Overexpression Of Cyclin Research Articles

The NFAT1 Transcription Factor is a Repressor of Cyclin A2 Gene Expression

The NFAT (Nuclear Factor of Activated T cells) family of transcription factors plays a central role in the regulation of several genes related to the immune response. Recently, NFAT proteins have been implicated in the proliferation and differentiation of different cell types. Previous studies have shown that NFAT1-deficient mice display lymphocyte hyperproliferation, shortened cell cycle duration, and cyclin overexpression. Here, we demonstrate that cyclin A2 expression is upregulated in the absence of NFAT1 in lymphocytes. Ectopic expression of NFAT1 in CHO cells decreases cyclin A2 levels. Indeed, NFAT1 binds to a consensus binding site found at the mouse cyclin A2 promoter in vitro and in vivo. Luciferase reporter assays show that NFAT1 downregulates cyclin A2 expression by directly binding to the cyclin A2 promoter. Together, these results indicate that the NFAT1 transcription factor represses cyclin A2 expression in lymphocytes, and may act as a silencer of gene transcription during the cell cycle.

Mip/LIN-9 can inhibit cell proliferation independent of the pocket proteins

Progression through the G1-phase of the cell cycle requires that cyclin D and CDK4 phosphorylate pRB and the other pocket proteins, p107 and p130. Cyclin E and CDK2 further phosphorylate pRB to complete its inactivation and allow the cell to enter S-phase. These phosphorylation events lead to the inactivation of the antiproliferative effect of the pocket proteins. The pocket proteins are the main targets of CDK4, and its unregulated activity can contribute to carcinogenesis. Mip/LIN9 is a recently described protein with growth suppressor, as well as growth promoting effects due to its ability to stabilize B-Myb and induce genes required for S phase and mitosis. The finding that a mutation that deletes the first 84 amino acids of Mip/LIN-9 corrects the defects of the CDK4 knockout mouse suggests that it should have a growth repressor effect that is blocked by CDK4. However, overexpression of cyclin D only partially blocks the inhibitory effect of Mip/LIN-9 on cell proliferation. Here, we performed experiments to further understand the antiproliferative effect of Mip/LIN-9 within the context of the pocket proteins. Our results suggest that there is a pocket protein-independent mechanism of the Mip/LIN-9 antiproliferative effect since it can be observed in cells with ablation of the three members of the family, and in NIH3T3 cells expressing the adenovirus E1A-12S protein. Altogether, the independence from the pocket proteins and the partial blockade of the antiproliferative effect produced by expression of cyclin D suggest that the role of Mip/LIN-9 downstream of CDK4 may be more closely related to the activation of B-Myb and the induction of S/M genes. Importantly, the regulatory effect of CDK4 is not due to direct phosphorylation of Mip/LIN-9 by this kinase or even CDK2, suggesting an indirect mechanism such as phosphorylation of the pocket proteins.

A phase 1 study of the selective cyclin dependent kinase inhibitor P276–00 in patients with advanced refractory neoplasms

14117 Background: In human cancers, genetic and epigenetic events result in over-expression of cyclins or absence or diminished levels of Cdk inhibitors, providing tumor cells with selective growth advantage. This has prompted the development of pharmacological Cdk inhibitors that could potentially produce anti-tumor effect. P276–00 is a selective Cdk4-D1 and Cdk1-B inhibitor. This study was designed to determine the maximum tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumour activity of P276–00 given intravenously to patients with advanced refractory solid tumours. Methods: P276–00 was administered in escalating doses to cohorts of eligible patients (pts), starting with a dose of 9 mg/m2 as a 30 minute iv infusion day 1 to 5, and day 8 to 12, q 3 weekly. To date 22 pts have been entered on the study (cohort 1 - 4 pts at 9 mg/m2, cohort 2 - 4 pts at 12.6 mg/m2, cohort 3 - 6 pts at 17.6 mg/m2, cohort 4 - 8 pts at 24.6 mg/m2) with PS 0–2, and mean age of 56 years. Pharmacokinetic profiles were obtained on cycle 1 days 1 and 5. Skin biopsies were obtained immediately prior to starting study treatment and on day 21 of cycle 2 and will be analyzed for Ki67, cleaved caspase 3, phospho-Rb, cyclin D1 and cdk4, and microarray. Results: To date dose limiting toxicity has occurred in one pt. Grade 3 fatigue occurred in 1 pt at 17.6 mg/m2. The most common drug-related adverse events, which were all grade 1 or 2, were fatigue, nausea, hypotension, sweating, and dry mouth. No Grade 3 biochemical toxicities have been reported so far. There have been no responses noted to date. 4 pts have stable disease after 2 cycles. Pharmacokinetic results: The Cmax, t1/2, and AUC0–8 on day 1 were as follows: 9 mg/m2- 315 ng/mL, 6.6 hr, 883 ng.h/mL; 12.6 mg/m2- 402 ng/mL, 5.5 hr, 848 ng.h/mL; 17.6 mg/m2- 589 ng/mL, 5.3 hr, 1289 ng.h/mL; 24.6 mg/m2- 621 ng/mL, 5.6 hr, 1286 ng.h/mL. Conclusions: P276–00 is well tolerated, but grade 3 fatigue has been noted in 1 pt at 17.6 mg/m2 dose level. We have observed confirmed stable disease in one patient. PK results indicate that at 9 mg/m2,12.6 mg/m2, 17.6 mg/m2 and 24.6 mg/m2 we are able to cross the cdk4 enzyme IC50 approximately 10, 13, 19 and 20 times and cross the anti-proliferative IC50 1.1, 1.4, 2.1 and 2.2 times respectively. Accrual continues at the 34.4 mg/m2 dose level. No significant financial relationships to disclose.

