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Toll-like Receptor 4-mediated Apoptotic Cell Death in Primary Isolated Human Cervical Cancers
Jinyoung Won(원진영),Yunkyung Hong(홍윤경),Sookyoung Park(박수경),Joo-Heon Kim(김주헌),Yonggeun Hong(홍용근) 한국생명과학회 2018 생명과학회지 Vol.28 No.6
Toll 유사수용체의 TLR4는 세포자연사(apoptosis)와 관련하여 세포의 생존과 증식에 영향을 미치는 것으로 알려져 있다. 본 연구에서는 TLR4의 활성이 부인과 질환 특이적 종양세포의 세포사멸기작에 어떠한 영향을 미치는지 살펴보았다. TLR4의 활성에 의한 세포자연사를 확인하기 위하여 부인암 조직(자궁경부암, 자궁내막암, 난소암)에서 종양세포를 분리하여 초대배양시스템을 구축하였고, lipopolysaccharide (LPS)에 의한 TLR4의 활성유도 과정에서 종양세포의 형태학적 변화를 살펴보았다. 또한, TLR4 매개성 세포사멸 기작을 확인하기 위하여 역전사 중합효소 연쇄반응(RT-PCR)을 통해 유전자 분석을 진행하였다. 연구 결과, 부인암의 초대배양세포에서 세포접촉저지(contact inhibition)현상이 감소되었고, 세포의 배가시간(doubling time)이 단축되어, 종양세포의 성장률 변화를 확인하였다(p<0.05). 자궁근육층(정상조직)의 초대배양세포에서는 민무늬근육 확인 인자인 ITGA5 (an alpha5 integrin marker)의 유전자 발현이 나타났으나, 자궁경부조직의 초대배양세포에서는 발현변화를 확인할 수 없었다. 종양세포의 유전자분석 결과에서 p53과 같은 종양억제인자의 발현이 유의적으로 증가한 반면(p<0.05), 세포사멸 신호기작과 관련하여 TLR4와 Caspase-3의 발현은 감소하였다(Caspase-3, p<0.05). LPS를 처리한 종양세포에서는 LPS 비처리군과 비교 시, TLR4의 발현증가와 함께 Caspase-3의 발현변화가 동반되었다. 이러한 결과들은 TLR4 매개성 apoptosis 유도가 종양세포의 증식억제에 중요한 영향을 미치는 것을 의미하며, TLR4 신호기작을 이용한 종양세포의 새로운 치료적 접근법을 제시할 것으로 기대한다. Toll-like receptor 4 (TLR4) has been implicated in cell proliferation and apoptosis in several types of cancer. In this study, the impact of TLR4 activation on apoptotic cell death in gynecologic cancers induced by lipopolysaccharide (LPS) was investigated. Cervical cancer cell lines were produced from isolated surgical specimens supplied by Paik Hospital. The primary cultures of normal myometrium and gynecologic cancers, including cervical, endometrial, and ovarian cancers, were used to examine the differences in morphological characteristics between normal and cancerous cells. A reverse transcription polymerase chain reaction analysis was used to determine the relative expression levels of TLR4 gene involved in apoptosis-associated signaling in cervical cancer cells. The cancer cell colonies showed a tendency to reach high levels of confluency compared with normal cells. In addition, an enhanced growth rate and loss of contact inhibition were observed in gynecologic cancer cells compared with normal cells (doubling times of 16.6 hr vs. 26 hr, respectively). The expression level of ITGA5, an alpha-5 integrin marker, was upregulated in normal myometrial cells, but this tendency was not exhibited in cervical cancer cells. Furthermore, p53 tumor suppressor gene expression was upregulated, whereas TLR4 and caspase-3 gene expressions were downregulated in cervical cancer cells. Notably, the expression levels of TLR4 and caspase-3 were increased significantly in LPS-treated cancer cells compared with those in non-LPS-treated cells. These results suggest that the TLR4-mediated caspase-dependent apoptotic signaling pathway could be suggested as a therapeutic target for the treatment of gynecologic cancers, including cervical cancers.
Intuitive Modification of the Friedewald Formula for Calculation of LDL-Cholesterol
Hong Jinyoung,Gu Hyunjung,Lee Juhee,Lee Woochang,Chun Sail,Han Ki Hoon,Min Won-Ki 대한진단검사의학회 2023 Annals of Laboratory Medicine Vol.43 No.1
Background: High LDL-cholesterol (LDL-C) is an established risk factor for cardiovascular disease and is considered an important therapeutic target. It can be measured directly or calculated from the results of other lipid tests. The Friedewald formula is the most widely used formula for calculating LDL-C. We modified the Friedewald formula for a more accurate and practical estimation of LDL-C. Methods: Datasets, including measured triglyceride, total cholesterol, HDL-cholesterol, and LDL-C concentrations were collected and assigned to derivation and validation sets. The datasets were further divided into five groups based on triglyceride concentrations. In the modified formula, LDL-C was defined as total cholesterol − HDL-cholesterol − (triglyceride/adjustment factor). For each group, the adjustment factor that minimized the difference between measured LDL-C and calculated LDL-C using modified formula was obtained. For validation, measured LDL-C and LDL-C calculated using the modified formula (LDL-CM), Friedewald formula (LDL-CF), Martin-Hopkins formula (LDL-CMa), and Sampson formula (LDL-CS) were compared. Results: In the derivation set, the adjustment factors were 4.7, 5.9, 6.3, and 6.4 for the groups with triglyceride concentrations <100, 101–200, 201–300, and >300 mg/dL, respectively. In the validation set, the coefficient of determination (R2) between measured and calculated LDL-C was higher for LDL-CM than for LDL-CF (R2=0.9330 vs. 0.9206). The agreement according to the National Cholesterol Education Program Adult Treatment Panel III classification of LDL-C was 86.36%, 86.08%, 86.82%, and 86.15% for LDL-CM, LDL-CF, LDL-CMa, and LDL-CS, respectively. Conclusions: We proposed a practical, improved LDL-C calculation formula by applying different factors depending on the triglyceride concentration.
