Breast cancer is the fifth most common cause of cancer death in the worldwide. In the past decades, tamoxifen has been used for clinical treatment for breast cancers. The derivatives of compound thiazolidinedione (TZDs) including troglitazone (rezulin) and rosiglitazone (avandia) are also in the stages of clinical trials. But in the earlier research, some studies reported that the use of these drugs was associated with some serious side effects. Cyclin D1 plays an important role in G1/S phase cell cycle progression and in growth factor- or estrogen-induced mammary epithelial cell proliferation. Cyclin D1 overexpression is also found in high percentage (over 30%) of human breast cancers, correlating with poor prognosis. In this study, we used a cyclin D1-ablative drug VGH No.47 to reduce the expression of cyclin D1 in human breast adenocarcinoma cell line MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) and to study its effect on cell proliferation. Our results demonstrated that VGH No.47 decreased the protein stability of cyclin D1. Conversely, VGH No.47 reduced cyclin D1 at both transcriptional level and protein stability in ER-negative MDA-MB-231 cells. We found that VGH No.47 caused G2/M arrested in both breast cancer cell lines. In addition, we tested whether cyclin D1-ablative drug could sensitize breast cancer cells to tamoxifen and TZDs. We expect to lower the dose of tamoxifen, troglitazone or rosiglitazone to reduce the side effects, but the results do not meet our expectation and do not exhibit synergistic effect.

Les cyclines D jouent un rôle essentiel dans les mécanismes du cycle cellulaire. Cette famille de protéines est composée de trois membres (D1,D2,D3) qui partagent un domaine très homologue de la « cyclin box » (codée par les exons 1-3). Ce domaine est responsable de leur activité redondante dans la phosphorylation de la protéine du rétinoblastome lors de l'association avec les kinases cycline-dépendantes CDK4/6. Parmi les trois cyclines, la cycline Dl, bien que faiblement exprimée dans les lymphocytes, est la cycline la plus impliquée dans les cancers lymphoïdes ou elle aurait une fonction de facteur de transcription indépendante de Cdk. Etant donné qu'après stimulation antigénique, les lymphocytes T et B ont une capacité remarquable de division, essentielle à la génération d’une réponse immunitaire efficace, nous avons porté un intérêt particulier au rôle des cyclines D dans la lymphopoïèse. Pour étudier le rôle de la cycline Dl dans la différenciation des lymphocytes, nous avons utilisé des souris déficientes pour les exons 1,2,3 de la « cycline box » Dl mais conservé les exons 4 et 5. Étonnamment, ces souris présentaient des phénotypes très différents que nous avons subdivisés en quatre groupes. Dans le groupe I, les souris avaient un thymus réduit car la différenciation de la lignée lymphoide est bloquée à un stade très précoce, avec un faible nombre de cellules progénitrices (CLP) dans la moelle osseuse. Dans le thymus, les progéniteurs des thymocytes (ETP) étaient pratiquement absents et les précurseurs CD4CD8CD3 (TN) immatures essentiellement constitués par des cellules CD44*CD25 (TN1) et CD44*CD25+ (TN2) les plus immatures. De plus, les CD4*CD8* (DP) qui donnent naissance aux thymocytes matures CD4+ et CD8+ étaient présents en très faible quantité. Dans la moelle osseuse, on observe un blocage majeur dans la différenciation de la lignée B au stade pré-proB. Dans les ganglions, la forte réduction du nombre de lymphocytes T observée était liée au faible nombre d'ETP et à l’absence du récepteur aux chimiokines CCR7. Dans le groupe II, les souris présentaient une diminution moins sévère des ETP et une atrophie modérée du thymus. La différenciation était bloquée à un stade ultérieur, soit dans la transition des étapes TN3 à TN4. Dans la moelle osseuse, les lymphocytes B ont subi un blocage partiel au stade pré-proB et une réduction des cellules pré-B. Le nombre de CLP est également réduit, mais dans une moindre mesure que dans les souris du groupe I. dans les groupes III et IV, les souris ont une répartition normale des thymocytes mais présentaient une augmentation du compartiment ETP. Alors que les souris du groupe III contenaient un nombre normal de thymocytes, les souris du group IV présentaient une hyperplasie thymique. Par ailleurs, en comparaison avec des souris normales, bien que la différenciation des lymphocytes B soit normale, on observe dans les deux groupes une augmentation des CLP et des progéniteurs hématopoïétlque (LSK). L’implication de la cycline Dl dans la transition de G1 à S nous a conduit à analyser les divisions cellulaires in vivo. De manière surprenante, les souris du groupe I étaient fortement dépourvues de cellules en cycle dans tous les compartiments lymphoïdes, ce qui peut