HLA-restricted and Antigen-specific CD8+ T Cell Responses by K562 Cells Expressing HLA-A*0201

Yun, Sun-Ok,Sohn, Hyun-Jung,Yoon, Sung-Hee,Choi, Hee-Baeg,Kim, Tai-Gyu The Korean Association of Immunobiologists 2006 Immune Network Vol.6 No.4

Background: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). Methods: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/ A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-${\gamma}$ enzyme linked immunospot (ELISPOT) assay. Results: The stable transfectant K562/ A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-${\gamma}$ secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-${\gamma}$ in both K562/ A*02 with peptide and without peptide. The number of IFN-${\gamma}$ secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/ A*02 showed similar antigen presenting function to live K562/ A*02. Moreover, K562/ A*02 could present antigenicpeptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. Conclusion: These results suggest that K562/ A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

K562 백혈병 세포주에서 방사선에 의해 유도되는 Apoptosis에 미치는 PTK Inhibitors의 영향

이형식(Hyung Sik Lee),문창우(Chang Woo Moon),허원주(Won Joo Hur),정수진(Su Jin J eong),정민호(Min Ho Jeong),이정현(Jeong Hyeon Lee),임영진(Young Jin Lim),박헌주(Heon Joo Pa rk) 대한방사선종양학회 2000 Radiation Oncology Journal Vol.18 No.1

목 적 : 방사선에 의해 유도되는 apoptos is에 내성을 가진 세포로 알려진 K562 세포를 대상으로, PTK inhibitors인 herbimycin A와 genist ein을 이용한 방사선에 의한 apoptos is의 내성 기전을 연구하고자 하였다. 대상 및 방법 : 6 MV 체외 X-선 방사선 치료기를 이용하여 200∼300 cGy/min의 선량률로 10 Gy의 X-선을 세포에 균일하게 조사하였다. Apoptosis의 관찰은 agarose gel elect rophores is를 이용하여 DNA frgment at ion의 지표인 ladder를 관찰하였고, TUNEL 염색을 이용하여 정량 분석을 시행하였다. Western blot 방법으로 apoptos is 관련 유전 단백인 bcl-2, bcl-XL 및 bax들의 발현을 관찰하였다. 방사선 조사 및 약물 처치 후의 세포 주기 분석은 flow cytometry로 분석하였다. 결 과 :Agarose gel elect rophores is 실험에서 방사선을 조사하지 않은 K562 세포와 방사선을 10 Gy 조사한 세포를 48시간에 걸쳐 12시간 간격으로 관찰하였을 때 DNA fragment at ion를 관찰할 수 없었다. 이러한 현상은 genistein을 투여한 세포들에서도 동일한 현상을 관찰할 수 있었지만, 방사선 조사 후 herbimycin A를 투여한 세 포들에서는 48시간째 확연한 DNA fragmentation을 관찰할 수 있었다. 이를 TUNEL assay에서 정량적으로 확인하였다. 방사선만 조사한 세포들과 방사선과 genistein 투여 후 48시간째 관찰한 세포들에서는 10%미만의 apoptosis 양성 세포의 빈도를 관찰할 수 있었지만, 방사선 조사 후 herbimycin A를 투여한 세포들에서는 30∼35%빈도로 apoptosis 양성 세포들이 관찰되었다. Western blot analysis에서 bcl-2의 경우 방사선을 조사하지 않았던 대조군에 비하여 전체적인 발현은 증가되었지만 방사선 및 약제간의 발현의 차이는 없었다. 그 외 bcl-XL과 bax는 대조군에 비해 방사선 및 약제간의 발현의 차이를 관찰할 수 없었다. K562 세포에 방사선을 10 Gy 조사하였을 때 나타나는 세포 주기의 변화는 시간이 경과함에 따라 전형적인 G2/Mblock의 소견을 보였다. 이러한 소견은 genistein을 투여했을 경우에는 특별한 변화를 보이지 않지만, herbimycin A를 투여했을 경우에는 12시간째부터 G2/Mblock이 소실되면서 세포가 세포 주기를 재 순환하는 양상을 보였고, 48시간째 관찰한 소견에서는 G2/Mblock이 거의 소실된 양상을 띠었다. 이러한 소견을 토대로 apoptosis 유도와의 상호 연관성을 유추할 수 있었다. 결 론 : herbimycin A는 방사선에 의해 유도되는 apoptosis가 억제된 K562 세포에서 apoptosis를 유도할 수 있었다. 이러한 유도 기전에 apoptos is 관련 유전 단백들인 bcl-2, bcl-XL 및 bax와 관련된 영향은 관찰되지 않았다. 세포 주기의 분석에서 G2/Mblock의 해소와 apoptos is 유도와의 연관성을 유추할 때, 세포 주기 관련 인자들에 대한 연구가 방사선에 의한 apoptosis의 내성의 극복 및 방사선에 의한 세포의 감수성 조절 약제로서의 역할에 이바지할 것으로 생각한다. Purpose :The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph- positive K562 leukemia cell line was investigated. Materials and Methods :K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X- ray irradiation and drug treatment, cultures were initiated at 2x106cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37℃ for 0∼48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-XL and bax protein levels. Results :Treatment with 10 Gy X- irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electrophoresis and TUNEL assay. Genistein failed to enhance the ability of X- irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bcl- 2 or bcl-XL anti- apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30∼40% at 48 h. On the other hand, cells exposed to 10 Gy Xirradiation alone or maintained with genistein did not show marked cell cycle redistribution. Conclusion :We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X- irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210bc r/a bl failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bcl- 2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation- induced apoptosis.

