Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export

The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lymphoblasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing a new mechanism to regulate the function of the FA protein complex.     

Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication

Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.     

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.     

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.     

The role of the cyclin D1-dependent kinases in ErbB2-mediated breast cancer

Intact cyclin D1 functions are essential for transformation by erbB2 in tissue culture and murine models. Because cyclin D1 may alter cell proliferation through a variety of mechanisms, we used transgenic models and human tumor samples to particularly address the role of cyclin D1-cyclin-dependent kinases in transformation by erbB2. The p16 tumor suppressor specifically blocks cyclin-dependent kinase 4 and 6 activity. Here we show that an MMTV-p16 transgene blocked tumorigenesis by erbB2, demonstrating that deregulation of the cyclin-dependent kinase partner of cyclin D1 is an essential target of erbB2. ErbB2 overexpression was a determining factor in deregulation of cyclin D1-cdk4/6 interactions because neither transgenic cyclin D1 nor loss of p16 accelerated tumorigenesis in MMTV-erbB2-transgenic mice. ErbB2 was also a deciding factor in deregulation of cyclin D1-cdk4/6 in human tumors because no loss of pRb or p16 was found in tumors overexpressing erbB2, although erbB2-negative invasive breast adenocarcinomas frequently lacked expression of p16 or pRb. We conclude that deregulation of cyclin D1-Cdk4/6 interactions is a critical target of erbB2 function in human and mouse breast tumors, and erbB2's overexpression may be sufficient to deregulate cyclin D1-cdk4/6 activity in breast cancer.     

The link between PKCalpha regulation and cellular transformation

Protein kinase Calpha (PKCalpha) is a serine/threonine protein kinase that has been implicated in the regulation of a variety of cellular functions such as proliferation, differentiation and apoptosis in response to a diverse range of stimuli. In order to execute these biological events PKCalpha activity is modulated by, and functionally interacts with, a number of proto-oncogenes, therefore it is perhaps unsurprising that dysregulation of PKCalpha is associated with a diverse range of cancers. Recently, PKCalpha has become a target for a number of anti-cancer therapies. The purpose of this review is to describe how PKCalpha regulates key biological events, to gain an insight into how PKCalpha-mediated cellular transformation may occur. In this way, it may be possible to design therapeutic tools to combat cancers specifically associated with PKCalpha dysfunction.     

beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

The immunohistochemical expression of beta-catenin, cyclin D1, Ki-67 and PCNA was Examined in 38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated beta-catenin within the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant correlation between beta-catenin accumulation and cyclin D1 overexpression (p=0.029). Each group with beta-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those without, the difference being statistically significant (p=0.007, p=0.004, respectively). Differential PCR was also performed to detect amplification of the cyclin D1 gene and mutational analysis was undertaken for exon 3 of the beta-catenin gene. Amplification of the cyclin D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of 18 cases (38.9%). The distribution of beta-catenin mutation fell within a wide range, from codon 21 to codon 67. In conclusion, beta-catenin nuclear expression correlated with cyclin D1 overexpression in sporadic desmoid tumours, which could be an in vivo model system for the APC-beta-catenin-Tcf pathway. In addition, beta-catenin mutations in desmoid tumours occurred at an unusually wide range of sites within the gene.     

Expression profile and molecular genetic regulation of cyclin D1 expression in epithelioid sarcoma.

