부산지역 유흥업소 종사여성으로부터 분리된 HPV16형의 발암유전자(E6/E7) 돌연변이 유형 분석

민상기(Sang-Kee Min),김성순(Sung Soon Kim),최병선(Byeong-Sun Choi),장대호(Dai-Ho Jang),이미옥(Mee-Ok Lee),최성화(Seung-Hwa Choi),김남호(Nam-Ho Kim),박연경(Yon-Koung Park),정영아(Yeong-A Jeong),김성준(Seong-Joon Kim),빈재훈(Jae-Hun Bi 한국생명과학회 2009 생명과학회지 Vol.19 No.6

HPV-16형의 염기배열 변이는 지역적, 인종적으로 특징적인 차이가 있으며 특히 HPV-16형 E6/E7 유전자의 특정 염기서열변이는 자궁경부암 및 자궁상피내 신생종양물의 발생을 일으키는 고위험 요인으로 알려져 있다. 본 연구는 2007년 부산지역 유흥업소 종사여성으로 분리된 HPV-16형 19건을 대상으로 E6/E7 유전자 영역(nt 34-880)을 표적으로 지역적 염기서열 변이를 조사하였다. nucleotide 수준에서 HPV16형 E6 유전자는 T178G (n=11), T178A (n=1), T350G (n=4), A442C (n=2), A104T, A111G, C116T, G145T, T183G, C335T, G522C 등 11종의 변이주가 발견되었고, E7 유전자는 A647G (n=12), A645C, A777C, G663A, T732C, T760C, A775T, T789C, T795G 등 9종의 변이주가 발견되었다. 아미노산 수준에서는 HPV-16형 E6 단백질의 경우 D25E (n=12), L83V (n=4), E113D (n=2), M1L, Q3R, P5S, Q14H, D25N, I27R, H78Y, C140S 등 11종의 변이주를, HPV16형 E7 단백질의 경우 N29S (n=12), L28F, T72S 등 3종의 변이주를 관찰할 수 있었다. 본 연구 결과, 부산지역의 HPV-16형 E6/E7 우점 돌연변이주는 E6 D25E (75%), E7 N29S (78%)로 각각 나타났다. 앞으로 자궁경부암 환자 및 일반여성을 포함한 더 많은 모집단을 대상으로 HPV-16형 E6/E7의 intratypic variants를 비교 조사하여 실제 HPV-16형 E6/E7 어떤 변이주가 자궁경부암 유발 위험성과의 관련성은 더 많이 연구되어져야 할 것으로 사료된다. Recent studies have reported that the distribution of HPV-16 sequence variation differs geographically, and more specifically that HPV-16 E6/E7 intratypic variants might carry a high risk for development of ICC (invasive cervical cancer) and CIN (cervical intraepithelial neoplasia) in a given population. To investigate the genetic diversities of HPV-16 E6/E7 oncogene by region, we collected nineteen HPV-16 isolates from sexually high-risk women in Busan, and analyzed the HPV-16 E6/E7 coding regions (nt 34 to 880) with HPV-16 E6/E7 specific PCR amplification. At the nucleotide level, eleven variants of the E6 genes and nine variants of the E7 genes were identified as follows: E6 T178G (n=11), E6 T178A (n=1), E6 T350G (n=3), E6 A442C (n=2), E6 A104T, E6 A111G, E6 C116T, E6 G145T, E6 T183G, E6 C335T, E6 G522C and E7 A647G (n=12), E7 A645C, E7 A777C, E7 G663A, E7 T732C, E7 T760C, E7 A775T, E7 T789C and E7 T795G, respectively. At the amino acid level, the isolated HPV-16 E6 and E7 genes showed eleven E6 variants: E6 D25E (n=12), E6 L83V (n=4), E6 E113D (n=2), E6 M1L, E6 Q3R, E6 P5S, E6 Q14H, E6 D25N, E6 I27R, E6 H78Y, E6 C140S and three E7 variants: N29S (n=12), L28F, T72S. HPV16 E6 L83V, the dominant variant in the Caucasian population, showed relatively low frequencies in our study population. We elucidated that the dominant HPV-16 E6/E7 variants were HPV-16 E6 D25E (63.2%) and HPV-16 E7 N29S (63.2%), which were phylogenetically included in Asian lineage. Further study is needed to evaluate the risk of cervical cancer related HPV-16 E6/E7 intratypic variants in the Korean population.