Preferential chemosensitivity to gemcitabine by cyclin E overexpression in human gastric cancers

21020 Background: Exploration of molecular determinants for chemosensitivity is the key element of personalized cancer therapy. Cyclin E is a major G1-phase cyclin, together with the CDK2, which mediates phosphorylation and functional inactivation of Rb protein. Cyclin E overexpression has been demonstrated in a variety of cancers, including human gastric cancers (GC), while its biologic significance in drug therapy remains unclear. Previously, we have demonstrated that cyclin D1 overexpression plays an important role in differential chemosensitivity of human GC cells (Proc Am Assoc Cancer Res 2004; 45: abstract 4888). In this study, we examine the roles between cyclin E overexpression and chemosensitivity of human GC cells. Methods: Compared human gastric cancer cells (NCI-N87) with stably transfected cells (N87-CyE), which have 2-fold overexpression of cyclin E, the IC50 for gemcitabine (2'-2'-difluoro- deoxycytidine, dFdC) was more than 1-log lower in N87-CyE (17.0 ± 2.7 nM) than N87 cells (113.7 ± 6.5 nM) by MTT colorimetric cytotoxicity assay. In contrast, the N87-CyE cells are only slightly more chemosensitive to taxanes (paclitaxel and docetaxel), and confer largely identical chemosensitivity to 5-FU, cisplatin, and irinotecan (CPT-11). We applied RNA interference (RNAi) of cyclin E to N87 cells. The stably transfected N87-CyE/RNAi cells readily confer gemcitabine resistance with the IC50 for gemcitabine of 124.5 ± 1.6 nM. Results: Gemcitabine-induced apoptosis in N87, N87-CyE, or N87-CyE/RNAi cells was shown in either caspase-3 or PARP (poly ADP-ribose polymerase) cleavage assay by Western blotting, and Annexin-V-FITC apoptosis detection by flowcytometry. The threshold concentration of gemcitabine for caspase-3 and PARP cleavage was 10–25 nM for N87-CyE, and 100–200 nM for N87 or N87-CyE/RNAi, respectively. Conclusions: Our data demonstrate that preferential chemosensitivity to gemcitabine by cyclin E overexpression in gastric cancer cells. Gemcitabine-induced apoptosis is enhanced by cyclin E overexpression, while is reduced by RNAi of cyclin E. Further studies for potential clinical use of gemcitabine in personalized chemotherapy for cyclin E or cyclin D1-overexpressing GC are warranted (supported by the grants of NHRI-CN-CA9201S, Taiwan). No significant financial relationships to disclose.

