Human Telomerase Reverse Transcriptase (hTERT): Cisplatin-내성 암의 치료를 위한 목적 단백질

박육필,김광동,강성호,윤도영,박주원,김종완,이희구 대한진단검사의학회 2008 Annals of Laboratory Medicine Vol.28 No.6

Background : Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. Methods : To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. Results : hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. Conclusions : These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy. (Korean J Lab Med 2008;28:430-7) Background : Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. Methods : To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. Results : hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. Conclusions : These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy. (Korean J Lab Med 2008;28:430-7)

시스플라틴 유도 세포사멸에서 Caspase 및 Bcl-2 Family 단백질에의한 끝분절효소 활성 및 인간끝분절효소 역전사효소 발현의 조절

박육필,최승철,조미영,송은영,김재화,백상기,김영권,김종완,이희구 대한진단검사의학회 2006 Annals of Laboratory Medicine Vol.26 No.4

배경 : 인간 끝분절효소(telomerase)는텔로미어(telomere) 반복서열을 합성하는 리보핵산 합성효소이며, 인간 hTERT (humantelomerase reverse transcriptase)는끝분절효소의 rate-limiting요소일 뿐 아니라 활성단위체로서 동정되었다. 본 연구에서는 끝분절효소 조절자를 동정하고, 시스플라틴 유도 세포사멸에서의분자적 메커니즘을 분석하고자 하였다. 방법 : 시스플라틴 유도 세포사멸에서의 끝분절효소의 기능을규명하기 위해, 끝분절효소 활성을 측정하고, 세포사멸을 PI 염색과 trypan blue 염색으로 분석하였다. 또한, hTERT 단백질 발현에 있어서의 효과를 결정하기 위해 Z-VAD-fmk로 caspase활성을 억제하였다. 마지막으로 HEK293 세포주에Bcl-2와 Bak유전자를 일시적으로 동시에 과발현시킨 다음, 끝분절효소 활성및 hTERT 발현을 분석하였다. 결과 :Bcl-2를 과발현하는 HeLa 세포에서는 끝분절효소 활성이 더욱 증가되었고, 세포사멸이 mock 대조군에 비해 40-50%정도 감소되었다. 이는 Bcl-2에 의해 유도된 끝분절효소활성이 시스플라틴-유도 세포사멸에서 항세포사멸적 효과를 발휘함을 제시한다. Z-VAD-fmk로 caspase의 활성화를 억제함으로써 Mock대조군 세포에서는 hTERT 단백질 발현이 회복되나, Bcl-2 발현세포에서는 변화가 없었다. 이는 hTERT의 발현이 caspase에 의해 조절될 수 있으나, Bcl-2는 상위 신호전달 체계에 존재하고 있음을 제시한다. 또한, HEK293 세포주에 Bcl-2와 Bak 유전자를동시에 과발현 시켰을 때, 끝분절효소 활성과 hTERT 단백질 발현이 뚜렷이 감소되었다.

Mac-2 결합단백질 발현에서의 STAT3의 역할

박육필,김준태,양영,임종석,윤도영,김종완,이희구 대한진단검사의학회 2008 Annals of Laboratory Medicine Vol.28 No.3

Background : Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level. Methods : To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA. Results : EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5′-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (-2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator. Conclusions : EGF signal is critical for the Mac-2BP expression, and more importantly, STAT3 could work as a negative regulator, while hTERT as a positive regulator in Mac-2BP transcription. (Korean J Lab Med 2008;28:230-8) Background : Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level. Methods : To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA. Results : EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5′-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (-2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator. Conclusions : EGF signal is critical for the Mac-2BP expression, and more importantly, STAT3 could work as a negative regulator, while hTERT as a positive regulator in Mac-2BP transcription. (Korean J Lab Med 2008;28:230-8)

주변식물의 유관속 관찰

육필상,박인근 충북대학교 과학교육연구소 1998 과학교육연구논총 Vol.14 No.1

This study was aimed to suggest that we used as science teaching material Cucurbitaceae plants which are easily obtainable for the formation of concept on the vascular bundle in the higher plants. We could not easily observe the vascular bundle from teaching materials as Balsam in the current textbook and also around plants as Black nightshade, Amaranthus, Egg plants, and etc. We could observe only vessels from the materials of the current textbook. However, in the case of the use of Cucurbitaceae plants it was easy to not only observation of vessels but also sieve tubes and companion cell. We could easily get the material of Cucurbitaceae plants and made a microscopic section by knife and simple process. Therefore, this investigator recommend to use Cucurbitacese plants as teaching material in primary and secondary school to make the student obtain the improvement of learning effect.

