The regulatory role of c-MYC on HDAC2 and PcG expression in human multipotent stem cells

Bhandari, Dilli Ram,Seo, Kwang-Won,Jung, Ji-Won,Kim, Hyung-Sik,Yang, Se-Ran,Kang, Kyung-Sun Blackwell Publishing Ltd 2011 JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Vol.15 No.7

<P><B>Abstract</B></P><P>Myelocytomatosis oncogene (c-MYC) is a well-known nuclear oncoprotein having multiple functions in cell proliferation, apoptosis and cellular transformation. Chromosomal modification is also important to the differentiation and growth of stem cells. Histone deacethylase (HDAC) and polycomb group (PcG) family genes are well-known chromosomal modification genes. The aim of this study was to elucidate the role of c-MYC in the expression of chromosomal modification <I>via</I> the HDAC family genes in human mesenchymal stem cells (hMSCs). To achieve this goal, c-MYC expression was modified by gene knockdown and overexpression <I>via</I> lentivirus vector. Using the modified c-MYC expression, our study was focused on cell proliferation, differentiation and cell cycle. Furthermore, the relationship of c-MYC with HDAC2 and PcG genes was also examined. The cell proliferation and differentiation were checked and shown to be dramatically decreased in c-MYC knocked-down human umbilical cord blood-derived MSCs, whereas they were increased in c-MYC overexpressing cells. Similarly, RT-PCR and Western blotting results revealed that HDAC2 expression was decreased in c-MYC knocked-down and increased in c-MYC overexpressing hMSCs. Database indicates presence of c-MYC binding motif in HDAC2 promoter region, which was confirmed by chromatin immunoprecipitation assay. The influence of c-MYC and HDAC2 on PcG expression was confirmed. This might indicate the regulatory role of c-MYC over HDAC2 and PcG genes. c-MYCs’ regulatory role over HDAC2 was also confirmed in human adipose tissue-derived MSCs and bone-marrow derived MSCs. From this finding, it can be concluded that c-MYC plays a vital role in cell proliferation and differentiation <I>via</I> chromosomal modification.</P>

Zygotic Expression of c-myc Gene in Mouse Early Embryos: Functional Role of c-myc Promoter

박기수,강해묵,심찬섭,선웅,김재만,이영기,김경진,Park, Ki-Soo,Kang, Hae-Mook,Shim, Chan-seob,Sun, Woong,Kim, Jae-man,Lee, Young-Ki,Kim, Kyung-jin The Korean Society for Integrative Biology 1995 동물학회지 Vol.38 No.4

c-myc proto-oncogene은 여러 세포들의 분화와 형질전화에 뿐만 아니라 정상세포의 분열조절에도 관여한다고 알려져왔다. 특히 생쥐의 초기배아에서 c-myc mRNA가 발현되고 antisense c-myc oligomer의 미세주입에 의해 배발생이 억제된다는 연구결과는 c-myc이 초기배아의 발생 및 분열에 관여하는 것을 시사한다. 그러나 최근까지 초기배아에 존재하는 c-myc promoter의 기능적 활성화에 관한 연구는 미진하였다. 이를 위하여, c-myc promoter와 대장균의 lacZ 유전자를 결합시킨 두 종류의 vector(pcmyc-Gall, pcmyc-Ga12)를 만들어 수정란의 전핵에 미세주입한 후, 배 발생에 따른 c-myc promoter의 활성화를 lacZ 유전자의 산물인 $\beta$-galactosidase 에 의한 X-gal 염색으로 조사하였다. 미세주입된 초기 배아는 2세포기 배아를 포함하는 여러 발생단계에서 $\beta$-galactosidase 의 활성을 보였다. 이는 c-myc 유전자가 배아의 게놈유전자로부터 발현되며, 또한 궁극적으로 초기 배아의 발생과정에 중요한 역할을 하고 있음을 시사하고 있다. The c-myc proto-oncogene is Involved In the control of normal cell proliferation and differentiation of many cell lineages. Although it has heen suggested that c-myc may play an important role in the mammalian early development, it Is unclear whether the embryonic c-myc mRNA is originated from zygotic gene expression or stored maternal message. Thus, we have construded expression vectors, In which the 5, flanking sequences including c-myc promoter region and a large non-coding exon I are fused 'sith E. coli lacZ gene that encedes $\beta$-galactosldase as a reporter. As c-myc exon I contains a modulatory sequence, we designed t, vo types of vectors (pcmyc.Gall and pcmyc-Ga12) to examine the role of exon I in c-myc expression. The former contains the complete exon I and the later has a deletion in 40 bp of modulator sequence located In the exon I of c-myc These vectors were microInjected into fertilized one-cell embryos and $\beta$-galactosidase activity was examined by X-gal staining during early embryogenesis. $\beta$-galactosidase activity derived from c-myc promoter was decreased at two-cell stage. The expression level directed by pcmyc- Ga12 was similar to that of pcmyc-Gal1, indicating that the medulatory sequence in exon I may not be Involved at least In the regulation of embryonic c-myc expression. In summary, the present study indicates that the c-myc promoter is functional at the early stage embryo, and the regulation of c-myc expression is under the control of "zygotic" clock of preimplantation mouse embryos.e embryos.