P141 Overexpression of cyclin E and centrosome hyperamplification in oral squamous cell carcinomas

P141 Overexpression of cyclin E and centrosome hyperamplification in oral squamous cell carcinomas

Actin cytoskeleton as a putative target of the neem limonoid Azadirachtin A

Limonoids isolated from the Indian neem tree ( Azadirachta indica) have been gaining global acceptance in agricultural applications and in contemporary medicine for their myriad but discrete properties. However, their mode of action is still not very well understood. We have studied the mode of action of Azadirachtin A, the major limonoid of neem seed extracts, using Drosophila melanogaster as the model system. Azadirachtin A induces moderate-to-severe phenotypes in different tissues in a dose-dependent manner. At the cellular level, Azadirachtin A induces depolymerization of Actin leading to arrest of cells and subsequently apoptosis in a caspase-independent manner. Azadirachtin A-induced phenotypes were rescued by the over-expression of Cyclin E in a tissue-dependent manner. Cyclin E, which caused global rescue of Azadirachtin A-induced phenotypes, also effected rearrangement of the actin filaments. These results suggest that probably actin is a target of Azadirachtin A activity.

Expression patterns of cyclin E, cyclin A and CDC25 phosphatases in laryngeal carcinogenesis

The aim of the research was to evaluate the expression levels of cyclin E/A and CDC25A/B during laryngeal carcinogenesis. The expression was demonstrated using immunohistochemistry in 46 cases of laryngeal cancer (LSCC), 23 epithelial dysplasias (ED) and 21 samples of normal mucosa (NM). The mean labeling indices (LI) for cyclin E in LSCC, ED and NM were 10.6, 4.9 and 0%; for cyclin A 27.2, 17.5 and 7%; for CDC25A 73.9, 53 and 32% and for CDC25B 36.5, 25.9 and 0%, respectively. A gradual increase in cyclin A and CDC25A expression levels from mild through moderate and severe dysplasia to in situ carcinoma were noted. Cyclin A LI significantly increased also from NM through ED to LSCC. Cyclin A and CDC25A LI significantly increased from NM to ED. Overexpression of cyclin A and CDC25A was significantly associated with proliferation among ED. Linear interdependency was detected in ED between the expression of CDC25A and cyclin A. Cyclin E and CDC25B overexpression occurs as a late event in neoplastic transformation. The progressive expression of proteins supports the multistep model of laryngealcarcinogenesis. The results indicate a possible role of cyclin A as a marker reflecting cell proliferation. The enhanced immunoexpression of cyclin A and CDC25A suggests the potential for malignant formation in preneoplastic lesions.

Nuclear cyclin B1 in human breast carcinoma as a potent prognostic factor

Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki-67, p53, c-myc or 14-3-3sigma, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki-67 or polo-like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease-free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo-like kinase 1 and 14-3-3sigma. Nuclear cyclin B1-positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients.

Frequency and distribution of trisomy 11 in multiple myeloma patients: relation with overexpression of CCND1 and t(11;14)

Multiple myelomas (MM) recently have been stratified into five groups (TC1–TC5) on the basis of the presence of the recurrent IgH chromosomal translocation and cyclin D expression. Cyclin D1 is detectable in up to one third of MM patients and plays crucial role in the regulation of G1–S transition in cell cycle. To evaluate the mechanisms of cyclin D1 overexpression, fluorescence in situ hybridization analysis with specific probes for the CCND1 gene and t(11;14)(q13;q32) were performed on highly purified plasma cells from bone marrow samples of 30 MM patients at diagnosis. CCND1 gene overexpression was detected in 14/30 cases (46.6%). Patients with evidence of the t(11;14) showed strong nuclear staining for cyclin D1 (TC1 group) and 7/8 demonstrated CCND1 overexpression. The remaining 7/15 cases with increased CCND1 gene copy numbers lacked the t(11;14) and showed low to negative levels of cyclin D1 protein (TC2 group). Trisomy 11 was demonstrated in 2/8 cases carrying the t(11;14) (TC1), 6/7 overexpressing cyclin D1 without the translocation (TC2), and 4/15 negative for both alterations (TC3–TC5). According to our data, trisomy 11 does not appear to directly cause CCND1 gene overexpression because it was present in 4/15 patients without the overexpression of the CCND1 gene and in 2/8 patients carrying the t(11;14). One patient belonging to the TC2 group overexpressed cyclin D1 and lacked both trisomy and translocation, suggesting that cyclin D1 can be dysregulated by additional mechanisms. In the TC2 group, trisomy 11 may probably be considered as a recurrent polisomy of the hyperdiploid status.