Analysis of Telomerase Activity by HPV E6/E7 Expression in SW13

김영권,박육필 대한의생명과학회 2006 Biomedical Science Letters Vol.12 No.4

Bcl-2 유사인자 Bfl-1과 결합하는 IKK- 의 C말단 Ser부위

윤현경,김진구,조희준,조미영,박육필,유관희,김종완,윤도영,최용경,이희구 대한진단검사의학회 2005 Annals of Laboratory Medicine Vol.25 No.3

Background : Bcl-2 family proteins play a central role in regulating apoptosis. In human, over 20 members of this family have been identified to date. Bfl-1, a member of the Bcl-2 family, has been known to retard apoptosis in various cell lines. However, the function of Bfl-1 remains unclear. Methods : In order to investigate the Bfl-1 function, we employed yeast two-hybrid system to identify the proteins which are capable of interacting with Bfl-1. The interaction of inhibitor kappaB kinase- (IKK- ) and Bfl-1 was confirmed using glutathione S-transferase pull down assays. To determine which regions of IKK- were required for interaction with Bfl-1, we constructed 12 deletion mutants of IKK- and 5 deletion mutants of Bfl-1. Results : Bfl-1 interacted with the C-terminal region of IKK- which is a subunit of IKK complex, and IKK- activity is very important in the NF- B related pathway. In addition, the amino acids 673- 745 of IKK- were important for Bfl-1 interactions, and amino acids 1-484 of Bfl-1, including Bcl-2 homology domains (BH1, BH2, BH3, BH4), were crucial for IKK- interactions. Conclusions : IKK C-terminus contains many serine residues as binding partner of Bfl-1. Our results suggested that Bfl-1 is involved in the NF- B activation through interaction of IKK- and Bfl- 1. Further studies need to be performed to understand functions of the IKK- and Bfl-1 associated with the regulation of the NF- B activation pathway. 배경 : Bcl-2 family 단백질은 세포사멸을 조절하는 데 중요한역할을 한다. 인체에서 현재까지 20여가지 이상의 Bcl-2 family가 알려지고 있다. Bfl-1은 Bcl-2 family member 중 하나로서여러 가지 세포주에서 세포사멸을 지체시키는 것으로 알려지고있으나 Bfl-1의 명확한 기능은 아직알려지지 않았다.방법 :Bfl-1의 기능을 분석하기 위하여 yeast two-hybrid sys-tem을 적용하여 Bfl-1과 결합할 수 있는 단백질을 확인하였다.GST pull down assay를 이용하여 Bfl-1과 IKK- 의 결합 반응을 확인하였다. 12가지의 IKK- deletion mutant와 5가지의Bfl-1 deletion mutant를 제조하여 Bfl-1과 결합하는 데 필요한IKK- 의 부위를 결정하였다.결과 : Bfl-1은 IKK- (IKK complex의 단위분자이면서 NF-B관련 신호전달에 중요한 역할을 함)의 C 말단 부위와 결합하였다. 또한 IKK- 의 673에서 745의 잔기가 Bfl-1과의 결합에 중요하였고, Bfl-1의 1에서 484의 잔기(Bcl-2 유사 부위 BH1, BH2,BH3, BH4 포함)가 IKK- 결합에 필수 적이었다. 결론 : IKK- 의 C말단은 Bfl-1의 결합파트너로서 여러 개의 serine잔기를 함유하고 있다. 본 연구의 결과를 미루어보면 IKK-와 Bfl-1과의 결합은 NF- B활성조절에 중요한 역할을 할 것으로 사료되었다. NF- B활성 신호 조절에 관련된 IKK 와 Bfl-1간의 기능을 좀 더 연구하기 위해서는 추가연구가 필요하다

인간 파필로마 바이러스 E6/E7에 의한 Telomerase 활성

김영권 ( Young Kwon Kim ),서충원 ( Choong Won Seo ),김상하 ( Sang Ha Kim ),박육필 ( Yuk Pheel Park ) 대한임상검사과학회 2007 대한임상검사과학회지(KJCLS) Vol.39 No.1

Cervical cancer is one of the most prevalent cancers developed in women worldwide, and human papillomavirus(HPV) type 16 is the most common agent linked to human cerivical carcinoma. Viral oncogenes E6 and E7 are selectively retained and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperate with each other in the immortalization and transformation of primary keratinocytes. Because the HPV oncogenesis mechanism was not completely solved, more thorough studies are required. In the present study, we investigated the telomere independent role of telomerase in HPV oncogenesis, we constructed the E6 mutant, E7, E6/E7 and hTERT over-expressed stable cells with a telomerase negative cell line, SW13. Expressions of inserted genes were measured by RT-PCR. E6, E7 and hTERT genes were well expressed in each cell lines when compared with the control groups. By analyzing the cell morphology under the microscope, hTERT clone size was a smaller than the mock control but oncogene expressed clones had a slightly lengthened marginal region. In addition, hTERT cells also has a tendency of brief dividing time compared to the mock control. To determine whether telomerase activity was associated with a HPV oncogenesis by oncoprotein expression, we performed the PCR based TRAP assay and a Northern blot analysis. In TRAP assay data, telomerase activities in hTERT and oncogene clones increased compared to the mock control. In addition, SW13/E6/E7 cells showed an extremely increased activity compared to the other clones. Induced hTERT mRNA by E6/E7 wasn``t, however, detected in Northern blotting. In conclusion, these findings suggest that telomerase activity is closely associated with the HPV oncogenesis and E6/E7 co-expression is a most important factor of telomerase activity.