白血病에서의 c-myc 腫瘍蛋白의 發現樣相에 관한 硏究

조윤정,이갑노 고려대학교 의과대학 1992 고려대 의대 잡지 Vol.29 No.3

To evaluate the relationship between the expression of c-myc oncoprotein and the development of leukemia, flow cytometric analysis (FACScan®) of c-myc oncoprotein using monoclonal antibody was performed in 23 clinical leukemic samples (4 cases of ANLL ; 15 cases of ALL; 3 cases of ALL in complete remission ; 1 case of chronic myelomonocytic leukemia).20 cases of control groups (non-leukemic bone marrow aspirates). & 4 ATCC leukemic cell lines(ATCC CCL 240 HL-60, acute promyelocytic leukemia ; ATCC CCL 213 Daudi, Burkitt lymphoma ; ATCC CCL 243 K-562. Chronic myelogenous leukemia in blast crises ; ATCC CRL 1582 Molt-4, acute T-cell leukemia). The results were summarized as follows. 1. The proportion of myelocytes, basophilic normoblasts. and polychromatophilic normoblasts of the bone marrow aspirate in control group showed weak, but significant correlation with the c-myc oncoprotein staining indices (% positivity of cell staining and/or corrected mean fluorescence intensity, CMFI) (r= 0.35, 0.35, 0.30 respectively). But the proportion of the other immature cells such as blasts, promyelocytes, pronormoblasts, and orthochromic nor-moblasts showed no correlation with the c-myc oncoprotein staining indices. 2. The difference of the percent positivity of c-myc staining in the cells among the immunologic classes of ALL was not significant. But the CMFI of B ALL is significantly higher than that of common ALL or T ALL (F ratio=3.85, p=0.05). 3. The extent of CDl3. CD33, CD34 expression in ANLL showed no correlation with the c-myc oncoprotein staining indices. But that of CD14 expression in ANLL showed negative correlation with CMFI (r= -1.0). 4. There is no significant differences in c-myc oncoprotein staining indices among ANLL, ALL. ALL in complete remission, CMMoL, leukemic cell lines, and control group. 5. The CMFI of c-myc protein staining is high in K-562, Molt-4, HL-60, and Daudi in a decreasing order. From the above findings, it was concluded that ; first, the malignant transformation of the hematopoietic cells require a complex phenomena in addition to the c-myc oncogene expression because the c-myc oncoprotein is present both in the malignant transformation and in the benign proliferation of hematopoietic cells. Secondly, in the hematopoietic malignancy c-myc oncogene expression is not only present in acute promyelocytic leukemia, but also in the other myeloid leukemia and lymphoblastic leukemia. Thirdly, c-myc oncogene expression is diminished in the terminal stage of monocytic differentiation.