The Significance of p53 and Retinoblastoma Pathways in Canine Hemangiosarcoma

To investigate whether inactivation of the p53 and retinoblastoma (Rb) protein pathways contributes to the development of canine hemangiosarcoma (HSA), we examined immunohistochemically the expression of p53, Rb, phosphorylated Rb (phospho-Rb), p16, and cyclin D1 in 39 spontaneous canine HSAs and 10 hemangiomas. In addition, mutations in the p53 gene were analyzed by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and PCR direct sequencing; furthermore, we quantified cyclin D1 mRNA by semiquantitative real-time reverse transcription-PCR. Positive immunoreactivity for p53 was observed in 17.9% of HSAs. However, mutations were not detected in these cases. The labeling indices for Rb, phospho-Rb, and cyclin D1 were markedly higher in all HSAs than in hemangiomas. Of the 7 cases with cyclin D1-positive immunoreactivity, 4 overexpressed cyclin D1 mRNA (to a level more than 10-fold higher than that of GAPDH mRNA). The p16 protein was clearly detected in all hemangiomas; however, 82% of the neoplastic cells in HSA showed a loss of or low immunoreactivity. These results suggest that alteration of the p16-cyclin D1-Rb pathway, rather than the p53 pathway, may be associated with the pathogenesis of canine HSA.

Involvement of GSK-3β and DYRK1B in Differentiation-inducing Factor-3-induced Phosphorylation of Cyclin D1 in HeLa Cells

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.

HuR Contributes to Cyclin E1 Deregulation in MCF-7 Breast Cancer Cells

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

Prognostic significance of cyclin A in gastric cancer

High level of cyclin A promotes carcinogenesis, and overexpression of cyclin A has been associated with poor prognosis of cancer patients. We validated the prognostic role of cyclin A in gastric cancer and evaluated its correlation with expression of an mRNA stability factor HuR. From 342 consecutive histologically confirmed gastric cancer patients were obtained 325 representative tissue specimens for cyclin A and 316 for HuR immunohistochemistry. Specimens were stained by cyclin A and HuR specific monoclonal antibodies. Nuclear immunostaining detected in > or =5% of the tumor cells was considered the cut-off for cyclin A positivity. Positive HuR immunoreactivity was scored as nuclear or cytoplasmic. Associations between scores, clinicopathological factors and survival were calculated by the chi2-test, Fisher's exact test, Kaplan-Meier test and Cox model. Cyclin A detected in the nuclei of cancer cells was positive in 55% (179 of 325) of the specimens; 40% (127 of 316) of the specimens had cytoplasmic and 88% (279 of 316) nuclear immunoreactivity of HuR. Cyclin A expression was an independent prognostic factor for poor survival. Cyclin A immunoreactivity was associated with old age, high stage, proximal location of the tumor, intestinal type, noncurative resection, advanced penetration depth and with nodal metastases but not distant metastases. Furthermore, cyclin A expression was associated with cytoplasmic HuR expression, whereas no association with nuclear HuR was evident. Cyclin A is an independent prognostic factor in gastric cancer, and one mechanism for its overexpression may depend on cytoplasmic localization of HuR.

Cyclin E, p16ink4a, and Ki67 Overexpression as Possible Diagnostic Markers in HPV-Related Cervical Neoplasial Cell

Objectives: To investigate cyclin E, pl6ink4a, and Ki67 as possible diagnostic biomarkers for cervical preneoplasia, and to evaluate its significance for screening human papillomavirus (HPV)-infected patients who are at high risk of developing cervical carcinoma. Methods: The expressions of cyclin E, pl6ink4a, and Ki67 were examined in 266 cervical exfoliated epithelial specimens using immunohistochemical analysis; simultaneously, HPV type 16/18 DNA status were detected by PCR. Results: Overexpression of cyclin E was observed in low-grade squamous intraepithelial lesion (LSIL) (92.4% P < 0.005), and pl6ink4a and Ki67 were overexpressed in high-grade squamous intraepithelial lesion (HSIL) (92.0% and 89.8%, respectively). Conclusions: Cyclin E, pl6ink4a, and Ki67 may be used as diagnostic biomarkers for HPV-related cervical neoplastic lesions. In addition, this technique can be used for screening patients at high risk of developing cervical carcinoma.

