• Journal of Korean Neurosurgical Society
• /
• 제30권sup1호
• /
• pp.13-19
• /
• 2001

Objective : We investigated the feasibility of a double suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase(TK) gene and the Escherichia coli cytosine deaminase(CD) gene, via a recombinant adenoviral vector and ganciclovir(GCV) and/or 5-fluorocytosine(5-FC) treatment, in C6 glioma cells. Methods : Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the ${\beta}$-galactosidase gene and then staining cells with 5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene(ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-${\Delta}E1$(as a control). After addition of a variety of concentrations of GCV and 5-FU, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. Result : C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity(>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30ug/ml or GCV at concentrations greater than 0.3ug/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after sinlge-prodrug treatments, indicating additive effects of the prodrug treatments. Conclusion : The administration of a double-suicide gene/prodrug therapy might have great potential in the treatment of brain tumors.

• 생명과학회지
• /
• 제20권5호
• /
• pp.789-793
• /
• 2010

복잡한 진핵생물에서의 물리적 지도 작성이나 기능해석에 효모인공염색체(YAC)를 이용하기 위해서는 원하는 target region의 인공염색체화 및 single-copy인 YAC의 복제수를 늘이는 것이 요구된다. 본 연구에서는 YAC manipulation system에 복제수 증폭시스템(copy number amplification system)을 도입한 Simultaneous YAC Manipulation-Amplification (SYMA) system을 구축하였다. 식물염색체를 가진 YAC clone의 splitting과 증폭을 위해 conditional centromere와 thymidine kinase (TK) 유전자를 가진 pBGTK plasmid를 구축하였고, splitting fragment의 PCR을 위한 주형으로 사용하였다. 590 kb의 YAC clone은 splitting과 동시에 copy number amplification element를 가진 100 kb YAC와 490 kb YAC로 분리되었고, 100 kb YAC는 유도기질로 3 mg/ml sulfanilamide와 $50\;{\mu}g/ml$ methotrexate (S3/M50)의 첨가에 의해 14.4배로 그 복제수가 증가하였음을 확인할 수 있었다.

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

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제27권4호
• /
• pp.314-320
• /
• 2001

Matrix metalloproteinases have long been viewed as ideal candidates for proteinases that enables tumor cells to permeated basement membrane defenses and invade surrounding tissue. There is growing evidence that the MMPs have an expanded role, as they are important for the creation and maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. MT-MMPs are not secreted but instead remaining attached to cell surfaces. Although not all of the MT-MMPs are fully characterized, MT-MMPs have important role in localizing and activating secreted MMPs. The MMP genes are transcriptionally responsive to a wide variety of oncogene, growth factors, cytokine, and hormones. Currently, a number of MMP inhibitors are being developed and some have reached clinical trials as anti-metastatic or anti-cancer therapies. MT1-MMP is involved in the activation of proMMP-2. MT1-MMP is significant not only as a tumor marker but as a new target for chemotherapy against cancer. The purpose of this study was to evaluate the effects of protein kinase C inhibitor(genistein) on the proliferation of HT1080 and expression of MT1-MMP mRNA. Human fibrosarcoma cell line HT1080 was cultured and divided 2 groups. The experimental group was treated with $100{\mu}M$ genistein and incubated 12h, 24h for $[3^H]-thymidine$ uptake assay and northern hybridization individually. And the control group was treated with same amount of PBS for the above procedures. $[3^H]-thymidine$ incorporation was measured with ${\beta}$ ray detector. And RT-PCR and northern blotting for MT1-MMP mRNA was performed. The results were as follows 1. $[3^H]-thymidine$ uptake was reduced in experimental group with statistical significance. 2. MT1-MMP mRNA expression was significantly reduced in experimental group. These results showed that protein kinase C inhibitor (genistein) inhibited proliferation of HT1080 and almost completely blocked transcription of MT1-MMP mRNA. So, it is possible to use the protein kinase inhibitor (genistein) as anti-metastatic and anti-proliferative agent.