Differential Tumorigenic Effects by C-Myc Mutants in Liver Cancer

( Daeyoung Kim ),( Hyuk Moon ),( Sook In Chung ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Liver cancer is a major health concern worldwide, ranking third in terms of cancer-related mortality. The c-Myc gene is epigenetically altered in almost 50% of human liver cancers, leading to persistent over-expression of cMyc. In addition to quantitative changes of cMyc protein in cancers, mutation leading to amino acid substitution of cMyc has been found in a certain type of cancers. In this study, we compared tumorigenic potentials among c-Myc mutants in the liver. Methods: Transgenic liver cancer mouse models expressing different c-Myc mutants were developed using hydrodynamic transfection. Transposon vectors encoding the wild-type c-Myc, c-MycT58A, and c-MycS71F were constructed. To induce liver cancer, 20 μg of transposons were mixed with plasmids expressing the Sleeping Beauty transposase and then diluted in 2.5 ml of 0.9% saline. The DNA mixtures were injected into the lateral tail veins of 6-week-old C57BL/6 mice. Mice were monitored at least twice per week and sacrificed when moribund. Tumor-bearing livers were formalin fixed for hematoxylin- eosin staining. Results: Hepatocellular carcinomas (HCC) were induced by the stable expression of c-Myc and shp53. Wild type c-Myc was less tumorigenic than c-Myc2T58A or c-Myc2S71F when co-expressed with shp53. The c-Myc mutant groups, c-Myc2T58A or c-Myc2S71F died earlier than the c-Myc wild type group (p< 0.05). There was no difference in phenotypes of malignant hepatocytes among tumors induced by cMyc mutants and wild-type. Conclusions: Co-expression of c-Myc and shp53 in the mouse liver promoted hepatocarcinogenesis. Wild type c-Myc was less tumorigenic than c-Myc2T58A or c-Myc2S71F under the condition that P53 was down-regulated.

T58A Mutation Does Not Enhance Tumorigenic Potentials of C-MYC in the Liver

( Kyungjoo Cho ),( Sook In Chung ),( Hyuk Moon ),( Daeyoung Kim ),( Do Young Kim ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Liver cancer is a major health concern worldwide, ranking the second among cancer-related mortality. The c-Myc gene is epigenetically altered in almost 50% of human liver cancers, leading to persistent over-expression of c-Myc. Mutation at codon 58 of c-Myc (c-Myc^T58A) can enhance oncogenic potentials of c-Myc through suppressing apoptotic signaling cascades or stabilizing the oncoprotein. In this study, we compared tumorigenic potentials between c-Myc^T58A and the wild-type (WT) c-Myc in the liver. Methods: Transgenic mouse models expressing c-Myc^T58A and WT c-Myc were developed using hydrodynamic transfection. Transposons encoding an activated from of human H-RAS were mixed with transposons encoding either c-Myc^T58A or WT c-Myc. The DNA mixtures were injected into the lateral tail veins of 6-week-old C57BL/6 mice. Mice were monitored at least twice per week and sacrificed when moribund. Tumor-bearing livers were formalin fixed for hematoxylin- eosin staining and immunohistochemistry. Results: Hepatocellular carcinomas (HCC) were induced by co- expression of HRAS with either c-Myc^T58A or WT c-Myc with 100% penetration. There was no significant difference in animal survivals between the c-Myc^T58A and WT c-Myc groups. The numbers and sizes of tumors were similar between the two groups. Cellular proliferation (determined by Ki-67 staining) and apoptosis levels (by TUNEL assay) were also similar between c-Myc^T58A and WT c-Myc groups. Finally, there was no difference in phenotypes of malignant hepatocytes between the two groups. Conclusions: T58A mutation does not enhance tumorigenic potentials of c-MYC in our transgenic mouse models. No downregulation of apoptosis was detected in c-Myc^T58A, compared with WT c-Myc.