Diffuse Large B-Cell Lymphoma with Overexpression of Cyclin E Substantiates Poor Standard Treatment Response and Inferior Outcome

Gold standard to predict survival and stratify patients for risk-adapted therapy in diffuse large B-cell lymphoma (DLBCL) is the international prognostic index, although it does not consider the molecular heterogeneity of DLBCL. Deregulation of cyclin E (CCNE) is a strong predictor of poor prognosis in some neoplastic diseases. In tumor cells, it induces chromosomal instability with an increased rate of aneuploidy/polyploidy. We analyzed in this retrospective study the prognostic value of immunohistochemical CCNE expression on a validated tissue microarray containing 101 de novo DLBCLs and, in 9 cases, the CCNE-induced chromosomal instability as assessed by cytometry. Forty-six of 98 evaluable DLBCLs expressed CCNE in a mean proportion of 20 +/- 29% of tumor cells; 38 cases expressed CCNE in >/=20% of tumor cells. CCNE-positive samples were aneuploid compared with near tetraploidy in CCNE-negative cases. Multivariate analysis showed CCNE expression in >/=20% of tumor cells to be an international prognostic index-independent, Adriamycin-based treatment-independent, and BCL2-independent prognostic factor for poor disease-specific survival. CCNE expression in >/=80% of tumor cells was associated with dismal short-term prognosis. CCNE expression in >/=50% of tumor cells emerged as an independent predictive factor for standard CHOP treatment resistance. CCNE expression assessment is easy on paraffin-embedded tissue. The high prognostic value of CCNE expression in DLBCL may be the basis for future prospective trials. In addition, a high CCNE expression hints at the presence of a possible target for individualized cancer therapy.

Tumor-specific hyperactive low-molecular-weight cyclin E isoform detection and characterization in non-metastatic colorectal tumors

Purpose: Several molecules involved in cancer biology have been studied as potential prognostic markers. Recently, overexpression of cyclin E and its low-molecular-weight (LMW) isoforms has been reported to be the most prominent prognostic marker in breast cancer, surpassing proliferation index, ploidy, and axillary nodal involvement. Furthermore, cyclin E and p53 are considered the main factors controlling the euploid equilibrium in human cells. We investigated the status of cyclin E and p53 in cell lines and tissue samples of colorectal cancer, one of the leading causes of death from a tumor in the Western world.Experimental Design: We analyzed colorectal cancer cells, from established cell lines and patient specimens, to determine the protein levels of cyclin E and p53, and to detect p53 and APC mutations, microsatellite and chromosome instability. In addition, we assessed the presence of cyclin E LMW isoforms and their enzymatic activity.Results: Colorectal cancer cells expressed hyperactive LMW forms both in vitro and in vivo. These tumor-specific isoforms are correlated to genomic instability even in p53-proficient cells, and represented a constant feature in the tumors analyzed.Conclusions: In colorectal cancer, the formation of cyclin E LMW forms is an early event leading to DNA-damage checkpoint-independent proliferation. Collectively, our results provide evidence that evaluation of LMW forms could represent a novel tool in the molecular characterization of colorectal tumors aimed at identifying sensitive prognostic factors and uncovering subsets of high-risk patients within the traditional categories.

The Prognostic Value and Overexpression of Cyclin A Is Correlated with Gene Amplification of both Cyclin A and Cyclin E in Breast Cancer Patient

Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3 and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle propulsion and are not previously evaluated in breast cancer. We have examined protein expression and gene amplification of cyclins in breast carcinomas and correlated the findings with clinical follow-up data. We have previously demonstrated that over-expression of cyclin A is associated with poor prognosis in breast cancer patients. In this study we wanted to evaluate the mechanisms behinde overexpression of cyclin A, as well as the impact of other cyclins, both at the gene level and at the protein level, on prognosis of breast cancer patients. The impact of TP53 gene mutations on gene amplification of cyclins was also evaluated. Methods: Real-Time Quantitative PCR was used to detect gene amplification of cyclins in tumour tissue from 86 patients operated for invasive breast carcinomas, while immunohistochemistry was applied to detect protein expression of the same cyclins. Result: Of the 80-breast tumour samples available for cyclin A gene amplification analyses, 26.7% (23/80) was defined to have cyclin A gene amplification. 37.2% (32/79) had cyclin B1 gene amplification, 82.6% (71/82) of the samples harboured amplification of cyclin C gene, 74.4% (64/82) had cyclin D1 gene amplification, 41.9% (36/86) had cyclin D3 gene amplification, 29.1% (25/81) of the patients had cyclin E gene amplification and 9.3% (8/86) of the samples showed amplification of the cyclin H gene. When correlation between gene amplification and protein expression was evaluated, we observed a statistical significant correlation between gene amplification and protein expression of cyclin A (p = 0.009) and cyclin D3 (p < 0.001). However, the correlation between gene amplification and protein expression of cyclin A, as well as the prognostic value of cyclin A overexpression, was affected by gene amplification of cyclin E. Gene amplification of none of the other cyclins was associated with patient prognosis. There was a statistical significant correlation between TP53 gene mutations and gene amplification of cyclins A, D3 and B1. No correlation was observed between gene amplification of secondary cyclins (H and C) and TP53 gene mutations. Conclusions: The overexpression of cyclin A is correlated to gene amplification of both cyclin A and cyclin E. Over-expression of cyclin A is associated with poor prognosis in breast cancer patients. When analysed in a multivariate analyses model, gene amplification as well as protein expression of none of the other cyclins than cyclin A are associated with patient prognosis in breast carcinomas. TP53 gene mutation seems to correlate with gene amplification of primary, but not secondary cyclins.