Cloning of E6/E7 Genes of Human Papillomavirus Type 16 DNA

Kook, Joong-Ki,Kim, Gwan-Shik,Min, Byung-Moo The Official Publication of Korean Academy of Oral 1996 International Journal of Oral Biology Vol.20 No.1

To obtain the specific probe which can detect human papillomavirus type 16(HPV-16)-specific transcripts encoding for E6 and E7 genes in cells or cell lines containing HPV-16 DNA, we cloned 0.57-kbp fragment (nucleotides 198-767) of the HPV-16 DNA into pUC18 and named pHPV-16-E6E7R. To test the specificity of the cloned probe compared with whole HPV-16 genome, we cultured normal human oral keratinocytes, and HOK-16B cells, human oral keratinocytes immortalized by transfection with recombinant HPV-16 DNA, in keratinocyte growth medium supplemented with pituitary extract, isolated poly(A^+)RNAs from the cells, and hybridized them with the cloned probe and whole HPV-16 genome. Northern blot analysis showed that multiple poly(A^+)RNAs hybridized to whole HPV-16 genome were expressed from the HPV-16-immortalized HOK-16B cells, while only two transcripts with sizes of 1.9-kb and 1.6-kb hybridized to HPV-16 E6/E7 gene fragment were detected from the cells. However, both probes did not detect the viral transcripts in normal numan oral keratinocytes which are not infected with HPV-16. There data indicate that 0.57-kbp fragment representing HPV-16 E6/E7 genes can be used to detect HPV-16-specific transcripts encoding for E6 and E7 genes in cells or cell lines containing HPV-16 DNA.

Effecs of Dexamethasone and Epidermal Growth Factor on Activity of Viral Promoter p97 and Expression of HPV-16 E6/E7 in Human Oral Keratinocytes Transformed with HPV-16 and Benzo(a)pyrene

Kook, Joong-Ki,Lee, Gene,Woo, Kyung Mi,Min, Byung-Moo The Official Publication of Korean Academy of Oral 1995 International Journal of Oral Biology Vol.19 No.2

Primary human oral keratinocytes were previously transformed by transfection with cloned human papillomavirus type 16(HPV-16)DNA and subsequent exposure to benzo(a)pyrene, and an oral cancer cell line, CTHOK-16B-BaP, was established. To determine the effects of dexamethasone and epidermal growth factor (EGF) on cell proliferation, the expression of HPV-16 E6/E7 and several proto-oncogenes, and activity of HPV-16 E6/E7 promoter, p97, the CTHOK-16B-BaP cells were exposed to either dexamethasone and EGF alone or together. After incubation for 3 days, the degrees of both cell proliferation and the expression of HPV-16 E6/E7, EGF receptor (EGFR), c-myc, and c-fos genes was determined. Dexamethasone and EGF, when added alone or together in the culture media, increased cell proliferation. Although dexamethasone did not affect the transcriptional levels of HPV-16 E6/E7, EGFR, or c-myc in the cells, it down-regulated c-fos mRNA expression. On the other hand, EGF, alone or in conjuction with dexamenthasone, down-regulated the transcriptional levels of HPV-16 E6/E7, c-myc, and c-fos genes, but it had little effect on the level of EGFR transcription. These results suggest that the increased proliferation of CTHOK-16B-BaP cells in the presence of dexamethasone and/or EGF may not depend on the extent of expression of such genes as HPV-16 E6/E7, EGFR, c-myc, and c-fos. In addition, the HPV-16 E6/E7 mRNA level was not changed and reduced by treatment with dexamethasone and EGF, respectively. Unexpectedly, however, dexamethasone and EGF enhanced the activity of the viral promoter p97 in CTHOK-16B-BaP line as analyzed by transient expression assays using the chloramphenicol acetyltransferase gene as a reporter. It appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to dexamethasone and EGF. Another EGF-responsive element (7454-7643 nt) which acts as an enhancer may be located within the HPV-16 LCR portion.