expliquer les blocages de la différenciation lymphoide. Par contre, dans les trois autres groupes, on observe une augmentation du nombre de divisions cellulaires. Ces résultats différents peuvent être dû à l'expression ou l'absence d'une protéine Dl tronquée qui contient cependant les exons 4-5. Alors que ces ARNm tronqués ne sont pas détectables dans les souris de groupe I, on observe des niveaux élevés d'expression dans les autres groupes. De plus, nous avons observé une corrélation entre l'absence d’expression des exons 4-5 et la très faible expression des gènes CCND2 et CCND3, ce qui attribue à cette protéine tronquée un rôle prépondérant dans la régulation des cyclines D et permet d’expliquer l'aplasie profonde et… / D Cyclins play an essential role connecting exogenous stimulation to the intrinsic cell cycle machinery. This family of proteins is composed of three members sharing a highly homologous domain, the cyclin box (coded by exons 1-3), which is responsible for their redundant role in the phosphorylation of the retinoblastoma protein upon association with cydin-dependent kinases Cdk4/6. Both mature T and B-cells have a remarkable division capability after antigen stimulation, essential to the generation of efficient immune responses, raising the interest of D Cyclins in lymphopoiesis. Cyclin Dl, although weakly expressed by lymphocytes, is the D Cyclin most commonly implicated in lymphoid cancers and as having a Cdk-independent transcriptional role. To study the role of Cyclin Dl, we used mice deficient for the Dl cyclin box but sparing exons 4-5. Surprisingly, individual mice have very different phenotypes that we subdivided into four arbitrary groups. Group I mice show the most precocious block in lymphoid lineage differentiation, illustrated by a low cellularity of common lymphoid progenitor cells (CLP). The thymi showed very few CD4*CD8*, double positive (DP) cells, while the CD4 CD8TCR, triple negative (TN) populations were found to be mostly constituted by the early CD44*CD25' (TNI) and few CD44*CD25* (TN2). TNl's early thymocyte progenitors (ETP) were virtually absent. At the B-cell lineage level in the bone marrow (BM) there was a major block in pre-proB differentiation. The number of peripheral T-cells was severely reduced, mainly in LN, since group I T-cells lack CCR7 expression. Group II mice presented moderate thymus atrophy. The block on TN differentiation occurs at a later stage, i.e., in the TN3 to TN4 transition, and the TNI population was characterized by a less severe depletion of the ETP. Group II mice showed a partial pre-proB block and a reduction in pre-B-cells. CLPs were also reduced but to a lesser extent than in group I mice. Group ill and group IV mice appear to have a normal thymocyte population distribution but showed an increase on ETP compartment. Group IV mice displayed thymic hyperplasia while group III mice possessed normal thymus cellularity. B-cell differentiation on both groups appeared to be normal but BM precursors had an increase in both CLP and early haematopoietic progenitor's (LSK) levels as compared with wild type mice. Cyclin Dl involvement in G1 to S transition led us to analyse in vivo division rates. Strikingly, group I mice were virtually devoid of cycling cellsin all lymphoid compartments, explaining why lymphoid lineage cells do not differentiate in these mice. In contrast, in all other groups we observed an increased BrdU incorporation. These contradicting phenotypes correlated with the expression or absence of a truncated Dl protein coded by exons 4-5. The presence of the cyclin Dl truncated mRNA was not found in group I mice but high levels of expression are consistently observed in the remaining groups. In the absence of the Dl truncated protein only trace values of Cyclins D2 and D3 were found, highlighting the role of this protein as a master D cyclin regulator, which further supports the profound aplasia and arrest in lymphoid lineage division on cells that predominantly express Cyclin D2. These results suggest that, while the function of the Dl cyclin box is redundant, the regulatory domain coded by exons 4-5 is fundamental for lymphopoiesis. Full Dl protein was also eliminated by RNA interference both in vitro and in vivo. These experiments reproduced the phenotype of group I mice. We have developed a lentiviral vector with a truncated Dl (exons 4-5) and conditional knockout (KO) mice by floxing exons 4-5 of cyclin Dl. These tools will allow us to show Cyclin Dl Cdk-independent role as a transcription regulator in lymphopoiesis and to attribute this function to exons 4-5. Understanding how exons 4-5 regulate different transcription factors might be a key in…

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The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.