Hydroxyurea에 의해 유도된 만성골수성백혈병 K562세포에서 Caspase 활성화

이영진,박래길,소홍섭,박지선,조지현,최삼임 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.5

Cytotoxicity of natural killer cells co-cultured with γ-irradiatedor mitomycin C-treated feeder cells

이세아,구나연,이시우,이지현,이윤희,현방훈 한국예방수의학회 2020 예방수의학회지 Vol.44 No.2

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

Cytotoxicity of natural killer cells co-cultured with γ-irradiatedor mitomycin C-treated feeder cells

Se-A Lee,Na-Yeon Gu,Siu Lee,Jienny Lee,Yoon-Hee Lee,Bang-Hun Hyun 한국예방수의학회 2020 예방수의학회지 Vol.44 No.2

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

K562 세포의 방사선 감수성 변화에 영향을 미치는 신호전달인자

양광모(Kwang Mo Yang),윤선민(Seon-Min Youn),정수진(Soo-Jin Jeong),장지연(Ji-Yeon Jang),조월순(Wol-Soon Jo),도창호(Chang-Ho Do),유여진(Yeo-Jin Yoo),신영철(Young-Cheol Shin),이형식(Hyung Sik Lee),허원주(Won Joo Hur),임영진(Young-Jin Li 대한방사선종양학회 2003 Radiation Oncology Journal Vol.21 No.3