• Journal of Microbiology
• /
• 제40권1호
• /
• pp.1-10
• /
• 2002

At the dose of 1000 p.f.u. per mouse,100% mortality occurred in mice inoculated with wild-type pseudorabies virus (PrV). In contrast, upon stable deletion of 10 bp nucleotides at the Bsrl site within the TK gene, PrV was rendered to be completely apathogenic. The deletion also caused the virus to be less capable of replicating in respiratory as well as in nervous system tissues. Although animals were exposed to high titers of TK-deleted PrVs, the virus failed to replicate to a high titer as compared to the pathogenic parental virus. In contrast to previous studies the deletion in the TK gene did not prevent the virus from establishing latency. Upon immunosuppression, the latent virus? however, reactivated but replicated at low titers. Interestingly, TK-deleted virus established latency and reactivation, that are occurred only in trigeminal ganglia and the cerebrums and no other tissues involved. Following reactivation, there was no indication of virus shedding in respiratory tissues as confirmed by virus isolation and polymerase chain reaction (PCR) technique targeting at the gB gene of PrV, The non-pathogenic virus with non-shedding characteristics, upon reactivation of the latent virus, would be the important feature of a live virus vaccine candidate.

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

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

• 대한핵의학회지
• /
• 제38권1호
• /
• pp.62-73
• /
• 2004

목적: 단순 헤르페스 제 1형 티미딘 키나제(herpes simplex virus type 1 thymidine kinase gene: HSV1-tk)는 GCV와 함께 유전자치료의 한 방법으로서 가장 활발하게 연구되어왔으며, HSV1-tk 효소에 대한 다양한 기질들이 연구되어서 이를 보고 기질로 한 비침습적인 HSV1-tk 유전자 영상시스템에서 가장 널리 사용되고 있다. 본 연구에서는 보고기질로서 방사성 요오드가 표지된 5-iodovinyl-2-deoxyuridine (IVDU) and 5 -iodovinyl-2-fluoro-2-deoxyuridine (IVFRU)를 보고 기질로 하여 HSV1-tk 유전자 영상시스템에서의 유용성을 확인하고자 하였다. 대상 및 방법 HSV1-tk 유전자 영상을 위하여 HSV1-tk 유전자를 레트로 바이러스 벡터를 이용하여 Morris hepatoma 세포주(MCA-tk)에 이입한 세포주를 제조하였으며, HSV1-tk 유전자의 발현을 확인하기 위하여 Northern blotting과 Western Blotting을 시행하였다. 대조세포주인 MCA와 제조된 MCA-tk 세포주에 방사표지 IVDU와 IVFRU를 이용하여 480분까지 세포내 섭취율을 평가하였으며, 또한 MCA-tk 세포주의 백분율을 증가시키면서 이에 따른 IVDU와 IVFRU의 섭취율을 평가함으로서 섭취율과 세포수와의 상관관계를 평가하였다. 결과: MCA-tk 세포주에서 HSV1-tk의 mRNA의 발현과 HSV1-TK 단백질의 발현을 확인하였다. 방사성 요오드 표지 IVDU와 IVFRU 모두는 MCA 세포주에서는 아주 낮은 섭취율을 나타내었으며, MCA-tk 세포주에서는 모두 증가된 섭취를 보였다. IVDU가 480분에서 IVFRU보다 4배 높은 섭취를 나타내었다(p<0.01). 방사표지 IVDU와 IVFRU 모두에서 MCA-tk의 백분율의 증가에 따라서 직선적인 상관관계($R^2>0.96$)를 나타내었다. 결론: 방사성 요오드 표지 IVDU와 IVFRU는 HSV1-tk유전자가 이입된 간암세포주에서 모두 특이적인 높은 섭취율을 나타내고 직선적인 상관관계가 나타나서 두 기질 모두 HSV1-tk유전자 영상시스템에서 보고 기질로서 유용하게 사용될 수 있을것으로 기대된다.

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

• Molecules and Cells
• /
• 제21권1호
• /
• pp.104-111
• /
• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.