Cyclin A1 Overexpression Research Articles

Cyclin A1 affects the invasion, metastasis, and prognosis of hepatocellular carcinoma

Objective: To investigate the effect of cyclin A1 on the invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). Methods: Immunohistochemistry (IHC) was used to detect the expressional condition of cyclin A1 in HCC and paraffin-embedded non-tumor adjacent tissues. Kaplan-Meier method was used for the survival analysis of patients with HCC. Western blot (WB) was used to detect the expression of cyclin A1 in HCCLM3 and QGY-7703 cells. Scratch wound healing assay, transwell migration, and invasion assay were used to detect the effect of cyclin A1 overexpression on cell migration and invasion ability. WB was used to detect changes in the expression of matrix metalloproteinase (MMP) 2, MMP9, and vascular endothelial growth factor (VEGF) after overexpression of cyclin A1. Measurement data were compared using a t-test and analysis of variance. Count data was measured using χ (2) test and the Log-rank method was performed for survival analysis. Results: Cyclin A1 expression rates were higher in the tissues of HCC patients with recurrent metastasis than in the tissues of patients without recurrent metastasis (60.42% vs. 46.81%, χ (2) = 4.711, P < 0.05). The overall postoperative survival time (OS) and disease-free survival (DFS) were shorter in patients with high cyclin A1 expression than those with low cyclin A1 expression (45.9 months vs. 53.1 months; 42.9 months vs. 51.3 months, and P < 0.01). The postoperative OS and DFS were shorter in patients with high cyclin A1 expression and recurrent metastasis than those with low cyclin A1 expression without recurrent metastasis (31.7 months vs. 43.9 months; 18.0 months vs. 31.5 months, and P < 0.05). HCCLM3 and QGY-7703 cells were higher in the cyclin A1-pEX group than in the empty vector (vector) group (1.56 ± 0.06 vs. 0.18 ± 0.01, t = 18.75, P < 0.001; 1.31 ± 0.05 vs.0.37 ± 0.02, t = 15.17, P < 0.001). The migrated distances of HCCLM3 cells in the cyclin A1-pEX group and the vector group were (536.7 ± 14.5) μm and (327.3 ± 9.3) μm, t = 11.84, P < 0.05, respectively, while the migrated distances of QGY-7703 cells in the two groups were (916.7 ± 35.3) μm and (320.0 ± 20.8) μm, t = 13.54, P < 0.01. The migrated numbers of HCCLM3 cells in the cyclin A1-pEX group and vector group were (37.3 ± 2.4) and (7.0 ± 1.2), t = 12.67, P < 0.001, and the number of invasive cells was (73.7 ± 4.1) and (12.6 ± 1.5), t = 12.36, P < 0.001, respectively. The migrated numbers of QGY-7703 cells in the two groups were (153.3 ± 6.0) and (17.7 ± 3.7), t = 17.59, P < 0.001, and the number of invasive cells was (45.0 ± 2.9) and (9.3 ± 1.5), t = 10.66, P < 0.001, respectively. The expression levels of MMP2, MMP9, and VEGF in HCCLM3 and QGY-7703 cells were significantly higher in the cyclin A1-pEX group than those in the vector group (P < 0.05). Conclusion: Cyclin A1 plays an important role in HCC invasion and metastasis, but HCC patients with high cyclin A1 expression have a poor prognosis. Hence, cyclin A1 has high guiding significance for evaluating patient prognosis.

1,6-Hexanediol regulates angiogenesis via suppression of cyclin A1-mediated endothelial function

