• Journal of Chest Surgery
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• 제36권1호
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• pp.7-14
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• 2003

Cyclin E는 Gl-S기로의 이행을 조절하는데 매우 중요한 역할을 하는데, 세포주기 중 $G_{0}$ / $G_{1}$기로부터 S기로의 이행의 조절이상은 종양형성에 중요한 요소로 알려져 있으므로, 여러 종류의 악성종양에서 cyclin E의 발현은 종양의 생물학적 양상과 관련되어 보고되고 있다. 또 cdk2-cyclin E 복합체의 활성도는 cdk 억제인자인 p27$^{kip1}$의 분해에 의하여 증가한다. 그러나 비소세포폐암에서 cyclin E와 p27의 발현에 관한 연구는 매우 드물다. 대상 및 방법: 81례의 절제된 제1병기 비소세포폐암 조직을 이용하여 cyclin E와 p27의 발현율을 조사하고, 조직학적 유형, 분화도, 종양의 크기, 늑막의 침범 여부, 생존율과의 상호관계를 비교 분석하였다. Cyclin E와 p27의 발현은 특이한 단클론 항체를 이용하여 ABC법으로 면역조직화학염색을 시행하였고, 생존율은 Kaplan-Meier법을 이용하였다. 결과: 제1병기 비소세포폐암에서 cyclin E와 p27의 발현율은 각각 29.6%,28.4%였다. Cyclin E는 늑막 침범례에서, p27은 종양의 직경이 3cm 이하인 경우에 각각 높은 발현율을 보였다(p=0.04, p=0.015). Cyclin E의 발현군의 5년 생존율은 44.4%로, 미 발현군 68.2%에 비해 낮았으며(p=0.015), p27의 발현군의 5년 생존율은 72.2%로, 미 발현군 56.2%에 비해 높은 경향을 보였다(p=0.09). Cyclin E가 발현되지 않고 p27이 발현되는 군의 5년 생존율은 73.5%로, cyclin E가 발현되고 p27이 발현되지 않는 군 36.3%에 비해 높게 나타났다(p=0.0029). 다변량 분석상 cyclin E은 불량한 예후를(RR=3.578, p=0.006), p27은 양호한 예후를 시사하는 독립적인 인자였다(RR=0.183, p=0.009). 결론: 제1병기 비소세포폐암에서 cyclin E의 발현은 불량한 예후를, p27은 양호한 예후를 나타내는 독립적인 예후인자가 될 수 있음을 확인하였다.

• 한국자원식물학회지
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• 제30권6호
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• pp.640-646
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• 2017

In this study, we elucidated anti-cancer activity and potential molecular mechanism of 70% ethanol extracts from Taxilli Ramulus (Taxillus chinensis (DC.) Danser) (TR-E70) against human colorectal cancer cells. Anti-cell proliferative effect of TR-E70 was evaluated by MTT assay. The effect of TR-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively. TR-E70 suppressed the proliferation of human colorectal cancer cell lines, HCT116 and SW480. Although TR-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by TR-E70 more dramatically occurred than that of cyclin D1 mRNA. Cyclin D1 downregulation by TR-E70 was attenuated in presence of MG132. In addition, TR-E70 phosphorylated threonine-286 (T286) of cyclin D1. TR-E70-mediated cyclin D1 degradation was blocked in presence of LiCl as an inhibitor $GSK3{\beta}$ but not PD98059 as an ERK1/2 inhibitor and SB203580 as a p38 inhibitor. Our results suggest that TR-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through $GSK3{\beta}$-dependent cyclin D1 degradation. From these findings, TR-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

• Radiation Oncology Journal
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• 제16권4호
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• pp.377-388
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• 1998