Cyclin E Overexpression Obstructs Infiltrative Behavior in Breast Cancer: A Novel Role Reflected in the Growth Pattern of Medullary Breast Cancers

Cell cycle deregulation is a prerequisite in tumor development and overexpression of cyclin E, a major G1-S regulator, is often observed in breast cancer and is further linked to poor prognosis. By overexpressing cyclin E in a retinoblastoma-inactivated breast cancer cell line, we induced significant alterations in the expression of genes associated with proliferation and cell adhesion. Rearrangements of the actin cytoskeleton in addition to increased adhesive properties, decreased motility, and invasive potential in functional assays, indicated an overall abrogated mobility. Consistent in vivo findings were obtained upon investigation of 985 primary breast cancers, where cyclin E-high tumors predominantly (67%) displayed a low infiltrative, pushing growth pattern. Furthermore, medullary breast cancers, a subtype defined by its pushing, delimited growth, exhibited a remarkable frequency of cyclin E deregulation (87%) compared with other histologic subtypes (5-20%). Taken together, our results suggest the novel role of cyclin E in modeling infiltrative behavior. The consequences of cyclin E overexpression in breast cancer seems to be multiple, including effects on proliferation as well as growth patterns, a scenario that is indeed observed in the archetype of cyclin E-overexpressing medullary breast cancers.

Safe from Degradation

DNA replication is initiated through the assembly of prereplication complexes (pre-RCs); replication does not occur in quiescent cells and is limited to once per cell cycle because, outside of the G 1 phase of the cell cycle, inhibitors prevent assembly of the pre-RC. During G 1 , APC Cdh1 (the form of the anaphase-promoting complex E3 ubiquitin ligase that contains Cdh1) targets the pre-RC assembly inhibitors geminin and cyclin A for degradation. Paradoxically, however, APC Cdh1 targets Cdc6, which is essential to pre-RC assembly, for destruction as well (see Ayad). Mailand and Diffley noted that, in cultured human cells treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine, Cdc6 abundance was reduced, whereas the abundance of geminin and cyclin A was unaffected. Knockdown of Cdk2 with small interfering RNA (siRNA) also decreased Cdc6 abundance. Roscovitine accelerated Cdc6 degradation, as did depletion of Cdk2 or cyclins E1 and E2, whereas overexpression of cyclin E (which forms an active CDK complex with Cdk2) promoted Cdc6 accumulation. Mutational analysis indicated that the effects of cyclin E on Cdc6 stability depended on Cdc6 phosphorylation. Cdc6 phosphorylation inhibited its APC Cdh1 -dependent ubiquitination and degradation; further, whereas wild-type Cdc-6 or an unphosphorylatable mutant coimmunoprecipitated with Cdh1, a phospho-mimicking Cdc6 mutant did not. In serum-starved (noncycling) cells, Cdc6, geminin, and cyclin A were undetectable. After serum stimulation, and reentry into the cell cycle, Cdc6 abundance increased 3 to 4 hours before the other APC Cdh1 targets became detectable. When Cdc6 was detected, it was phosphorylated, and cyclin E-associated kinase activity appeared simultaneously with Cdc6. Thus, the authors propose that, after entry into the cell cycle, cyclin E-Cdk2-dependent phosphorylation of Cdc6 allows it to accumulate (and promote pre-RC assembly) before inactivation of APC Cdh1 and the ensuing accumulation of pre-RC assembly inhibitors such as geminin and cyclin A. N. Mailand, J. F. X. Diffley, CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122 , 915-926 (2005). [PubMed] N. G. Ayad, CDKs give Cdc6 a license to drive into S phase. Cell 122 , 825-827 (2005). [PubMed]