Purpose The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncopro-tein, whose deregulated protein tyrosine kinase activity antagonizes the induction of apoptosis via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HMA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosis of p210bcr/abl protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the induction of a number of transcription factors and the differential gene expression in this model were investigated. Materials and Methods K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 MeV Linac(Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with 0.25 M o f H MA and 25 M of genistein, and the expressions and the activities of ablkinase, MAPK family, NF-B, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. Results: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-ablkinase activity. The signaling responses through the MAPK family of proteins, were not involved either. In association with the radiation-induced apoptosis, which is accelerated by HMA, the expre-ssion of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF-Bactivity and the TK1 expression and activity. Conclusion: The effects of HMA and genistein on the radiosensitivity of the K562 cells were not related to the bcr-abl kinase activity. In this study, another signaling pathway, besides the MAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection. 목 적: 만성 골수성 백혈병 세포인 K562 세포주는 방사선 및 다양한 항암제에 대한 apoptosis에 저항성을 가진다. 지난 연구에서 K562 세포는 방사선에 대하여 내성반응을 보이며, 세포내 PTK의 작용을 억제하고 자방사선조사와 함께 투여한 herbimycin A (HMA)에 의하여 방사선에 대한 apoptosis와 같은 감수성 반응이 유도되는 반면, genistein에 의하여 방사선에 대한 apoptosis 반응이 저해 됨을 확인하였다.본 연구에서는 타이로신 인산화 효소억제에 의한 K562세포의 방사선 반응변화를 조절하는 신호전달경로를 조사하였다. 대상 및 방법 : K562 세포를 지수증식기의 세포 들만 선택하여 실험에 이용하였다. 방사선조사는 6 MeV 선형가속기 (Clinac 1800C, Varian)를 이용하여 200˜300 cGy/min 선량률로 0.5˜12 Gy를 균일하게 조사하였다. HMA와 genis-tein은 각각 0.25μM, 25μM을 방사선 조사 후 즉시 투여하였다. 실험에서 신호전달 경로로 abl kinase, MAPK family, NF-κB, c-fos, c-myc, thymidine kinase1 (TK1)등에서의 단백질 또는 유전자 발현 및 활성을 조사하였다. 또한 약제 투여에 따른 유전자 발현차이(differential gene expression)를 조사하였다. 결 과: Abl kinase의 발현 및 활성 변화를 조사하였으나 PTK 저해제에 의한 방사선 유도 세포사의 변화와의 연관성을 찾을 수 없었다. 세포생존 및 사멸의 신호전달 체계에서 주요조절과 정인 MAPKfamily의 관여 여부확인에서 방사선으로 인한 SAPK/JNK의 활성화의 유도가 관찰되었으나, PTK 저해제에 따른 변화는 없었으며,또한 MAPK/ERK와 p38MAPK활성은 모든 조건에서 변함없이 일정하였다.전사인자 활성화에 대한 조사에서 방사선 조사와 함께 genistein을 투여한 경우에NF-κB 활성이 증가하였다. 유전자 발현 차이의 조사에서 genistein 투여에 의한 TK1유전자 발현 및 단백질 활성이 증가하였다. 결 론: PTK 억제에 의한 K562 세포의 방사선에 대한 반응 변화는 bcr-abl kinase활성과는 무관하게 진행되며, MAPK family경로 외의 다른 경로를 통한 전사인자 활성화 과정이 연관되어 있음을 확인하였다.

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

Bae, Duk Seong,Lee, Jae Kwon Korean Society of Hematology; Korean Society of Bl 2014 Blood Research Vol.49 No.3

<P><B>Background</B></P><P>Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, <I>ex vivo</I> NK cell expansion is one of the important steps for developing NK cell therapeutics.</P><P><B>Methods</B></P><P>CD3<SUP>+</SUP> depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines.</P><P><B>Results</B></P><P>We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naïve NK cells.</P><P><B>Conclusion</B></P><P>Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.</P>

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

Duk Seong Bae,이재권 대한혈액학회 2014 Blood Research Vol.49 No.3

Background Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. Methods CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. Results We compared feeder activities of three different cells−PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naïve NK cells. Conclusion Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.

K562 세포주와 IL-2를 이용하여 말초혈액 단핵구로부터 자연살세포의 선택적 증폭

조덕,신시원,박정선,강현규,김상기,Nguyen Pham,TNZhu XW,신명근,서순팔,양동욱,남종희,김영진,이제중 대한혈액학회 2006 Blood Research Vol.41 No.1

