4.44%, 6.25% and 13.57% in LSIL, HSIL, K-SCC, and NK-SCC, respectively. Statistically, immunoreactive positive cell rate of NK-SCC was significantly higher than those of LSIL (p=0.007) and HSIL (p=0.031). But, there was no significant difference of immunoreactive cell rate between K-SCC and HSIL. Conclusion: These results suggested that bcl-2 may play a role in a cervical tumorigenesis and prognostic relationship with NK-SCC.

Anti-proliferative and Asnti-telomerase Activity of Curcuma Rhizome Extract on Oral Squamous Cell Carcinoma and Osteosarcoma Cells

Kim, Kyung Jin,Kim, Hee Jeong 대한구강생물학회 2007 International Journal of Oral Biology Vol.32 No.4

Anti-proliferation of methanol extract of Curcuma rhizomeon oral squamous cell carcinoma (KB) and osteosarcoma(HOS) cells were investigated. In order to elucidate theinvolvement of telomerase inhibitory activity as a part ofanti-proliferative effect of Curcuma rhizome on cancer cells,we measured telomerase activity in Curcuma rhizomeextract-treated cancer cells. The concentration inhibitedcell proliferation to 50% (IC50) of the methanol extract ofCurcuma rhizome against oral squamous cell carcinoma(KB) cells and osteosarcoma (HOS) cells were 21.30µg/mland 39.3µg/ml, respectively. The methanol extract ofCurcuma rhizome showed inhibitory telomerase inhibitoryeffect which is required for cancer cell immortality.Therefore, it seems that the anticancer effect of methanolextract of Curcuma rhizome is at least partially due totelomerase inhibitory effect. Five fraction samples wereprepared according to its polarity differences and analyzedanti-proliferative effects of each fraction samples on oralsquamous cell carcinoma and osteosarcoma cells. Anticancereffect was observed in dichloromethane, and ethylacetatefractions. The highest anticancer effect was found indichloromethane fraction which had IC50 value of 23.3µg/ml and 10.5µg/ml against oral squamous cell carcinoma(KB) cells and osteosarcoma (HOS) cells, respectively.