Activities of E6 Protein of Human Papillomavirus 16 Asian Variant on miR-21 Up-regulation and Expression of Human Immune Response Genes

Chopjitt, Peechanika,Pientong, Chamsai,Bumrungthai, Sureewan,Kongyingyoes, Bunkerd,Ekalaksananan, Tipaya Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.9

Background: Variants of human papillomavirus (HPV) show more oncogenicity than do prototypes. The HPV16 Asian variant (HPV16As) plays a major role in cervical cancer of Asian populations. Some amino acid changes in the E6 protein of HPV16 variants affect E6 functions such as p53 interaction and host immune surveillance. This study aimed to investigate activities of HPV16As E6 protein on modulation of expression of miRNA-21 as well as interferon regulatory factors (IRFs) 1, 3, 7 and c-fos. Materials and Methods: Vectors expressing E6 protein of HPV16As (E6D25E) or HPV16 prototype (E6Pro) were constructed and transfected into C33A cells. HCK1T cells expressing E6D25E or E6Pro were established by transducing retrovirus-containing E6D25E or 16E6Pro. The E6AP-binding activity of E6 and proliferation of the transfected C33A cells were determined. MiR-21 and mRNA of interesting genes were detected in the transfected C33A cells and/or the HCK1T cells, with or without treatment by culture medium from HeLa cells (HeLa-CM). Results: E6D25E showed binding activity with E6AP similar to that of E6Pro. Interestingly, E6D25E showed a higher activity of miR-21 induction than did E6Pro in C33A cells expressing E6 protein. This result was similar to the HCK1T cells expressing E6 protein, with HeLa-CM treatment. The miR-21 up-regulation significantly corresponded to its target expression. Different levels of expression of IRFs were also observed in the HCK1T cells expressing E6 protein. Interestingly, when treated with HeLa-CM, IRFs 1, 3 and 7 as well as c-fos were significantly suppressed in the HCK1T cells expressing E6D25E, whereas those in the HCK1T cells expressing E6Pro were induced. A similar situation was seen for IFN-${\alpha}$ and IFN-${\beta}$. Conclusions: E6D25E of the HPV16As variant differed from the E6 prototype in its activities on epigenetic modulation and immune surveillance and this might be a key factor for the important role of this variant in cervical cancer progression.

B Cells Transduced with HPV16 E6/E7-expressing Adenoviral Vector Can Efficiently Induce CTL-dependent Anti-Tumor Immunity

Kim, Yun-Sun,Ko, Hyun-Jeong,Kim, Yeon-Jeong,Han, Seung-Hee,Lee, Jung-Mi,Chang, Woo-Sung,Jin, Hyun-Tak,Sung, Young-Chul,Kang, Chang-Yuil The Korean Association of Immunobiologists 2007 Immune Network Vol.7 No.3

Background: Human papillomavirus (HPV) infection is responsible for cervical cancer, a common cancer in women. Since HPV infection and cancer development are controlled by the host immune system, immunotherapy against HPV can be helpful in preventing or treating HPV-associated cervical cancer. Two oncoproteins of HPV16, E6 and E7, are promising targets for immunotherapy against cervical cancer, because they are constitutively expressed in cervical cancer. Methods: Since cellular vaccines using B cells as well as dendritic cells offer an efficient approach to cancer immunotherapy, we opted to use B cells. We evaluated the immunogenicity and anti-tumor effects of a B cell vaccine transduced with HPV16 E6/E7-expressing adenovirus. Results: Vaccination with HPV16 E6/E7-transduced B cells induced E6/E7-specific $CD8^+$ T cell-dependent immune responses and generated anti-tumor effects against E6/E7-expressing TC-1 tumor. The anti-tumor effect induced by this B cell vaccine was similar to that elicited by DC vaccine, showing that B cells can be used as an alternative to dendritic cells for cellular vaccines. Conclusion: Thisstudy has shown the feasibility of using B cells as immunogenic APCs and the potential for developing prophylactic and therapeutic vaccines against HPV-associated cervical cancer using a B cell vaccine transduced with adenovirus expressing HPV16 E6/E7.

Situation of HPV16 E2 Gene Status During Radiotherapy Treatment of Cervical Carcinoma

Kahla, Saloua,Kochbati, Lotfi,Maalej, Mongi,Oueslati, Ridha Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.6

Background: Human papillomavirus (HPV) integration within the E2 gene has been proposed as a critical event in cervical carcinogenesis. This study concerned whether HPV16 status and E2 gene intactness are predictive of radiation response in patients with cervical cancer. Materials and Methods: Biopsies of 44 patients with cervical cancer were collected before or after radiotherapy. The presence of HPV16 was assessed by polymerase chain reaction (PCR) using specific primers for the L1 region. E2 disruption was detected by amplifying the entire E2 gene. Results: HPV16 DNA was found in 54.5% of the clinical samples. Overall, 62.5% of the HPV16 positive tumors had integrated viral genome and 37.5% had episomal genome. There was a tendency of increase of HPV16 E2 negative tumors compared with HPV16 L1 ones in advanced stages (75% versus 20% in stage III respectively). Detection of E2 gene appeared influenced by the radiotherapy treatment, as the percentage of samples containing an intact HPV16 E2 was more frequent in pretreated patients compared to radiotherapy treated patients (66.6% versus 20%). The radiation therapy caused an eight-fold [OR= 8; CI=1.22-52.25; p=0.03] increase in the risk of HPV16 genome disruption. The integration status is influenced by the irradiation modalities, interestingly E2 disruption being found widely after radiotherapy treatment (75%) with a total fractioned dose of 50Gy. Conclusions: This study reveals that the status of the viral DNA may be used as a marker to optimize the radiation treatment.