BackgroundAngiogenesis plays important roles in physiological and pathologic conditions, but the mechanisms underlying this complex process often remain to be elucidated. In recent years, liquid–liquid phase separation (LLPS) has emerged as a new concept to explain many cellular functions and diseases. However, whether LLPS is involved in angiogenesis has not been studied until now. Here, we investigated the potential role of LLPS in angiogenesis and endothelial function.ResultsWe found 1,6-hexanediol (1,6-HD), an inhibitor of LLPS, but not 2,5-hexanediol (2,5-HD) dramatically decreases neovascularization of Matrigel plug and angiogenesis response of murine corneal in vivo. Moreover, 1,6-HD but not 2,5-HD inhibits microvessel outgrowth of aortic ring and endothelial network formation. The endothelial function of migration, proliferation, and cell growth is suppressed by 1,6-HD. Global transcriptional analysis by RNA-sequencing reveals that 1,6-HD specifically blocks cell cycle and downregulates cell cycle-related genes including cyclin A1. Further experimental data show that 1,6-HD treatment greatly reduces the expression of cyclin A1 but with minimal effect on cyclin D1, cyclin E1, CDK2, and CDK4. The inhibitory effect of 1,6-HD on cyclin A1 is mainly through transcriptional regulation because proteasome inhibitors fail to rescue its expression. Furthermore, overexpression of cyclin A1 in HUVECs largely rescues the dysregulated tube formation upon 1,6-HD treatment.ConclusionsOur data reveal a critical role of LLPS inhibitor 1,6-HD in angiogenesis and endothelial function, which specifically affects endothelial G1/S transition through transcriptional suppression of CCNA1, implying LLPS as a possible novel player to modulate angiogenesis, and thus, it might represent an interesting therapeutic target to be investigated in clinic angiogenesis-related diseases in future.

Cyclin A1 Expression in Leukemia and Normal Hematopoietic Cells

Human cyclin A1 is a newly cloned, tissue-specific cyclin that is prominently expressed in normal testis. In this study, we showed that cyclin A1 was highly expressed in a subset of leukemia samples from patients. The highest frequency of cyclin A1 overexpression was observed in acute myelocytic leukemias, especially those that were at the promyelocyte (M3) and myeloblast (M2) stages of development. Cyclin A1 expression was also detected in normal CD34(+) progenitor cells. The expression of cyclin A1 increased when these cells were stimulated to undergo myeloid differentiation in vitro. Taken together, our observations suggest that cyclin A1 may have a role in hematopoiesis. High levels of cyclin A1 expression are especially associated with certain leukemias blocked at the myeloblast and promyelocyte stages of differentiation.

CCNA1 gene as a potential diagnostic marker in papillary thyroid cancer

The malignancy that most affects the endocrine system is thyroid neoplasm, with an increasing incidence over the years. The most prevalent histological type of the carcinomas that affect the thyroid gland is papillary carcinoma with a prevalence of 80 % worldwide. The current diagnostic methodology may present inconclusive results, emphasizing the need for new effective and sensitive techniques to aid the diagnosis. For this, it is necessary to understand molecular and protein mechanisms in the identification of diagnostic and predictive markers in the lesions. The Cyclin A1 protein, encoded by the CCNA1 gene, is an important cell cycle regulator, belonging to the MAPK/ERK signaling pathway directly involved with thyroid cancer. The aim of this study was to evaluate the CCNA1 gene and Cyclin A1 protein expression in papillary thyroid carcinoma, follicular thyroid carcinoma, and benign thyroid lesions, by real time quantitative PCR and immunohistochemistry analysis, respectively, to verify their roles as potential diagnostic and predictive markers to future applications in the clinical routine. Overexpression of CCNA1 gene was observed in the papillary carcinoma group compared to the normal group (P = 0.0023), benign lesions (P = 0.0011), colloid goiter (P = 0.0124), and follicular carcinoma (P = 0.0063). No differential expression was observed in the papillary primary tumor group from negative lymph nodes compared with the one from positive lymph nodes (P = 0.3818). Although an increased expression of Cyclin A1 was observed in the PTC group compared to the other one in the IHC analysis, no significant difference was observed (Fisher’s exact Test). A Cyclin A1 overexpression was detected with weak to mid-moderate immunoreactivity in the benign group (k = 0.56), (score 1.5); mid-moderate to moderate in the goiter group (k = 0.58); weak in the FTC group (k = 0.33); and mid-moderate to moderate in the PTC group (k = 0.48). Due to the small sample size in the IHC analysis and to the fact that not all RNA is translated into protein, the diagnostic potential of Cyclin A1 could not be assessed. However, these findings highlight the potential of the CCNA1 gene as a diagnostic marker for papillary thyroid carcinoma.