목적 : 방사선조사에 의한 apoptosis에서 나타나는 각종 세포주기관련 유전자들의 발현 양상을 RNA와 단백 수준에서 분석하여 방사선조사에 의한 apoptosis에서의 세포주기 조절의 변화를 규명함으로서 방사선치료의 기전에 대한 분자생물학적 이해를 도모하고자 본 연구를 시행하였다. 대상 및 방법 : promyelocytic leukemia 세포주인 HL60 세포주를 배양하여 선형가속기(6MV X-선)로 세포에 8Gy의 방사선을 조사하였다. 조사후 다양한 시간 간격으로 Apoptotic DNA Fragmentation Assay법을 이용하여 apoptosis를 확인하고 동시에 세포주기관련 유전자들(cyclinA, cyclin B, cyclin C, cyclin Dl, cyclin E, cdc2, CDK2, CDK4, $p16^{INK4a}$, $p21^{WAF1}$, $p27^{KIP1}$, E2F, PCNA와 Rb)을 단백질과 RNA 수준에서 분석하기위해 western blot analysis와 반정량적 RT-PCR을 시행하였다. 결과: 8 Gy의 방사선 조사에 의해 HL60세포에서 apoptosis가 관찰 되었다. 방사선 조사군에서 cyclin A단백은 조사후 48시간까지 시간이 갈수록 증가하였으며, cyclin E, E2F, CDK2 및 Rb 단백은 증가되었다가 다시 감소를 보였다. Rb단백의 증가는 대부분 비활성형인 ppRb (phosphorylated Rb protein)의 양적변화에 의한 것이었다. cyclin Dl, PCNA, COC2, CDK4, $p16^{INK4a}$단백은 발현의 차이를 보이지 않았으며 $p21^{WAF1}$과 $p27^{KIP1}$ 단백은 검출되지 않았다. cyclin A, B, C mRNA는 방사선 조사 직후 감소하였다가 12시간부터 발현이 증가되었으며 cyclin Dl mRNA는 조사후 바로 증가하여 48시간에 다시 감소하였다. cyclin E mRNA는 조사 후 시간이 경과함에 따라 감소하였다. CDK2 mRNA는 3시간째는 감소하다가 6시간부터 많은 증가를 보였으며 CDK4 mRNA는 조사후 6-12시간에 급격한 발현증가를 보였다. $p16^{INK4a}$ RNA는 발현의 변화가 없었으며, $p21^{WAF1}$ 및 $p27^{KIP1}$ RNA의 발현은 관찰되지 않았다. 결론 : 이상의 결과로 미루어볼 때, 방사선 조사에 의한 HL60세포의 apoptosis와 세포의 Gl/S transition는 밀접한 관계가 있는 것으로 생각되며 Rb단백의 증가와 활성형 Rb단백의 감소 현상도 관련이 있는 것으로 사료된다. 이는 E2F의 비정상적인 과발현 및 cyclin E/CDK2의 발현 증가와 관련이 있는 것으로 추측된다. 또한 $p21^{WAF1}$ 및 $p27^{KIP1}$는 방사선에 의한 apoptosis에는 관여되지 않는 것으로 사료된다.

• 한국약용작물학회지
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• 제25권5호
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• pp.328-334
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• 2017

Background: In this study, we evaluated the anti-cancer activity and potential molecular mechanism of 70% ethanol extracts of the root of Aralia cordata var. continentalis (Kitagawa) Y. C. Chu (RAc-E70) against human colorectal cancer cells. Methods and Results: RAc-E70 suppressed the proliferation of the human colorectal cancer cell lines, HCT116 and SW480. Although RAc-E70 reduction cyclin D1 expression at the protein and mRNA levels, RAc-E70-induced reduction in cyclin D1 protein level occurred more dramatically than that of cyclin D1 mRNA. The RAc-E70-induced downregulation of cyclin D1 expression was attenuated in the presence of MG132. Additionally, RAc-E70 reduced HA-cyclin D1 levels in HCT116 cells transfected with HA-tagged wild type-cyclin D1 expression vector. RAc-E70-mediated cyclin D1 degradation was blocked in the presence of LiCl, a $GSK3{\beta}$ inhibitorbut, but not PD98059, an ERK1/2 inhibitor and SB203580, a p38 inhibitor. Furthermore, RAc-E70 phosphorylated cyclin D1 at threonine-286 (T286), and LiCl-induced $GSK3{\beta}$ inhibition reduced the RAc-E70-mediated phosphorylation of cyclin D1 at T286. Conclusions: Our results suggested that RAc-E70 may downregulate cyclin D1 expression as a potential anti-cancer target through $GSK3{\beta}$-dependent cyclin D1 degradation. Based on these findings, RAc-E70 maybe a potential candidate for the development of chemopreventive or therapeutic agents for human colorectal cancer.