O câncer de próstata é o tumor maligno mais freqüente nos homens com idade superior a 50 anos, excetuando-se os tumores cutâneos. No Brasil estima-se para o ano de 2012 cerca de 60.180 casos novos deste tipo de neoplasia. Os marcadores tumorais permitem fazer o rastreamento do câncer, o diagnóstico diferencial entre uma neoplasia benigna e maligna, a avaliação de prognóstico e o acompanhamento terapêutico, assim como a detecção da recidiva tumoral. Dentre estes marcadores tumorais, tem-se dado muito atenção para as proteínas que mediam e participam da progressão do ciclo celular. A ciclina D1 é uma proteína nuclear de vida curta que é destruída pela via da ubiquitina ATP dependente, e está envolvida na transição celular da fase do ciclo G1 (repouso) para a fase S (síntese) tanto em células normais como em células neoplásicas. A super expressão de ciclina D1 remove a regulação normal do ciclo celular causando proliferação celular descontrolada, um crescimento anormal dos tecidos e a transformação para um fenótipo neoplásico, atuando como oncogene. No presente trabalho foi estudado a expressão de ciclina D1 em adenocarcinomas de próstata, tendo como objetivo avaliar a relação desta proteína com parâmetros epidemiológicos, clínicos e histopatológicos. Adicionalmente também foi feita comparação de escore de Gleason e lateralidade tumoral entre biópsias prostáticas com agulha e de prostatectomias radicais. No ensaio para ciclina D1 foram analisados 85 casos através de imunoistoquímica (IHQ) de material proveniente de prostatectomias radicais diagnosticados com adenocarcinoma de próstata entre os anos de 2005 e 2010 em nosso serviço. O método de avaliação se utilizou de microscopia ótica comum e contagem semi-quantitativa, comparado-se a expressão com achados clínicos, epidemiológicos e histopatológicos utilizando-se Teste T de Fisher, Qui Quadrado, Mann-Whitney, Curva ROC e correlação de Spearman. Os resultados demonstraram correlação positiva de ciclina D1 com escore de Gleason (p<0,05), com volume prostático (p=0,01) e uma tendência a correlação positiva com invasão perineural (p=0,07). Não houve correlação estatística entre ciclina D1 e o aumento de PSA, assim como outros achados histopatológicos. As biópsias prostáticas com agulha apresentaram subestimação em 40% dos casos para escore de Gleason e de 62,3% dos casos para lateralidade tumoral quando comparadas a prostatectomia radical. Já que as taxas de subestimação de escore de Gleason e lateralidade tumoral são relativamente altas e visto a urgência em se padronizar novos biomarcadores para o câncer prostático, sugerimos que ciclina D1 pode ser utilizada como biomarcador em patologia cirúrgica da próstata auxiliando numa gradação histológica mais precisa em biópsias com agulha colaborando para melhor vigilância e escolha terapêutica. / Prostate cancer is the most common malignant tumor in men older than 50 years, except for skin tumors. In Brazil it is estimated for the year 2012 about 60,180 new cases of this type of neoplasm. Tumor markers allow to cancer screening, differential diagnosis between a benign and malignant, assessment of prognosis and therapeutic monitoring, and detection of tumor recurrence. Among these tumor markers, has been given much attention for proteins that mediate and participate in cell cycle progression. Cyclin D1 is a short-lived nuclear protein that is destroyed by the ATP ubiquitin dependent pathway, and is involved in the transition of cell cycle G1 phase (resting) to the S phase (synthesis) cells both in normal and neoplastic cells. The overexpression of cyclin D1 removes the normal regulation of cell cycle causing uncontrolled cell proliferation, abnormal growth of tissues and transformation to a neoplastic phenotype, acting as an oncogene. In the present work we studied the expression of cyclin D1 in prostate adenocarcinomas, and to evaluate the relationship of this protein with epidemiologic factors, clinical and histopathological features. Additionally comparison was also made of Gleason score and laterality between tumor biopsies and prostate needle radical prostatectomies. In the assay for cyclin D1 were 85 cases analyzed by immunohistochemistry (IHC) of material from radical prostatectomies