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

Cyclin A1 and P450 Aromatase Promote Metastatic Homing and Growth of Stem-like Prostate Cancer Cells in the Bone Marrow

Bone metastasis is a leading cause of morbidity and mortality in prostate cancer. While cancer stem-like cells have been implicated as a cell of origin for prostate cancer metastasis, the pathways that enable metastatic development at distal sites remain largely unknown. In this study, we illuminate pathways relevant to bone metastasis in this disease. We observed that cyclin A1 (CCNA1) protein expression was relatively higher in prostate cancer metastatic lesions in lymph node, lung, and bone/bone marrow. In both primary and metastatic tissues, cyclin A1 expression was also correlated with aromatase (CYP19A1), a key enzyme that directly regulates the local balance of androgens to estrogens. Cyclin A1 overexpression in the stem-like ALDH(high) subpopulation of PC3M cells, one model of prostate cancer, enabled bone marrow integration and metastatic growth. Further, cells obtained from bone marrow metastatic lesions displayed self-renewal capability in colony-forming assays. In the bone marrow, cyclin A1 and aromatase enhanced local bone marrow-releasing factors, including androgen receptor, estrogen and matrix metalloproteinase MMP9 and promoted the metastatic growth of prostate cancer cells. Moreover, ALDH(high) tumor cells expressing elevated levels of aromatase stimulated tumor/host estrogen production and acquired a growth advantage in the presence of host bone marrow cells. Overall, these findings suggest that local production of steroids and MMPs in the bone marrow may provide a suitable microenvironment for ALDH(high) prostate cancer cells to establish metastatic growths, offering new approaches to therapeutically target bone metastases. Cancer Res; 76(8); 2453-64. ©2016 AACR.

A Limited Role for the Cell Cycle Regulator Cyclin A1 in Murine Leukemogenesis.

The quest for novel therapeutic targets in acute myeloid leukemia (AML) is still ongoing. One of such targets, cyclin A1, was shown to be overexpressed in AML including AML stem cells. However, the function of cyclin A1 in AML is largely unknown, and the data on its impact on patients´ survival remain controversial. Therefore, we developed a transgenic mouse model of stem cell-directed inducible cyclin A1 overexpression and crossed these mice with PML-RARα-knockin mice, which develop an AML M3-like phenotype. To observe the effects of cyclin A1 loss-of-function, we also crossed PML-RARα-knockin mice to cyclin A1-knockout mice. Neither overexpression nor loss of cyclin A1 significantly altered leukemogenesis in PML-RARα-knockin mice. These findings imply that upregulation of cyclin A1 is not essential for leukemogenesis. Our data suggest that cyclin A1 does not represent a suitable target for AML therapy.

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

Altered Expression of Cyclin A 1 In Muscle of Patients with Facioscapulohumeral Muscle Dystrophy (FSHD-1)

ObjectivesCyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD).MethodsMuscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy.ResultscDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients.ConclusionsIn most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease.

Cyclin A1 Modulates the Expression of Vascular Endothelial Growth Factor and Promotes Hormone-Dependent Growth and Angiogenesis of Breast Cancer

Alterations in cellular pathways related to both endocrine and vascular endothelial growth factors (VEGF) may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, molecular mechanisms by which endocrine-related pathways and VEGF signalling cooperatively promote breast cancer progression remain poorly understood. In the present study, we show that the A-type cyclin, cyclin A1, known for its important role in the initiation of leukemia and prostate cancer metastasis, is highly expressed in primary breast cancer specimens and metastatic lesions, in contrasting to its barely detectable expression in normal human breast tissues. There is a statistically significant correlation between cyclin A1 and VEGF expression in breast cancer specimens from two patient cohorts (p<0.01). Induction of cyclin A1 overexpression in breast cancer cell line MCF-7 results in an enhanced invasiveness and a concomitant increase in VEGF expression. In addition, there is a formation of protein–protein complexes between cyclin A1 and estrogen receptor ER-α cyclin A1 overexpression increases ER-α expression in MCF-7 and T47D cells. In mouse tumor xenograft models in which mice were implanted with MCF-7 cells that overexpressed cyclin A1 or control vector, cyclin A1 overexpression results in an increase in tumor growth and angiogenesis, which is coincident with an enhanced expression of VEGF, VEGFR1 and ER-α Our findings unravel a novel role for cyclin A1 in growth and progression of breast cancer, and suggest that multiple cellular pathways, including cell cycle regulators, angiogenesis and estrogen receptor signalling, may cooperatively contribute to breast cancer progression.