Pentylenetetrazol Kindling에 의한 백서 해마부위의 c-myc mRNA 발현의 변화

이장철,손은익,김인홍,이상도 대한신경외과학회 1996 Journal of Korean neurosurgical society Vol.25 No.11

전신성 간질의 모형인 pentylenetetrazol(PTZ) kindling 동물모형에서 PTZ kindling에 의한 신경계 가소성과 c-myc mRNA 발현사이의 상관관계를 알아보기 위하여 백서의 해마에서 PTZ kindling의 발달단계에 따른 c-myc mRNA의 발현양상을 관찰하였다. Sprague-Dawley종 백서를 대상으로 51마리는 15~25㎎/㎏의 PTZ을 2~3일 간격으로 복강내 주사하여 PTZ kindling을 형성하였고, 28마리는 30~60㎎/㎏의 PTZ를 일회 복강내 주사하여 PTZ 발작을 유발하였으며, 18마리는 양측 귀에 연결된 전극을 통하여 electroconvulsive shock(ECS) 자극을 가하여 발작을 유발하였다. 발작 양상을 관찰하여 각 단계별 발작을 보인 후 30분, 1시간 및 48시간 경과 후에 단두하여 희생시킨 다음 뇌를 척출하였다. 대조군 18마리는 0.9% 생리식염수를 복강내에 주사하였으며 주사 후 30분, 1시간 및 48시간 경과 후에 실험군과 같은 방법으로 뇌를 적출하였다. 적출한 뇌의 해마조직에서 RNA를 분리하여 slot-blot hybridization 방법으로 c-myc mRNA를 정량하였다. PTZ kindling에서 c-myc mRNA의 발현유도는 발작 후 30분경과 후에 대조군에 비하여 Ⅰ, Ⅲ, Ⅴ 단계에서 3~4배 증가되었으며 Ⅱ, Ⅳ 단계에서는 6~8배 증가되었으며 이들 각 군간의 차이는 통계적으로 유의성이 있었지만 kindling 단계인 발달성도에 따라 c-myc mRNA의 발현이 일관성 있게 증가되지는 않았다. 발작 후 1시간에 대조군에 비해 Ⅰ, Ⅱ, Ⅴ 단계에서 4~5배 증가되었지만 통계적 유의성은 없었다. 발작 후 48시간에 대조군에 비해 2~3배 증가하였지만 통계적 유의성은 없었다. PTZ 1회 투여 후 유발발작에 의한 단계별 c-myc mRNA의 발현은 발작 후 30분에 5~6배 증가되었으며 각 군가의 차이는 통계적으로 유의하였으나 발작 강도에 딸서 c-myc mRNA의 발현이 일관성 있게 증가되지 않았다. 발작 후 1시간에슨 s4~6배 증가되었으나 이는 통계적 유의성은 없었다. 발작 후 48시간에는 약 2배 증가되었으나 역시 통계적 유의성은 없었다. ECS 유발발작에서 e-myc mRNA의 발현은 가벼운 발작 후 30분 및 1시간에 약 4배 증가되었으며 48시간 후에는 약 2배 증가하였으나 전신 성긴장성간대성 발작 후 30분 및 1시간에는 모두 대조군보다 감소된 양상을 보였으며 48시간 후에 1.6배 증가된 양상을 보였다. 이러한 결과는 PTZ kindling, PTZ 혹은 ECS 유발발작과 c-myc mRNA의 발현사이에 의미있는 상관관계가 없다는 것을 시사한다. 그러므로 PTZ kindling 후에 보이는 c-myc mRNA의 발현증가는 신경세포의 비특이적인 활성화일 뿐이며 kindling의 발달 기전에 반드시 필요한 master switch와 같은 중요한 역할을 하지는 않는다고 할 수 있다. Kindling development is a good animal model of epilepsy and neural plasticity. It is induced by repeated subconvulsive electrical or chemical stimulations. This leads to progressive and permanent amplification of seizure activity resulting in permanent brain changes. Immediate early genes(IEGs) are proposed as the master switch for turning on molecular events in long term neural plasticity. The role of c-myc, an IEG, in the development of kindling is not known. This study was conducted to investigate the role of c-myc in the neural plastic changes underlying kindling. Among 115 adult male Spargue-Dawley rats. 51 were kindled by repeated administrations of subconvulsive doses(15-25㎎/㎏) of pentylenetetrazol(PTZ). Twenty-eight rats experienced various degree of convulsions induced by a single injection of convulsive dose(30-60㎎/㎏) of PTZ. Eighteen rats experienced mild or severe convulsion by a single electroconvulsive shock(ECS). Eighteen rats received normal saline as a control group. Animals were sacrificed in 30 minutes. 1 hour and 48 hours after convulsion. C-myc mRNA levels in the hippocampus were quantified using slot-blot hybridization analysis. In the experiment of PTZ kindling, c-myc mRNA expression 30 minutes after convulsion was elevated about 3-8 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated about 4 times at stage Ⅰ, Ⅱ, and Ⅴ, but was not elevated at stage Ⅲ and Ⅳ. C-myc mRNA expression 48 hours after convulsion was elevated about 2-3 times compared with controls. In the experiment of PTZ-induced seizures, c-myc mRNA expression 30 minutes after convulsion was elevated 5-6 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated 4-6 times. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. In the experiment of ECS-induced seizures, c-myc mRNA expression was elevated 4 times at 30 minutes and 1 hour after mild convulsion, but decreased at 30 minutes and 1 hour after severe convulsion compared with control. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. These results suggest that the enhanced expression of c-myc mRNA is a non-specific consequence in the development of PTZ kindling. In addition, c-myc does not seem to play an important role in turning on a molecular program underlying kindling.

