• Laboratory Medicine Online
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• 제6권1호
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• pp.25-30
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• 2016

배경: Thymidine kinase 1 (TK1)은 세포 주기의 중요한 조절 효소로 암세포의 증식 시에 증가하는 것으로 알려져 있으며, 현재까지 혈액종양과 다양한 고형암에서 진단 또는 치료 후 모니터링과 예후 예측에 중요한 표지자로 보고되고 있다. 본 연구에서는 한국인에서 혈청 TK1 정량분석을 통하여 건강인의 혈청 TK1 참고치를 설정하고자 하였으며 B세포림프종 환자를 대상으로 임상적 악성도 표지자로서 혈청 TK1 검사의 유용성을 평가하고자 하였다. 방법: 72명의 B세포림프종 환자와 143명의 건강대조군의 혈청검체에서 화학발광면역측정법으로 혈청 TK1 농도를 측정하였다. 건강대조군에서 혈청 TK1의 참고치를 설정하였고, 환자군과 건강대조군에서 측정된 혈청 TK1 결과를 이용해 ROC 분석을 통한 기준치를 구하여 상대적으로 높은 혈청 TK1 정량값과 B세포림프종의 임상 지표들과의 상관성을 비교하였다. 결과: 전체 건강대조군의 혈청 TK1의 95 percentile에 따른 참고범위는 5.4-21.8 U/L였다. B세포림프종 환자군과 건강대조군의 혈청 TK1 수치 비교에서 평균${\pm}$표준편차는 각각 $40.6{\pm}68.5U/L$와 $11.8{\pm}4.4U/L$로 나타났으며 두 그룹 간에 유의한 차이를 보였다(P<0.001). ROC 분석 후, 혈청 TK1 수치 15.2 U/L를 이용하였을 때 민감도 59.7%, 특이도 83.2%, AUC 0.73을 보여 B세포림프종 환자를 선별할 수 있는 기준치로 설정하였다. 상대적으로 높은 혈청 TK1 수치(${\geq}15.2U/L$)는 병기, 골수침범, IPI 점수, LD 수치, 낮은 Hb (<12 g/dL), 림프구 수와 상관성이 있는 것으로 나타났다. 결론: B세포림프종 환자에서 측정되는 혈청 TK1 수치는 B세포림프종의 임상적 악성도 표지자로서 유용하게 사용될 수 있을 것이다.

• IMMUNE NETWORK
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• 제1권1호
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• pp.45-52
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• 2001

Background: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. Methods: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. Results: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X $10^6CFU/ml4$. After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. Conclusions: The results suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.

• 대한핵의학회지
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• 제36권2호
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• pp.102-109
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• 2002

목적: 유전자 치료에서, 종양세포에 herpes simplex virus thymidine kinase 유전자를 발현시키고 이에 민감한 prodrug을 전신투여하여 발현된 종양세포에 특이적으로 집적되는 방법은 매우 효율적인 방법이다. 대표적인 prodrug인, IVDU, IVFRU는 herpes simplex virus type 1의 비침습적 영상화 방법에 응용되는 방사옥소 표지가능 화합물이다. 재료 및 방법: Trans-l-trimethylsilyl-2-tri-n-butylstannylethylene과 uridine 변형체상의 iodo-uracil을 Pd촉매하에서 반응시켜 표지전구체인 5-trimethylvinyl-deoxy-uridine과 5-trimethylsilylvinyl-deoxy-fluororibofuranosyl uracil을 합성하였다. 이것을 column chromatography의 방법으로 분리 정제하였다. 합성한 각 전구체를 ICI 담체를 이용하여 높은 수율로 표지하였고, HPLC를 사용하여 분리하였다. Radiohalogen exchange 방법은 비방사능을 낮게 할수록 표지에 효율적으로 알려져 있다. 이 방법으로 ICI 방법을 사용하여 담체를 포함하는 높은 방사능의 화합물을 얻을 수 있다. 결과: IVDU와 IVFRU 표지를 위한 전구체 합성수율은 각각 43, 72% 였다. ICl로 담체를 이용한 표지방법을 사용하여 IVDU와 IVFRU 표지수율은 98% 이상이었다. 결론: HSV1-tk를 이용한 유전자 영상용 기질의 전구체를 성공적으로 합성하였고. 95% 이상의 높은 표지수율의 유전자 영상용 방사성 추적자를 성공적으로 제조하였다.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2004년도 춘계학술대회
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• pp.82-82
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• 2004