Cyclin A1 shows age-related expression in benign tonsils, HPV16-dependent overexpression in HNSCC and predicts lower recurrence rate in HNSCC independently of HPV16

BackgroundPromoter methylation of the tumor suppressor gene Cyclin A1 could be associated with Human Papillomavirus 16 (HPV16) induced Head and Neck Squamous Cell Carcinoma (HNSCC) and Cervical Carcinoma. There is disagreement about the impact of this epigenetic event on protein expression of Cyclin A1 in malignant and non-malignant tissue and there hardly exists any information about possible relationships between Cyclin A1 expression and clinicopathological characteristics in HNSCC.MethodsWe analyzed protein expression of Cyclin A1 in 81 HNSCC and 74 benign tonsils by immunohistochemistry and correlated it to Cyclin A1 methylation status, presence of HPV16 infection and other clinicopathological characteristics.ResultsOverexpression of Cyclin A1 was more present in HNSCC than in tonsils (p < 0.001). In both entities, HNSCC and benign tonsils, expression of Cyclin A1 significantly correlated with the expression of Cyclin-dependent kinase-inhibitor p16 (p = 0.000672 and 0.00495). In tonsils, expression of Cyclin A1 was inversely proportional to age (p = 0.00000396), and further correlated with expression of tumor suppressor gene p53 (p = 0.000228). In HNSCC Cyclin A1 expression was associated with the presence of HPV16 DNA (p = 0.0014) and a lower recurrence rate in univariate and multivariate analysis (p = 0.002 and 0.013). Neither in HNSCC nor in tonsils Cyclin A1 expression correlated with promoter methylation.ConclusionsCyclin A1 is an important cell cycle regulator with age-related increased expression in tonsils of children. HPV16 induces overexpression of Cyclin A1 in HNSCC despite promoter methylation. Overexpression of Cyclin A1 predicts a lower recurrence rate in HNSCC independently of HPV16.

Constitutive Cyclin A1 Expression Induces Abrogation of Cell Cycle Check Points Associated with Ara-C Resistance in Myeloid Leukemia Cells.