자궁경부 상피내종양 및 자궁경부암 조직에서 bcl-2 및 c-myc 암유전자 발현과 세포증식 및 apoptosis와의 상관관계에 관한 연구

김병기(Byoung Gie Kim),이효표(Hyo Pyo Lee) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.8

bcl-2 암유전자는 여러 가지 자극에 의한 apoptosis를 차단함으로써 유전자 이상을 가진 세포가 계속 생존하면서 유전자 변이가 누적되는 결과를 초래한다고 알려져 있다. 한편 c-myc 암유전자는 세포증식과 apoptosis를 유도하는 이중적 기능을 가지고 있으며 생존 신호가 결여될 경우에는 오히려 세포의 apoptosis를 유발한다고 알려져 있다. 그러나 c-myc과 bcl-2가 동시에 발현되면 bcl-2는 c-myc의 세포증식 작용은 영향을 주지 않고 apoptosis만을 선택적으로 차단함으로써 유전자 변이 세포의 생존 뿐만 아니라 증식을 촉진하는 것으로 관찰되었다. 자궁경부암에서 c-myc과 bcl-2 발현에 관한 개별적 보고는 있었으나 이들 두 유전자의 동시발현 및 이들 유전자들이 실제 암조직상에서 세포증식 및 apoptosis에 어떠한 영향을 미치는가에 관한 연구는 보고된 것이 없다. 따라서 본 연구에서는 자궁경부암 발생 과정중에서 bcl-2 및 c-myc 발현과 세포증식, apoptosis와의 상관관계를 알아보고자 하였다. 본 연구에서는 10개의 정상 자궁경부조직, 30개의 자궁경부 상피내종양 조직, 20개의 자궁경부암조직에서 bcl-2와 c-myc에 대한 면역조직화학 검사를 시행하였으며 세포증식과 apoptosis는 각각 Ki-67 면역조직화학적 방법과 TUNEL 방법으로 확인하였다. 또한 환자의 임상병리학적 인자들과의 상관관계도 알아보았다. 정상 자궁경부, 자궁경부 상피내종양, 자궁경부암조직 중 자궁경부암조직에서만 bcl-2와 c-myc 단백이 각각 35%와 50%에서 관찰되었으며, 또한 bcl-2와 c-myc의 동시발현이 25%에서 관찰되었다. 세포증식 지수(상피세포 100개중 Ki-67양성 세포수)는 정상 자궁경부, 상피내종양, 자궁경부암으로 진행되면서 10.2, 24.1, 59.7, 71.2로 유의하게 증가하는 양상을 보였으며(p<0.01), apoptosis 지수(상피세포 100개중 apoptosis 세포수)도 0, 0.33, 1.85, 3.89로 점차 증가하는 양상을 보였다(p<0.01). 또한 세포증식 지수와 apoptosis 지수와는 높은 상관관계(r=0.7451, p=0.0002)를 나타내었다. 그러나 자궁경부암 조직중 bcl-2 발현군과 비발현군간에 apoptosis지수에는 차이가 없었으며(p=0.4765), c-myc 발현군과 비발현군간에도 세포증식 지수에는 차이가 없었다(p=0.6891). 또한 bcl-2와 c-myc의 동시 발현군과 나머지 군간에도 증식지수와 apoptosis 지수에 차이가 없었다(각각 p=0.6311 및 p=0.7600). 한편 bcl-2와 c-myc의 동시 발현과 잘 알려져 있는 자궁경부암의 임상병리학적 예후인자들(종양 크기, FIGO 임상병기, 림프절 전이등)과는 유의한 상관관계가 없었다. 이상과 같은 결과에서 자궁경부암발생 과정에서 세포증식과 apoptosis는 병변의 등급과 비례하여 증가하고 apoptosis는 세포증식과 관련된 변화로 사료되었다. 한편 bcl-2와 c-myc과 발현은 자궁경부암에서만 관찰되는 유전자 변이로서 자궁경부 상피내종양의 발생과 진행과정에는 영향을 미치지 않으며, 또한 자궁경부암 조직에서도 암조직 전체의 세포증식 및 apoptosis와는 관련이 없을 것으로 사료되었다. bcl-2 prevents cell death from a wide variety of stimuli and provides survival of cells with accumulated genetic alterations and c-myc can promote both cell proliferation and cell death through the transcriptional regulation of target genes. Although several studies have been reported on the expression of bcl-2 or c-myc separately, little has been known about the role of coexpression of bcl-2 and c-myc to cell proliferation and apoptosis, as well as the frequency of these coexpression in cervical cancer specimens. In this study, we have examined the expression of bcl-2 and c-myc in cervical cancer specimens and cervical intraepithelial neoplasia(CIN) to determine the role of