Epithelioid sarcoma is a distinctive, aggressive soft tissue tumor typically presenting as a subcutaneous or deep dermal mass in the distal extremities of young adults. Molecular genetic data of well-characterized cases of epithelioid sarcoma are sparse. A recent cytogenetic study of epithelioid sarcoma by conventional metaphase comparative genomic hybridization reported recurrent gains at chromosome 11q13, a region containing many genes, including the cyclin D1 gene. Cyclin D1 is a positive cell cycle regulator that is overexpressed in a variety of neoplasms, including mantle cell lymphoma and breast carcinoma. The objective of this study was to examine cyclin D1 expression in epithelioid sarcoma. Of 24 cases evaluated, 23 (96%) displayed cyclin D1 nuclear expression using immunohistochemical evaluation. Eight cases, which expressed cyclin D1 by immunohistochemistry, were evaluated by fluorescence in situ hybridization (FISH) and RNA in situ hybridization (RISH) for amplification of the cyclin D1 gene and messenger RNA (mRNA) expression, respectively. Seven of eight cases showed a typical eusomic state. One case showed pseudoamplification due to aneusomy/polysomy. There was no evidence of cyclin D1 gene amplification or messenger RNA overexpression detected by FISH or RNA in situ hybridization analyses, respectively. Our data clearly demonstrate that cyclin D1 protein is upregulated in epithelioid sarcoma, suggesting a role for this cell cycle regulator in the pathogenesis of epithelioid sarcoma. The high level of cyclin D1 protein expression in epithelioid sarcoma appears to be regulated by translational and/or post-translational mechanisms.     

Prognostic role of cyclin D1 in lung cancer. Relationship to proliferating cell nuclear antigen

We developed an immunohistochemical assay specific for cyclin D1 and suitable for formalin-fixed and paraffin-embedded sections, to evaluate cyclin D1 expression in a group of 135 surgically resected lung-cancer patients for the purpose of investigating the prognostic role of this protein in lung cancer. In addition, we compared cyclin D1 expression with the expression of proliferating cell nuclear antigen (PCNA), considered to be a reliable index of the proliferation rate. We found cyclin D1 expressed in more than 60% of the neoplastic cells in 26.5% of our specimens. A total of 24.5% of the specimens showed cyclin D1 expression in a percentage of cells ranging from 30 to 60%; 36.7% of the specimens expressed cyclin D1 in less than 30% of the cells; and 12.2% of the specimens expressed cyclin D1 in less than 1% of the evaluated cells. Western blot analyses confirmed the specificity of this assay by correlating statistically in a highly significant fashion with the immunohistochemical results (P = 0.0003). Furthermore, we found a direct relationship between cyclin D1 and PCNA immunodetection (P = 0.0004), which correlated cyclin D1 overexpression with a higher tumor proliferation rate. When we analyzed our data statistically, cyclin D1 expression was found to be a negative prognostic marker (P < 0.00005) whose expression correlates with a shorter patient survival time.     

Searching for nuclear export elements in hepatitis D virus RNA

AIM: To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export.METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway.CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.     

Cytoplasmic cyclin D1 regulates glioblastoma dissemination

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4–retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

D cyclins in CD5+ B-cell lymphoproliferative disorders: cyclin D1 and cyclin D2 identify diagnostic groups and cyclin D1 correlates with ZAP-70 expression in chronic lymphocytic leukemia

We analyzed protein expression of cyclin D1, cyclin D2, and cyclin D3 using high-resolution enzymatic amplification staining and flow cytometry in the neoplastic cells from 80 patients with CD5+ B-cell lymphoproliferative disorders. The D cyclins were expressed differentially in chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), and mantle cell lymphoma (MCL) with strong staining of cyclin D1 and D2 in MCL, strong staining of cyclin D1 but weak staining of cyclin D2 in 4 of 5 PLLs, and low-level staining for both cyclins in most CLLs. No correlation between cyclin D1 and D2 and growth rates or CD38 expression was observed. However, cyclin D1 levels were significantly higher in ZAP-70+ CLL cases, although no association between ZAP-70 and cyclin D2 was detected. The results indicate that flow cytometric analysis of D cyclins may help in classification of CD5+ B-cell lymphoproliferative disorders.     