Abstract 4801Cyclin A1, an alternative A-type cyclin, is frequently over-expressed in acute myeloid leukemia (AML). To explore role(s) of cyclin A1 in leukemia cells, we studied AML cell line, K562.A1, which constitutively express cyclin A1 gene. K562.A1 showed prolonged doubling time in culture (1.3 fold) and decreased colony formation (25.6±6% of those of K562.C, which carries pcDNA3.1Hisc). flow cytometry (FCM) analysis of cells stained with PI showed that decreased cell growth of K562.A1 was accompanied with G1 accumulation and significant decrease in percentage of cells in the G2M (G1:49.5±2.6,S:30.3±2.3,G2M:18.7±1.1% for K562.C and G1:65.7±2.4,S:22.4±2.1,G2M:9.5±0.5% for K562.A1).Immunoblot analysis showed that k562.A1 constitutively over-expressed checkpoint kinase 1(chk1) protein (3.4±0.2 fold evaluated by densitometric analysis) but not chk2 compared with K562.C. Cdk1/Cdc2 protein, but not Cdk2, was also over-expressed in K562.A1 cells (3.9±0.3 fold). In addition, both chk1 and Cdk1/Cdc2 were constitutively phosphorylated at Ser345 and Tyr15 residues, respectively, while phosphorylation of those proteins was not detected in K562.C. In K562.A1, cell division cycle 20 homolog (Cdc20) was also over-expressed (2.7±0.1 fold), suggesting that de-regulation of spindle checkpoint was also involved in cell cycle inhibition in this cell line. Incubation with Gö6976, a specific chk1 inhibitor, partly restored cell growth of K562.A1 (53±1.2% in medium and 72±3.0% in 1μM Gö6976,% of cell number of K562.C at day3. p<0.01), while Gö6976 showed negligible effect on cell growth of K562.C. Restored cell growth was accompanied with decreased in percentage of cells in the G1 and increased incells in the G2M in the cell cycle. This suggest that a role of inappropriately activated Chk1-Cdk1/Cdc2 pathway in cell growth inhibition of K562.A1.Small increase of subG1 DNA content portion were observed in K562.A1 compared with K562.C(K562.A1=2.4±0.1% and K562.C=0.7±0.2%), suggesting the presence of low proliferating or apoptosis-prone G1 cells in K562.A1. However, K562.A1, compared with K562.C showed significantly impaired cytosine arabinoside (CA)-induced apoptosis (K562.C=50.9 ±1.5% and K562.A1=29.6±1.2%, p<0.01, Annexin V-FITC/PI stained cells, 1μMCA at day 3). STI571 (1nM-1000nM), a specific abl kinase inhibitor, showed similar cytotoxic effects on both K562.C and K562.A1. It suggests that K562.A1 have an alteration of apoptotic pathway(S) triggered by genotoxic agent. We found significant up-regulation of activated NF-κB in K562.A1 compared to K562.C in response to CA as judged by quantitative enzyme immunoassay. (K562.C=1.0±0.3 and 2.1±0.7 pg/μg (CA) K562.A1=7.9±1.3 and 23.6±0.9pg/μg (CA), P=0.01). Helenalin (NF-κB inhibitor), however, only slightly increased apoptotic cells in k562.A1 in the presence of CA (1μM CA =30.1 ±3.6% and 1μM CA plus 1μM helenalin=34.7±4.2%, p=NS), suggesting a role of activated NF-κB are limited for impaired CA-induced apoptosis in K562.A1. CA induced significant increase of cell surface expression of CD95 in K562.C (53.5±2.9 fold, mean signal intensity), while the increase in K562.A1 was only 1.7±0.1 fold, suggesting that activation of the FAS apoptotic pathway are impaired in K562.A1. Incubation with 1μM CA for 3 days further augmented protein expression of Cdc20 in K562.A1 cells (2.7±0.5 fold compared to those without CA), while the change in K562. C cells was negligible.The present study shows that de-regulated expression of molecule(s) responsible for transition through G2 and spindle checkpoint is evident in myeloid leukemia cells which constitutively express cyclin A1. The de-regulated activation of Chk1-Cdk1/Cdc2 pathway and abrogation of G2/M checkpoint in K562.A1 cells not only resulted in inhibition of CA-induced erythroid differentiation and cell cycle arrest, but also possible inhibition of Fas/CD95-mediated apoptosis. Association between ATM (ataxia teleangiectasia mutated) DNA damage response pathway and Fas/CD95-mediated apoptotic pathway is recently reported. Furthermore over-expressed Cdc20protein, a potential cancer therapeutic target, may be associated mitotic abnormalities and induction of aneuploidy in leukemia cells. It suggests that, in addition to activated NF-κB, abrogation of cell cycle checkpoint(s) are involved in chemo-resistant myeloid leukemia with over-expression of cyclin A1. Disclosures:No relevant conflicts of interest to declare.

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.

Cyclin A1, the alternative A-type cyclin, contributes to G1/S cell cycle progression in somatic cells

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1-/- cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1-/- mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.