Inhibition of Cervical Cancer Cell Growth by Gene Silencing of HPV16 E6 Induced by Short-interfering RNA

( Sang-muk Park ),( Sun-kyung Lee ),( Yoon-sik Kim ) 대한임상검사과학회 2011 대한임상검사과학회지(KJCLS) Vol.43 No.3

The Human Papilloma Virus (HPV) infection has been strongly associated with pathogenesis of uterine cervix carcinoma. HPV type 16, a causative agent of uterine cervix carcinoma, encodes the E6 and E7 oncogenes, expression of which is pivotal for malignant transformation and maintenance of malignant phenotypes. To develop a gene therapy for HPV-related carcinoma, We investigated the effect of E6 short-interfering RNA (E6 siRNA) on the expression of this oncogene and on the growth of HPV 16-related uterine cervix carcinoma cells. SiHa cells, a uterine cervix carcinoma cell line, which contain a single copy of HPV 16 integrated in the chromosome and express the E6 and E7 oncogenes. Before 24 hr of transfection, cells were seeded and transfected with control plasmid or E6 siRNA-expressing plasmid. The mRNA was analysed by reverse transcriptase polymerase chain reaction (RT- PCR). The cell growth rate was investigated by MTT method. The E6 mRNA level in SiHa cells was decreased in HPV 16 E6 siRNA-expression vector transfected cells and a decrease in the growth of these cells was also observed. From these results. it is evident that E6 siRNA played a role in suppression of growth of SiHa cells and has a fair chance as a candidate for gene specific therapy for HPV related uterine cervix carcinoma.

cDNA microarray를 이용한 HPV-16 E6와 연관된 유전자의 발현 분석

명진 ( Jin Myeong ),라선영 ( Sun Young Rha ),이명진 ( Myoung Jin Lee ),엄수종 ( Soo Jong Um ),남궁성은 ( Sung Eun Namkoong ),박종섭 ( Jong Sup Park ) 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.12

목적 : 이 연구는 자궁경부암의 주요 발암 원인 인자로 알려진 HPV-16 E6의 발현이 세포내 유전자들의 전사조절에 미치는 영향을 관찰하기 위해서 시행되었다. 연구 방법 : HPV-16 E6를 안정적으로 발현하는 A549E6와 RC10.1 세포주를 실험군으로 사용하여 cDNA microarray 실험을 시행하였다. 결과 : A549E6 세포주에서 HPV-16 E6의 세포내 발현으로 증가된 발현양상을 보인 주요 유전자들로는 GTPase-activatin Objective : To examine the effect of HPV-16 E6 expression on the transcription of cellular genes, we used cDNA microarray in HPV-16 E6 transfected stable cancer cell lines. Methods : Using cDNA microarray consisting of 1,024 genes, we have performed a sys

유방의 침윤성 관암종에서 pRb, p16, cyclin D1, Cyclin E의 발현

허혜경,노미숙,정진숙,나서희,허기영,홍숙희 대한병리학회 2001 Journal of Pathology and Translational Medicine Vol.35 No.4

한국인 자궁경부암 조직에서 분리한 HPV16형 E6 유전자의 대장균에서의 발현

이영희,진승원,윤희식,박병철,임종석,이희구,정현호,최인성,박순희 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.2

Human papillomaviruses, especially type 16 and 18, have been reported to be closely associated with human cervical lesions and carcinoma. One of the open reading frames, E6 has been identified as a transforming gene. We have isolated the HPV16 E6 oncogene from Korean cervical carcinoma tissues, subcloned into an expression plasmid pET-3a, and produced 16E6 proteins in E. coli under the control of T7 promoter. Recombinat E6 protein was successfully produced in high level upto 30% of total proteins and specifically recognized by anti-16E6 polyclonal antibodies produced by rabbit immunized with TrpE-16E6 fusion protein. 16E6 protein was produced in insoluble form. Currently we are establishing purification procedure of solubilized E6 protein.