Jinyoung Hong,Hyunki Kim,홍용상,이우창,임석병,변정식,전사일,민원기 대한임상검사정도관리협회 2019 Journal of Laboratory Medicine And Quality Assuran Vol.41 No.4
A 26-year-old man underwent colonoscopy to investigate weight loss and a lesion suspicious of colorectal cancer was detected. He had a family history of colorectal cancer and hepatocellular carcinoma. The biopsy result of the lesion showed a well-differentiated adenocarcinoma of the sigmoid colon and he underwent curative anterior resection of the colon. A microsatellite instability (MSI) test was performed on the resected tumor tissue specimen and it was found to be MSI-high. A next-generation sequencing (NGS)-based hereditary tumor panel test was performed on his peripheral blood to detect the causative germline variant. Neither a pathogenic variant nor a variant of uncertain significance was found in the single nucleotide variant (SNV) and small indel variant analyses. However, a copy number variation (CNV) detection algorithm identified a variant compatible with the deletion of exon 7 to exon 19 of the MLH1 gene. This finding was confirmed to be a true deletion by multiplex ligation-dependent probe amplification. Therefore, the deletion of exon 7 to exon 19 of the MLH1 gene was regarded as the causative pathogenic genetic variant for his colorectal cancer and familial genetic testing was recommended. Therefore, patients with suspected cancer syndromes, including hereditary colorectal cancer, should be tested for germline mutations including CNVs, SNVs, and indels. NGS is a technique that can simultaneously detect SNVs and CNVs and therefore, it has clinical utility for genetic testing for hereditary diseases.
Jinyoung Hong,Ji Hyun Kim,Seungman Park,Sang Gon Lee,이우창,전사일,민원기 대한임상검사정도관리협회 2021 Journal of Laboratory Medicine And Quality Assuran Vol.43 No.1
Background: DNA extracted from mutant cell lines is frequently used as an external quality assessment (EQA) material for genetic testing of solid tumors because it is easy to obtain. However, the proportion of mutations in cell lines is different from that in actual tumor samples. In this study, mixtures of mutant DNA and wild-type DNA mimicking patient samples were analyzed to optimize the amount of mutant DNA in EQA specimens. Methods: Four cell lines harboring the selected mutation were cultured, and genomic DNA was extracted from cultured cells. Wild-type cell line DNA was prepared in the same manner. Diluted samples were prepared by mixing each mutant cell line DNA and wild-type cell line DNA at different ratios. Sanger sequencing of target variants was performed. For reliability, sequencing was repeated three times and read by two readers. The cutoff was based on the lowest proportion of mutant DNA that was determined to be positive in all three experiments. Results: The cutoffs of mutant cell line DNA ratios were 10%, 5%, 25%, and 25% for KRAS G12C, EGFR exon 19 deletion, EGFR T790M, and BRAF V600E, respectively. For the cell lines harboring EGFR T790M and BRAF V600E, the mutant fraction was not 100%. Conclusions: When manufacturing EQA material for solid tumor genetic testing, consistent results can be obtained if the mutant proportion is 10% or more. In addition, the mutant allele frequency of the cell line should be checked in advance to guarantee that EQA materials contain enough mutant DNA.
Jinyoung Hong,Joonsang Yu,Hyunjung Gu,Juhee Lee,Woochang Lee,Sail Chun,Won-Ki Min 대한임상검사정도관리협회 2022 Journal of Laboratory Medicine And Quality Assuran Vol.44 No.3
Background: Next-generation sequencing (NGS)-based liquid biopsy testing using peripheral blood is a minimally invasive technique that can identify the characteristics of tumor-derived circulating tumor DNA (ctDNA) in cellfree DNA (cfDNA). External quality assessment (EQA) should be implemented to ensure the reliability of NGS-based liquid biopsy tests. This study aims to establish a method for producing EQA materials for NGS-based liquid biopsy tests. Methods: Eight cell lines harboring clinically important somatic mutations were selected for further analysis. Genomic DNA from the cell lines was extracted and fragmented using an ultrasonicator (Covaris Inc., USA). Two EQA materials were produced by spiking fragmented DNA into fresh frozen plasma and frozen at –70℃. The manufactured EQA materials were evaluated using a cfDNA gene panel (Dxome, Korea) using NextSeq Dx (Illumina, USA). Results: After sonication, the average sizes of the fragmented DNA were 203 and 201 bp, respectively. The results of the cell-free NGS panel showed a combination of different variants between the two EQA materials, and clinically important somatic mutations were detected as intended. Conclusions: In this study, a method for manufacturing materials for an NGS-based liquid biopsy test EQA scheme is presented. EQA materials with conditions similar to ctDNA clinical specimens can be produced at a relatively low cost using cell line-derived DNA and an ultrasonicator. The distribution of adequate EQA materials can improve the reliability of NGS-based liquid biopsy tests.