• 농약과학회지
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• 제6권3호
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• pp.157-165
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• 2002

화학물질의 안전성확보를 위한 여러 노력들이 경주되고 있다. 최근에 들어 이러한 안전성평가기법은 고전적인 안전성평가기법으로부터 분자생물학 기술의 발전에 힘입어 세포, 분자 독성학(Cellular and Molecular Toxicology) 으로 발전되어오고 있다. 여기에서는 최근에 이루어지고 있는 이러한 발전에 따라 국제적으로 널리 사용되고 있는 진보된 안전성평가 기법들을 중심으로 소개하고자 한다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.185-188
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• 2001

We have developed a synthetic method for dTDP-4-keto-6-deoxy-D-glucose with four enzyme system. We have used crude extracts from cultures of Escherichia coli BL21 strains harboring plasmids containing different sources. dTDP-4-keto-6-deoxy-D-glucose was synthesized by the combination of thymidine-monophosphate kinase, acetate kinase, dTDP-glucose synthase and dTDP-D-glucose 4,6-dehydratase in a batch system, starting the reaction with dTMP. The enzymatic synthesis strategy allowed a dTMP conversion with a 95%.

• BMB Reports
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• 제37권4호
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• pp.503-506
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• 2004

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.

• Journal of Pharmaceutical Investigation
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• 제33권4호
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• pp.267-272
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• 2003

Herpes simplex virus (HSV) thymidine kinase (TK) has been widely used for suicidal gene therapy in combination with nucleoside analogs such as ganciclovir (GCV). The use of HSV-TK is limited due to the side effect of GCV at high concentrations. Previous studies showed that stable transfectants of mutant HSV-TK with enhanced affinity to GCV were killed at lower GCV concentrations. In this study, we tested whether mutant HSV-TK can provide enhanced suicidal effect when transiently transfected with Epstein-Barr virus (EBV)-based plasmid. EBV-based plasmid which contains OriP and EBNA-1 sequence is well known for a stable episomal maintenance in human cells. Optimal transfection condition was assessed for SNU-638 gastric cancer cell line using polyetylnimine (PEI). Maximum transfection efficiency was achieved when DNA:PEI was 1:3 (w/v). Cytotoxicities of mutant and wild type HSV-TK were compared before and after partially selecting transfected cells. The cells were sensitive to $100\;{\mu}g/ml$ hygromycin. Following GCV treatment, more cells were killed after hygromycin selection than before selection. The mutant HSV-TK showed enhanced cytotoxicity compared with the wild type HSV-TK. Our results suggest that the EBV-based plasmid encoding mutant HSV-TK may be useful to treat the diseases caused by uncontrolled cell proliferation such as cancer and rheumatoid arthritis.

• Biomolecules & Therapeutics
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• 제22권2호
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• pp.114-121
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• 2014

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

• 대한한의학회지
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• 제20권3호
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• pp.94-104
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• 1999

Objectives: Hepatoma is a very serious disease in Korea and worldwide. Hepatitis B virus (HBV) has proved the most significant cause of hepatoma. We carried out this study to investigate the effect of Injinchunggan-tang (Yinchenqinggan -tang) on inhibiting cell proliferation and DNA synthesis in HepG2.2.15 cell lines and on inhibiting phosphorilation of oncogene (MAP kinase) in NIT /3T3-HEx cells. Methods: First we confinned the Hepatitis B virus producing ability of HepG2.2.15 cells. To investigate the anti-cancer effect of Injinchunggan-tang (Yinchenqinggan-tang), we did the NTS/PMS assay, [3H]-thymidine incorporation assay and transfection of pcDNA-X. We also measured the gene expression through western blotting. Results: Injinchunggan-tang (Yinchenqing gan tang) showed the suppressing effect of HepG2.2.l5 increase in the MTS/PMS assay and the inhibiting effect of DNA synthesis of HepG2.2.15 in the [3H] thymidine incorporation assay. Injinchunggan-tang (Yinchenqinggan-tang) also showed the inhibiting phosphorilation effect of MAP kinase in HBV -X genes. Conclusions: From the above Injinchunggan-tang (Yinchenqinggan-tang) is thought to have an anti-cancer effect on the hepatoma from HBV. It is suggested that further studies on this prescription would give us a better medicine with an anti-cancer effect.