Cyclin A1 is highly expressed in aggressive testicular germ cell tumors

Cyclin A1 is a tissue-specific A-type cyclin that is essential for spermatogenesis. Overexpression of cyclin A1 was found in acute myeloid leukemia and cyclin A1 induced leukemia in a transgenic mouse model. We used quantitative real-time reverse transcription–polymerase chain reaction to analyze cyclin A1 expression in solid tumors. Cyclin A1 expression was very low in breast cancer, non-small cell lung cancer and in cervical carcinoma. However, substantial expression of cyclin A1 was found in testicular and ovarian cancer and in endometrial cancer. In testis specimens, cyclin A1 expression was much higher in testicular tumors compared to Sertoli cell only syndrome that lacks spermatogenesis. Compared to normal spermatogenesis, testicular cancers expressed on average lower levels of cyclin A1. Among the different histological subtypes of testicular tumors, embryonal cell carcinomas and immature teratomas expressed the highest levels of cyclin A1. The cyclin A1 levels in these tumors were similar to those seen in normal testis. Seminomas and yolk sac tumors expressed intermediate levels, whereas cyclin A1 expression was very low in mature teratomas. These findings indicate that cyclin A1 is expressed in selected solid tumors. Its known oncogenic function and the high expression levels in aggressive testicular tumors suggest a role for cyclin A1 in germ cell tumorigenesis.

Altered myelopoiesis and the development of acute myeloid leukemia in transgenic mice overexpressing cyclin A1.

A mammalian A-type cyclin, cyclin A1, is highly expressed in testes of both human and mouse and targeted mutagenesis in the mouse has revealed the unique requirement for cyclin A1 in the progression of male germ cells through the meiotic cell cycle. While very low levels of cyclin A1 have been reported in the human hematopoietic system and brain, the sites of elevated levels of expression of human cyclin A1 were several leukemia cell lines and blood samples from patients with hematopoietic malignances, notably acute myeloid leukemia. To evaluate whether cyclin A1 is directly involved with the development of myeloid leukemia, mouse cyclin A1 protein was overexpressed in the myeloid lineage of transgenic mice under the direction of the human cathepsin G (hCG) promoter. The resulting transgenic mice exhibited an increased proportion of immature myeloid cells in the peripheral blood, bone marrow, and spleen. The abnormal myelopoiesis developed within the first few months after birth and progressed to overt acute myeloid leukemia at a low frequency ( approximately 15%) over the course of 7-14 months. Both the abnormalities in myelopoiesis and the leukemic state could be transplanted to irradiated SCID (severe combined immunodeficient) mice. The observations suggest that cyclin A1 overexpression results in abnormal myelopoiesis and is necessary, but not sufficient in the cooperative events inducing the transformed phenotype. The data further support an important role of cyclin A1 in hematopoiesis and the etiology of myeloid leukemia.

The aberrant fusion proteins PML-RARα and PLZF-RARα contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

The aberrant fusion proteins PML-RARα and PLZF-RARα contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

AbstractCyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

Cyclin A1 regulates WT1 expression in acute myeloid leukemia cells

Cyclin A1 is a cell cycle protein that is expressed in testes, brain and CD34-positive hematopoietic progenitor cells. Cyclin A1 is overexpressed in a variety of myeloid leukemic cell lines and in myeloid leukemic blasts. Transgenic cyclin A1 overexpressing mice develop acute myeloid leukemia with low frequency. In this study, we looked for putative target genes of cyclin A1 in hematopoietic cells. Microarray analysis of U937 myeloid cells overexpressing cyclin A1 versus conrol cells detected 35 differential expressed genes, 21 induced and 14 repressed ones upon cyclin A1 overexpression. Among the differentially expressed genes WT1 was chosen for further analysis. Repression of WT1 expression was confirmed on the mRNA and protein level. In addition, WT1 expression was higher in bone marrow, liver and ovary of cyclin A1-/- mice. Isoform analysis showed a profound change of the WT1 isoform ratio in U937 cyclin A1-overexpressing versus control cells. Functional analysis revealed an inhibition of colony growth when WT1 isoforms were transfected into U937 cells, which was not affected by the overexpression of cyclin A1. In addition, overexpression of the WT1-/+ isoform induced a G1 cell cycle arrest which was abrogated upon cotransfection with cyclin A1. This study identified WT1 as a repressed target of cyclin A1 and suggests that the suppression of WT1 in cyclin A1-overexpressing leukemias might play a role in the growth and suppression of apoptosis in these leukemic cells.