diagnosed with prostate adenocarcinoma between the years 2005 and 2010 at our institution. The evaluation method utilized were light microscopy and semi-quantitative score, comparing the cyclin D1 expression with clinical, epidemiological and histopathological features using Fisher\'s exact test, chi square test, Mann-Whitney test, ROC curve and Spearman correlation. The results showed a positive correlation of cyclin D1 with Gleason score (p <0.05), prostate volume (p = 0.01) and a trend toward positive correlation with perineural invasion (p = 0.07). There was no statistical correlation between cyclin D1 and increased PSA, as well as other histopathologic features. Prostate needle biopsies showed underestimation in 40% of cases for Gleason score and 62.3% of cases for tumor laterality when compared to radical prostatectomy. Since the rates of underestimation of Gleason score and tumor laterality are relatively high and the urgency to standardize new biomarkers for prostate cancer, we suggest that cyclin D1 may be used as biomarkers in surgical pathology of the prostate assisting more accurate histological grading in needle biopsies and collaborating for better surveillance and therapeutic choice.

O câncer de próstata é o tumor maligno mais freqüente nos homens com idade superior a 50 anos, excetuando-se os tumores cutâneos. No Brasil estima-se para o ano de 2012 cerca de 60.180 casos novos deste tipo de neoplasia. Os marcadores tumorais permitem fazer o rastreamento do câncer, o diagnóstico diferencial entre uma neoplasia benigna e maligna, a avaliação de prognóstico e o acompanhamento terapêutico, assim como a detecção da recidiva tumoral. Dentre estes marcadores tumorais, tem-se dado muito atenção para as proteínas que mediam e participam da progressão do ciclo celular. A ciclina D1 é uma proteína nuclear de vida curta que é destruída pela via da ubiquitina ATP dependente, e está envolvida na transição celular da fase do ciclo G1 (repouso) para a fase S (síntese) tanto em células normais como em células neoplásicas. A super expressão de ciclina D1 remove a regulação normal do ciclo celular causando proliferação celular descontrolada, um crescimento anormal dos tecidos e a transformação para um fenótipo neoplásico, atuando como oncogene. No presente trabalho foi estudado a expressão de ciclina D1 em adenocarcinomas de próstata, tendo como objetivo avaliar a relação desta proteína com parâmetros epidemiológicos, clínicos e histopatológicos. Adicionalmente também foi feita comparação de escore de Gleason e lateralidade tumoral entre biópsias prostáticas com agulha e de prostatectomias radicais. No ensaio para ciclina D1 foram analisados 85 casos através de imunoistoquímica (IHQ) de material proveniente de prostatectomias radicais diagnosticados com adenocarcinoma de próstata entre os anos de 2005 e 2010 em nosso serviço. O método de avaliação se utilizou de microscopia ótica comum e contagem semi-quantitativa, comparado-se a expressão com achados clínicos, epidemiológicos e histopatológicos utilizando-se Teste T de Fisher, Qui Quadrado, Mann-Whitney, Curva ROC e correlação de Spearman. Os resultados demonstraram correlação positiva de ciclina D1 com escore de Gleason (p<0,05), com volume prostático (p=0,01) e uma tendência a correlação positiva com invasão perineural (p=0,07). Não houve correlação estatística entre ciclina D1 e o aumento de PSA, assim como outros achados histopatológicos. As biópsias prostáticas com agulha apresentaram subestimação em 40% dos casos para escore de Gleason e de 62,3% dos casos para lateralidade tumoral quando comparadas a prostatectomia radical. Já que as taxas de subestimação de escore de Gleason e lateralidade tumoral são relativamente altas e visto a urgência em se padronizar novos biomarcadores para o câncer prostático, sugerimos que ciclina D1 pode ser utilizada como biomarcador em patologia cirúrgica da próstata auxiliando numa gradação histológica mais precisa em biópsias com agulha colaborando para melhor vigilância e escolha terapêutica. / Prostate cancer is the most common malignant tumor in men older than 50 years, except for skin tumors. In Brazil it is estimated for the year 2012 about 60,180 new cases of this type of neoplasm. Tumor