coexpression of bcl-2 and c-myc during progression into cervical cancer. Proteins and transcripts of bcl-2 and c-myc were evaluated by immunohistochemistry in 60 clinical specimens(20 cervical cancer, 30 CIN, and 10 normal cervix). In addtion, we evaluated kinetic indices of cell proliferation and apoptosis simultaneously. The cell proliferation index was determined by detection of the Ki- 67 in immunohistochemistry. Apoptotic index was determined by the detection of apoptotic cells with TUNEL staining. Medical records including pathologic reports were reviewed. Overexpression of bcl-2 and c-myc was identified in 7(35%) and 10(50%) of 20 cervical cancer specimens respectively, but none in normal cervix and CIN samples. In addition, coexpression of bcl-2 and c-myc was found in 5(25%) of 20 cervical cancer specimens. The cell proliferation index increased with progression from normal to CIN and invasive cancer(normal cervix, 10.2; CIN 1, 24.1; CIN 2/3 59.7; cervical cancer, 71.2; p <0.01). The apoptotic index also increased with grade of lesions(normal cervix, 0; CIN 1, 0.33; CIN 2/3, 1.85; cervical cancer, 3.89; p <0.01) and showed a significant correlation with proliferation index(r=0.7451, p=0.0002). However, there was no significant difference in apoptotic index between bcl-2 positive and bcl-2 negative group in cervical cancer(p=0.4765). In addition, there was also no significant difference in cell proliferation between c-myc positive and c-myc negative group(p=0.6891). Furthermore, there was no significant difference in cell proliferation and apoptosis between bcl-2 and c-myc positive group and others in cervical cancer(p=0.6311 and p=0.7600 respectively). The well-known clinicopathologic parameters, including tumor diameter, FIGO clinical stage, lymph node metastasis, did not correlate with simultanuos positive immunoreactivity for bcl-2 and c-myc proteins in cervical cancer. In conclusion, the cell proliferation and apoptosis increase with increasing lesion grade of cervical neoplasia and apoptosis correlates with cell proliferation. In addition, overexpression of bcl-2 and/or c-myc may be genetic alteration found only in cervical cancer and may not play a role in the development and progression of CIN. However, neither bcl-2 nor c-myc immunoreactivity correlated with the proliferation index or apoptotic index. These results suggest that other factors may also play a role in controlling the cell proliferation and apoptosis of cervical cancer.