배경: NK 세포를 이용한 면역치료를 위해서 사람의 말초혈액 단핵구로부터 NK 세포를 증폭시키려는 많은 연구가 있었다. 저자들은 말초혈액 NK 세포를 IL-2와 K562 세포주를 활용하여 선택적 증폭, 증폭된 NK 세포의 활성화 및 억제성 수용체의 발현 양상, 그리고 이들 NK 세포의 세포독 성능을 평가해 보았다.방법: 정상 성인 7명의 말초혈액 단핵구를 Cellgro SCGM 배지에 IL-2 (500IU/mL)와 feeder cell로 K562 세포주를 사용하여 14일간 배양을 하였고, NK 세포의 활성화 및 억제성 수용체를 분석하기 위해 배양을 하기 전의 NK 세포와 IL-2 단독배양법과 K562와 IL-2를 함께 사용한 배양법으로 72시간 배양한 NK 세포의 활성화 및 억제성 수용체를 분석하였다. 세포 독성능 평가는 증폭 전과 7일간 증폭된 NK 세포를 이용하여 K562 세포주를 표적세포로 하여 유세포를 활용한 독성 검사로 실시하였다. 결과: NK 세포는 7일간 배양 후 4.5배(범위 2.5~10) 증폭되었으며, CD3-CD56+ 세포는 56.5%를 보였다. IL-2 단독 혹은 IL-2와 K562를 함께 사용한 배양법에 의한 NK 세포의 수용체 중 CD158b (mean florescence intensity, MFI), CD158e1/e2 (MFI), NKp44 (MFI) 및 NKp46 (%)는 그 발현이 증가하였고, NKp30 (%), CD16 (MFI) 및 2B4 (MFI)는 그 발현이 감소하였다. 세포독 성능 평가에서 비 증폭된 NK 세포는 표적세포인 K562 세포주와 1:1 및 5:1에서 각각 9.0%와 27.6%를 살생했으며, 7일간 증폭된 NK 세포는 표적세포를 각각 36.9%와 57.2%를 살생했다.

종양세포(腫瘍細胞)의 염색체(染色體)에 대한 오크라톡신 A의 독성(毒性)에 관한 연구(硏究)

윤화중,노민희,김강련,Yoon, Wha-jung,Roh, Min-hee,Kim, Kang-ryun 대한수의학회 1989 大韓獸醫學會誌 Vol.29 No.2

This study was performed to investigate the toxicity of ochratoxin A (OA) to the chromosomes of $K_{562}$ tumor cell-line in vitro. The results of this experiment were as follows: 1) Chromosomes of $K_{562}$tumor cell-line resulted in pseudotriploidy on the control group. Chromosomes of $K_{562}$ tumor cell-line treated with OA resulted in heteroploidy compared with the control group. The mean number of chromosomes in the karyotype of the control group (60) were 7 in the A group, 5 in the B group, 20 in the C+X group, 7 in the D group, 9 in the E group, 6 in the F group, and 6 in the G+Y group respectively. The number of chromosomes were increased as follows: Treating with $0.7{\mu}M$ OA, the number of chromosomes were increased one in E and F group, two in G+Y group compared with control group. In treated with $1.5{\mu}M$ OA, the increasing number of chromosome was one in E and F group. In treated with $3{\mu}M$ OA, E and F group was increased one and G+Y group were increased two chromosomes compared with control group. But in treated with $6{\mu}M$ OA, the number of chromosome in G+Y group was decreased one. 2) $K_{562}$ tumor cell line treated with OA showed Philadelphia-Chromosome in the long arm of the G group karyotype chromosome. The rate of chromosome aberration in $K_{562}$ tumor cell-line treated with OA was 77% in $0.7{\mu}M$ OA group, 71% in $1.5{\mu}M$ OA group, 82% in $3{\mu}M$ OA group and 94% in $6{\mu}M$ OA group respectively. The rate of chromosome aberration of $K_{562}$ tumor cell-line treated with OA was high in the high dose level of OA, and chromosome aberration of $K_{562}$ tumor cell-line treated with OA showed deletion, minute, dicentric-chromosome and translocation in the long arm of the C-group karyotype. As a result of this study, the toxicity of OA showed deletion, minute, dicentric-chromosome and translocation in the long arm of the C-group karyotype, and then, the toxicity of OA resulted in the damage to RNA and protein synthesis in $K_{562}$ tumor cell-line, and the C-group karyotype of $K_{562}$ tumor cell-line was target of the toxicity of OA.