• 대한골관절종양학회지
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• 제14권2호
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• pp.106-118
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• 2008

목적: 세포주기의 조절곤란은 종양의 발생과 진행에 영향을 미친다. 몇가지 알려진 연부조직 육종에 대한 면역 조직화학적 예후인자 들이 있지만 상반된 연구도 있고 G1/S phase관계되는 인자에 대한 연구는 거의 없는 상태이다. 따라서 저자는 연부조직 육종의 재발 및 전이와 관계된 G1/S phase 세포조절주기 단백의 면역화학적 예후인자를 알아보고자 하였다. 대상 및 방법: 1998년 1월부터 2005년 12월까지 연부조직 육종으로 진단된 환자 중 최소한 1년이상 추시관찰이 가능하고 파라핀 블록의 보존상태가 비교적 양호한 43예의 환자를 대상으로 연구하였다. 지방육종 15예, 악성 섬유성조직구증 13예, 횡문근육종 5예, 활막육종 5예, 평활근육종 3예, 섬유육종이 2예였다. 모든 환자의 조직은 전 절제술로 제거된 조직을 대상으로 Cyclin D1, Cyclin E, CDK4, CDK, p16, p27, Rb, E2F-1, p53, Ki-67 등의 면역 조직화학적 발현을 조직 microarray 방법을 사용하여 측정하고 환자의 국소 재발 및 전이에 따른 예후를 비교 분석하였다. 결과: 국소 재발은 8예(19%)에서 일어났으며 이것은 Cyclin E(p=0.024), E2F-1(p=0.046)의 발현과 연관이 있었다. 전이는 16예(37%)에서 일어났으며 CDK4의 증가와 연관이 있었다(p=0.031). 결론: Cyclin E와 E2F-1이 연부조직 육종의 국소 재발과 관련있는 예후를 제공해주었고, CDK4가 전이를 예측할 수 있는 독립적인 예후를 제공해주었다. 따라서 조직 검사시 이들 표식자들의 적절한 사용이 환자의 예후를 예측하고 치료의 범위를 결정 짖는데 중요한 도움을 줄 수 있을 것이다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.419-422
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• 2013

Conventional classifications of gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are rather unsatisfactory because of the variation in survival within each subgroup. Molecular markers are being found able to predict patient outcome in more and more tumours. The aim of this study was to characterize the expression of the proteins cyclin D1, cyclin E and P53 in GEP-NETs and assess any prognostic impact. Tumor specimens from 68 patients with a complete follow-up were studied immunohistochemically for cyclin D1, cyclin E and P53 expression. High cyclin D1 and cyclin E immunostaining (${\geq}$ 5% positive nuclei) was found in 48 (71%) and 24 (35%) cases, and high P53 staining (${\geq}$ 10% positive nuclei) in 33 (49%). High expression of P53 was more common in gastric neuroendocrine tumors and related to malignant behavior, being associate with a worse prognosis on univariate analysis (RR=1.9, 95%CI=1.1-3.2). High expression of cyclin E was significantly associated with shorter survival in the univariate analysis (RR=2.0, 95%CI=1.2-3.6) and multivariate analysis (RR=2.1, 95%CI=1.1-4.0). We found no significant correlation between the expression of cyclin D1 and any clinicopathological variables. Our study indicated a prognostic relevance for cyclin E and P53 immunoreactivity. Cyclin E may be an independent prognostic factor from the 2010 WHO Classification which should be evaluated in further studies.

• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3817-3823
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• 2014

Background: The MDM2 oncogene, a negative regulator of p53, has a functional polymorphism in the promoter region (SNP309) that is associated with multiple kinds of cancers including non-melanoma skin cancer. SNP309 has been shown to associate with accelerated tumor formation by increasing the affinity of the transcriptional activator Sp1. It remains unknown whether there are other factors involved in the regulation of MDM2 transcription through a trans-regulatory mechanism. Methods: In this study, SNP309 was verified to be associated with overexpression of MDM2 in tumor cells. Bioinformatics predicts that the T to G substitution at SNP309 generates a stronger E2F1 binding site, which was confirmed by ChIP and luciferase assays. Results: E2F1 knockdown downregulates the expression of MDM2, which confirms that E2F1 is a functional upstream regulator. Furthermore, tumor cells with the GG genotype exhibited a higher proliferation rate than TT, correlating with cyclin D1 expression. E2F1 depletion significantly inhibits the proliferation capacity and downregulates cyclin D1 expression, especially in GG genotype skin fibroblasts. Notably, E2F1 siRNA effects could be rescued by cyclin D1 overexpression. Conclusion: Taken together, a novel modulator E2F1 was identified as regulating MDM2 expression dependent on SNP309 and further mediates cyclin D1 expression and tumor cell proliferation. E2F1 might act as an important factor for SNP309 serving as a rate-limiting event in carcinogenesis.