Cyclin G1在小鼠卵巢颗粒细胞的表达及调控

目的:探讨cyclinG1在小鼠卵巢颗粒细胞的表达以及促性腺激素对其表达的调控作用。方法:以免疫组化方法检测小鼠卵巢颗粒细胞中cyclinG1的表达情况;取性成熟小鼠的颗粒细胞进行体外培养,分别加入卵泡刺激素(FSH)和绒毛膜促性腺激素(hCG)作用12h后,收集细胞,提取细胞总RNA,采用半定量RTPCR检测cyclinG1表达水平的变化。结果:免疫组化结果显示cyclinG1在不同发育阶段卵泡的颗粒细胞和卵丘细胞中均有较强表达,而黄体细胞中表达较少。RTPCR结果显示,FSH与hCG分别作用于体外培养的颗粒细胞12h后,与对照组比较,hCG可使cyclinG1的表达水平明显降低(P<0.01),FSH对其表达水平影响不明显(P>0.05)。结论:小鼠卵巢颗粒细胞中存在cyclinG1表达;hCG可降低cyclinG1在颗粒细胞中的表达水平,提示cyclinG1在颗粒细胞增殖分化过程中可能发挥正调节作用。     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Overexpression of cyclin D1 in nonmelanocytic skin cancer

Although the overexpression of cyclin D1 has been believed to play important roles in neoplastic transformation of some tumors, little is known about the function of cyclin D1 protein in carcinogenesis in human skin. A total of 307 patients with nonmelanocytic skin cancer, being 46 with Bowen’s disease (BOD), 134 with squamous cell carcinoma (SCC) and 127 with basal cell carcinoma (BCC), were investigated immunohistochemically using monoclonal antibody to cyclin D1 by the LSAB method, to assess the expression of cyclin D1 in skin cancer including its precursors. The positive rates of cyclin D1 immunostaining in BOD, SCC and BCC were 63.0%, 69.4% and 54.3%, respectively. The positive rates in dysplasia adjoining BOD, SCC and BCC were 43.6%, 67.9% and 59.8%, respectively. In morphologically normal skin, however, only 2 cases, 1 of SCC and 1 of BCC, exhibited positive staining. These findings suggested that overexpression of cyclin D1 is an early event in dysplastic lesions of skin. Overexpression of cyclin D1 was related to sun exposure, especially in dysplasia of SCC. The score for cyclin D1 expression in dysplasia of BCC was correlated with age. Expression of cyclin D1 markedly increased from normal skin through dysplasia to BOD, but was not significantly related to the degree of SCC differentiation. These findings demonstrate that the effect of cyclin D1 overexpression is restricted to proliferation of cells, so that they gain a growth advantage, but their differentiation is not increased. Comparison with the results for p53 protein expression in these tumors, a significant correlation with cyclin D1 expression was found in dysplasia in BOD and SCC, and in patients with BCC who were less than 74 years old. These findings suggested the hypothesis that prior aberrant p53 expression may affect or regulate the overexpression of cyclin D1. Received: 6 September 1999 / Accepted: 10 November 1999     

Lack of cyclin D1 overexpression in gastric carcinogenesis

AIMS: Cyclin D1 overexpression was examined in early gastric carcinomas and precursor lesions with the following aims; (1) to assess the chronology of cyclin D1 overexpression in various stages of gastric carcinogenesis, (2) to correlate cyclin D1 overexpression with the Lauren type, the grade of differentiation and the type of growth pattern of the tumours and (3) to correlate cyclin D1 overexpression with clinical parameters, in particular lymph node metastasis and overall prognosis. METHODS AND RESULTS: Forty-five paraffin-embedded gastrectomy specimens from early carcinomas were examined for the presence of various precursor lesions. The Lauren type, the grade of differentiation and the type of growth pattern were reassessed for all early carcinomas. Cyclin D1 overexpression was examined using the monoclonal antibody DCS-6. Cyclin D1 overexpression was absent from all precursor lesions. Ten early carcinomas (22%) were cyclin D1 positive without significant differences when stratified according to Lauren type, grade of differentiation, type of growth pattern or lymph node status. Univariate analysis failed to show a significant difference in 5-year surival rate between cyclin D1 positive and negative early carcinomas (90% vs. 94%). CONCLUSIONS: Cyclin D1 protein overexpression does not play a role in the progression from normal to neoplastic gastric mucosa and does not discriminate between intestinal and diffuse type early gastric carcinomas of Caucasian origin. Moreover, mechanisms other than cyclin D1 protein overexpression underlie the reported difference in biological behaviour of early gastric carcinomas with different types of growth pattern. Finally, although it appears that cyclin D1 does not have prognostic significance, studies on larger numbers, including advanced carcinomas, are warranted.