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- 주제어 : 12-O-Tetradecanoyl-phorbol-13-acetate (검색결과 3 건)
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白血病細胞 U-937에서 Interleukin-1β遺傳子 發現에 미치는 12-O-tetradecanoyl phorbol-13-acetate와 體內因子의 影響에 관한 硏究
사람 골수 단핵구성 백혈병세포인 U-937에 세포분화유도물질인 12-O-tetradecanoyl phorbol-13-acetate (TPA) 단독 또는 1,25-dihydroxy-vitamin D_(3) (DHVD_(3)), interferon-γ (γIFN), dexamethasone과 병용 처치시 세포의 분화정도 및 interleukin-1β(IL-1β) 유전자의 발현정도를 알아 보고자 세포의 형태학적 관찰, flow cytometry, Northern blot hybridization를 수행하여 다음과 같은 결과를 얻었다. 1. U-937세포를 배양하면서 TPA로 48시간 처치하면 세포들이 모이는 경향을 보이며, 불규칙한 모양을 보이거나 죽어가는 세포들이 증가하였으나, DHVD_(3), γIFN 또는 dexamethasone으로 각각 처치하면 세포의 형태에는 변화가 없었다. TPA와 세가지 약제를 각각 병용 처치할 때, DHVD_(3)와의 병용시에 세포들이 길어지는 현상이 나타났다. 이때 U-937 세포의 증식은 TPA 단독 처치시, TPA와 DHVD_(3) 또는 γIFN 병용 처치시에 대조군에 비하여 감소되었고 세포생존율도 감소하였다. 2. U-937 세포 배양시 세포표면 항원인 CDl4 항원의 발현정도를 평균 형광강도로 비교하여 보면 대조군(6.17)에 비하여 TPA와 DHVD_(3) 병용 처치군에서 78.66으로 가장 높았는데, 이는 대조군의 양성 세포율을 2.9%로 기준하면 61.2%가 되었다. CDl5 항원에 대한 평균 형광강도는 대조군(292.09)에 비하여 TPA와 γIFN 병용 처치군에서 42.73으로 가장 낮았는데, 이는 대조군의 양성 세포율을 51.3%로 기준하면 2.5%가 되었다. 3. U-937 세포를 여러 물질로 처치하여 18시간 배양한 다음, mRNA를 추출하여 IL-lβ gene probe와 Northern blot hybridization을 실시한 결과, TPA 단독 처치군에 비하여 TPA와 DHVD_(3) 병용 처치군에서 IL-1β 발현이 현저히 증가하였으나, TPA와 γIFN 또는 dexamethasone 병용 처치군에서는 IL-1β 발현이 감소하였다. 4. U-937 세포를 여러 물질로 처치하여 48시간 배양한 다음, 그 상청액내의 IL-1 활성도를 측정한 결과, TPA 단독 처치군과 TPA와 DHVD_(3) 또는 γIFN병용 처치군에서 IL-1 활성도가 증가하였다. 이상의 결과는 U-937세포에서의 분화 정도는 CD14 항원과 IL-lβ의 발현 증가와 밀접한 관련이 있는 것을 보여 주었다. The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA), 1,25 dihydroxy-vitamin D_(3) (DHVD_(3)), interferon-γ (γIFN), dexamethasone alone or in combination on the degree of cellular differentiation and the level of interleukin-1β(IL-1β) expression was assessed in human myelomonocytic leukemic cell line, U-937, by morphological observation, flow cytometry and Northern blot hybridization. l. The TPA-treated U-937 cells showed to be gathered, to shape irregularly and to die, while either DHVD_(3)-, γIFN-, or dexamethasone-treated cells showed no change in the morphology. Among three combinations of TPA and other agents, the cells treated with combination of TPA and DHVD_(3) were partly changed into long cells. Compared with control, the proliferation and cell viability of U-937 cells treated with TPA, or combination of TPA and DHVD_(3) or γIFN was suppressed. 2. The mean fluorescence intensity of CD14 molecules on the cell surface and the percentage of the CD14^(+) cells were increased up to 78.66 and 61.2%, respectively, in the cells treated with combination of TPA and DHVD_(3) in comparison with the control cells(6.17, 2.9%). The mean fluorescence intensity of CD15 molecules on the cell surface and the percentage of the CD15^(+) cells were decreased to 42.73 and 2.5%, respectively, in the cells treated with combination of TPA and γIFN in comparison with the control cells(292.09, 51.3%). 3. Poly(A^(+)) mRNA was extracted from cells treated with the various agents alone or in combination for 18 hours. Northern blot analysis showed that compared with TPA-treated cells, the IL-1β expression of cells treated with combination of TPA and DHVD_(3) significantly increased, while that of cells treated with combination of TPA and γIFN or dexamethasone decreased. 4. Culture media of cells treated with the various agents alone or in combination for 48 hours was assessed for IL-1 activity. Compared with control cells, cells treated with TPA alone or combination of TPA and DHVD_(3) or γIFN showed some IL-1 activity. These results indicate that the differentiation of U-937 cells may be related considerably to the increase of CD14 antigen expression and IL-1β gene expression.
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DNA topoisomerase IIa plays an important role in events leading to mitosis and Topo IIa gene is regulated at cell-cycle dependent manner. To gain insight on the mechanism of transcriptional repression of Topo IIa gene, the binding pattern of nuclear proteins to various cis-elements in the Topo IIa gene upstream region has been investigated with DNase I footprinting and DNA mobility shift assay. HL-60 cells were arrested at G_/G_ phase after TPA treatment in FACS analysis. The level of Topo IIa mRNA and unknotting activity were reduced at 24 hours after exposure with TPA. The unclear factors, were which newly induced during reduction of Topo IIa mRNA after TPA treatment, bound with at least four sites : I-CCAAT-1 at -68 bp, I-CCAAT-2 at -108 bp, I-CCAAT-4 at -259 bp and novel cis-element at -196 (Topo IIa specific sequence, TSS : upper strand, 5-TAGTCCCGCCTCCCTAACCT-3). The DNase I protection pattern showed the same result with the control after addition of competitors including TSS and I-CCAAT. DNA mobility shift assays using oligonucleotides containing the I-CCAATs showed one specific DNA-protein complex and the competition studies indicated apparent binding of ICB-1~4 to the same protein while ICB-5 bound to a different protein. DNA mobility shift assays using oligonucleotide containing the TSS showed two specific DNA-protein complexes and the TSSBP bound to TSS-specific and novel trans-acting factor. TPA-dependet induction of TSS binding protein (TSSBP) reduced after pretreatment of staurosporin and completely vanished after pretreatment of cycloheximide. These results suggest that the transcriptional repression of Topo IIa gene during TPA-induced differentiation of HL-60 cells may be mediated by four transacting factors that are newly induced by TPA and TSSBP is a novel trans-acting factor.
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