c-myc gene expression by ceramide-mediated pathway in U-937 cells

Kim,Mie Young,Choi,Kyung Hee,Kim,Keun Cheol 中央大學校 遺傳工學硏究所 1994 遺傳工學硏究論集 Vol.7 No.1

본 연구에서는 sphingomyelin 가수분해 생성물인 ceramide에 의한 c-myc 유전자 발현 경로를 알아보기 위해 U-937세포에 ceramide를 처리하여 다음과 같은 실험결과를 얻었다. 첫째, U-937 세포에 0.5~6μM의 ceramide를 1시간 처리하면 농도 의존적으로 c-myc 유전자 발현이 감소되었으며 둘째, PKC의 활성 유도제인 500nM을 1시간 처리하였을 때의 c-myc protooncogene 발현이 증가되나 6μM mide를 19시간 전처리하였을 경우 1시간 이내에 감소하였다. 셋째, PMA를 19시간 전처리하면 ceramide에 의한 c-myc 유전자의 발현은 더욱 감소되었다. 이상의 결과로서 PKC 의존적 경로에 위한 c-myc 유전자 발현에 ceramide에 의한 신호 전달 경로가 조절 작용을 나타냄을 알 수 있었다. The present study examines the possibility that ceramide-mediated pathway may serve to regulate the c-myc gene expression by PKC-dependent pathway in U-937 cells. Treatment of U-937 cells with a cell permeable ceramide caused a decrease in c-myc mRNA levels in a dose-dependent manner. A 60% reduction occurred at 3μM. The level of c-myc mRNA was reduced to 76% of control in the cells pretreated with ceramide for 19 hours and then treated with PMA for 1 hour suggesting that ceramide-mediated pathway antagonized the c-myc gene expression by PKC-dependent pathway However, in the combinatory pretreatment of PMA for 19 hours and ceramide for 1 hour c-myc gene expression was drastically reduced, possibly reflecting that PKC-dependent pathway had a synergistic effects on c-myc gene expression by ceramide-mediated pathway.

Genomic changes of c-myc, c-H-ras in benzo(a)pyrene and dimethylbenz(a)anthracene treated human lymphoblast NC-37 cells

조무연,어완규,이상욱,정인철,Cho, Moo Youn,Eo, Wan Kyu,Lee, Sang Uk,Jeong, In cheol Korean Society of Life Science 1995 생명과학회지 Vol.5 No.3