• Animal cells and systems
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• 제9권4호
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• pp.191-198
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• 2005

The effects of 6-aminonicotinamide (6-AN) on viability of IMR32 neuroblastoma cells in the presence of ATP or $NAD^+$ have been investigated. 6-AN caused marked reduction in cell viability and similar observations were also made with cells treated with 6-AN+ATP. However, cells treated with $6-AN+NAD^+$ showed cell viability similar to untreated cells. Morphologically, 6-AN and 6-AN+ATP treated cells showed loss of neurites, polyhedric shapes, shrinkage of cell bodies and formation of lysed cells, while $6-AN+NAD^+$ cells did not show any such changes. The flow cytometry analysis demonstrated that 6-AN increased cell population in $G_0/G_1$ phase and decreased cell population in Sand $G_2/M$ phase following a 72 h exposure. Western blot analysis showed that 6-AN stimulated a substantial increase in the level of the cdk inhibitor $p27^{kip1}$, but lowered the levels of cdk2, cyclin E and p-Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of $p27^{kip1}$, cdk2, cyclin E and p-Rb revealed close correlation between the signal observed in the Western blot analysis. 6AN+ATP treated cells showed similar results obtained with 6-AN treated cells in expression of cdk2, cyclin E, p-Rb proteins and $p27^{kip1}$, $6-AN+NAD^+$ cells showed greater expression of cdk2, cyclin E and p-Rb than those in 6-AN and 6-AN+ATP treated cells. The results suggest that 6-AN induced the $G_0/G_1$ phase arrest in IMR32 neuroblastoma cell lines through the increase of $p27^{kip1}$ and the decrease of cdk2, cyclin E and p-Rb.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제44권4호
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• pp.182-190
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• 2018

Objectives: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Inconsistency in various histopathologic features for predicting nodal metastasis and overall prognosis and a better understanding of molecular mechanisms of tumourigenesis have shifted the focus to a search for more definitive predictive markers. To identify the role of two immunohistochemical (IHC) markers, E-cadherin and cyclin D1, as predictive markers of aggressiveness in HNSCC and to assess clonal expansion of tumour cells. Materials and Methods: A total of 66 cases of HNSCC with neck node dissection were studied. IHC was performed on primary tumour sections and lymph nodes showing metastatic deposits. Histopathological parameters such as tumour grade and TNM stage together with nodal status were compared according to expression of the two markers. Fischer's chi-square test was used to assess the correlation between the two markers and histopathological parameters. Results: Out of 66 cases studied, 37 showed LN metastasis. Most of the patients were male, and the most common tumour site was buccal mucosa. We found a significant association between loss of E-cadherin and node metastasis (P<0.001) and higher TNM stage (P<0.001). Cyclin D1 overexpression was significantly associated with only nodal metastasis (P=0.007). No significant association with tumour grade was found for either marker. The subgroup of E-cadherin loss with cyclin D1 overexpression was associated with the maximum incidence of nodal metastasis and higher TNM stage, highlighting the importance of using a combination of these two markers. A significant association was noted between the expression of markers at the primary site and at nodal deposits, indicating clonal expansion. Conclusion: A combination of the two markers E-cadherin and cyclin D1 can predict prognosis in HNSCC, although tumour heterogeneity may affect this association in some cases.

• 약학회지
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• 제41권1호
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• pp.94-98
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• 1997

Ceramide. a product of sphingomyelin hydrolysis, has been proposed as a lipid second messenger mediating antiproliferative activation. In this study, we examined the role of the cell cycle-related proteins in the ceramide-mediated growth suppression. Treatment of U-937 cells with C$_2$-ceramide(N-acetylsphingosine) resulted in growth suppression in a time- and concentration dependent manner. Ceramide induced concentration dependent dephosphorylation of retinoblastoma gene product (Rb). Rb remains hypophosphorylated in synchronized cells even after serum stimulation in the presence of ceramide. Ceramide decreased the expression of cyclin D$_1$ and cyclin E levels. These results suggest that antiproliferative effect of ceramide is associated with hypophosphorylation of Rb and decreased expression of cyclin D1 and cyclin E.