markers allow to cancer screening, differential diagnosis between a benign and malignant, assessment of prognosis and therapeutic monitoring, and detection of tumor recurrence. Among these tumor markers, has been given much attention for proteins that mediate and participate in cell cycle progression. Cyclin D1 is a short-lived nuclear protein that is destroyed by the ATP ubiquitin dependent pathway, and is involved in the transition of cell cycle G1 phase (resting) to the S phase (synthesis) cells both in normal and neoplastic cells. The overexpression of cyclin D1 removes the normal regulation of cell cycle causing uncontrolled cell proliferation, abnormal growth of tissues and transformation to a neoplastic phenotype, acting as an oncogene. In the present work we studied the expression of cyclin D1 in prostate adenocarcinomas, and to evaluate the relationship of this protein with epidemiologic factors, clinical and histopathological features. Additionally comparison was also made of Gleason score and laterality between tumor biopsies and prostate needle radical prostatectomies. In the assay for cyclin D1 were 85 cases analyzed by immunohistochemistry (IHC) of material from radical prostatectomies diagnosed with prostate adenocarcinoma between the years 2005 and 2010 at our institution. The evaluation method utilized were light microscopy and semi-quantitative score, comparing the cyclin D1 expression with clinical, epidemiological and histopathological features using Fisher\'s exact test, chi square test, Mann-Whitney test, ROC curve and Spearman correlation. The results showed a positive correlation of cyclin D1 with Gleason score (p <0.05), prostate volume (p = 0.01) and a trend toward positive correlation with perineural invasion (p = 0.07). There was no statistical correlation between cyclin D1 and increased PSA, as well as other histopathologic features. Prostate needle biopsies showed underestimation in 40% of cases for Gleason score and 62.3% of cases for tumor laterality when compared to radical prostatectomy. Since the rates of underestimation of Gleason score and tumor laterality are relatively high and the urgency to standardize new biomarkers for prostate cancer, we suggest that cyclin D1 may be used as biomarkers in surgical pathology of the prostate assisting more accurate histological grading in needle biopsies and collaborating for better surveillance and therapeutic choice.

By Western blotting and immunohistochemical analyses, we have demonstrated that Pin1 was overexpressed in 71.4% of hepatocellular carcinoma (HCC) and its levels correlated with the clinical survival rate. This conclusion was supported by the results from examining Pin1 protein in HCC cancer cell lines. RT-PCR was performed to examine the Pin1 transcription level in tumor part and was compared with that in non-tumor part. Our results indicated that pin1 overexpression was due to the upregulation of Pin1 transcription. Interestingly, most of the cases with upregulation of Pin1 have been shown to correlate with £]-catenin and Cyclin D1 accumulation in HCC specimens. These results were consistent with the previous studies that Pin1 caused £]-catenin and Cyclin D1 elevation in breast cancer. The concordance between hepatitis virus chronic infection and Pin1 overexpression of HCC patients was also analysis. Taken together, these data indicated that Pin1 overexpression leading to £]-catenin and Cyclin D1 accumulation might play a critical role in hepatocellular carcinogenesis and tumor progression. Pin1 levels therefore can be used as a prognostic marker for HCC, and our results suggested that Pin1 is a potential target for therapeutic intervention in hepatocellular carcinoma.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G1-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho β-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of β-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of β-catenin and CREB pathways.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第19654号 / 薬博第824号 / 新制||薬||240(附属図書館) / 32690 / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 松原 和夫, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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