To investigate genomic changes in c-myc gene by a chemical carcinogen, human lymphoblast NC-37 cells were exposed to benzo(a)pyrene(BP) and dimethylbenzanthracene(DMBA), and the c-myc gene expression was evaluated by Northern and Southern blot hybridization techniques. The results are as follows: When the genomic DNA of NC-37 cells exposed to several concentrations(1.25, 2.5 and 5ug/ml) of BP concentration. However, the c-myc gene was most significantly enhanced with 2.5ug/ml of BP. The expressions of c-myc gene in NC-37 cells was stimulated by BP and DMBA. Addition of TPA reduced the gene expression BP-treated cells, whereas it enhanced the gene expression in DMBA-treated cells. The expression of c-H-ras gene was slightly increased by treatment with BP and DMBA alone and in combination with TPA, however the magnitude of increase was not significantly different between each other. The expressions of c-myc c-H-ras genes in Burkitt's lymphoma cells were greater than those in NC-37 cells. When the DNA extracted from NC-37 cells exposed to various concentrations of BP were amplified by polymerase chain reaction using a primer set containing c-myc exon I, the amplified products were of the same size in all groups. To evaluate the BP toxicity in E.coli to which human c-myc gene-cloned pBR322 vector was inserted, Southern blot hybridization was conducted on c-myc genes digested with EcoRI/HindIII and Smal/Xbal restriction enzymes, and observing that in 2 ug/ml BP-treated cells a 3.5kb fragment was generated in addition to 1.3kb fragment which can be observed in normal cells. Direct nucleotide sequence analysis of polymerase chain reaction products showed a mutation of G$\longrightarrow$A transition at the Smal recognition site.

L1210 암세포에서 Multidrug Resistance-associated Protein(MRP),c-myc 및 c-fos 유전자의 발현양상

김성용 영남대학교 의과대학 1997 Yeungnam University Journal of Medicine Vol.14 No.1

항암제에 대한 내성은 내인성 또는 획득한 내성 모두가 암의 치료에 장애가 된다. P-당단백질을 encode 하고있는 mdrl 유전자의 발현이 항암제에 대해 내성을 가지고 있는 암세포에서 많이 관찰되고 있으며, 최근에는 시험관적으로 항암제에 대한 내성이 유도된 암세포주들에서 mdrl 유전자가 발현되지 않는 암세포들이 보고되고 있다. 다제내성에 관계하는 또 하나의 유전자인 MRP 발현정도를 L1210세포와 내성인 L1210변이주들에서 조사하였으며, c-myc과 c-fos 유전자의 발현변화를 관찰하였다. RT-PCR을 시행하여 L1210, L1210AdR, L1210VcR에서 MRP 유전자 발현을 확인하였으며, Northern hybridization 한 결과 L1210세포에 비하여 L1210AdR은 유전자 발현이 40% 정도 감소하였으며, L1210Cis는 90% 정도의 유전자 발현감소가 관찰되었다. c-myc과 c-fos유전자의 Northern hybridization 한 결과 L1210에 비하여 L1210AdR은 발현감소가 나타났으나, L1210VcR과 L1210Cis의 경우는 오히려 발현증가가 관찰되었다. The occurrence of multidurg resistance (MDR) is one of the main obstacles in the successful chemotherapeutic treatment of cancer. In this study the gene expressions of multidrug resistance-associated protein (MRP), c-myc and c-fos were investigated in L1210 cells. Adriamucin- or vincristine-resistant L1210 cells, L1210AdR or L1210VcR, respectively, has been indentiified to overexpression of mdrl gene. The expression leve of MRP gene in L1210AdR and L1210Cis was more decreased than that in L1210 cells. The c-myc and c-fos gnens were expressed boty in L1210 and resistant subines. In L1210AdR, the expressions level of c-myc and c-fos genes were decreased than in L1210. However, in L1210VcR and L1210Cis, c-myc and c-fosgene expressionwere rather increased than L1210. There results suggested that MRP does not contribute in resistance of drug-resistant L1210 cells and there is no relations between MRP and mdrl gene expression. The expression of c-myc and c-fos gene may be changed during transformation of L1210 to drug-resistant sublines.