Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.     

A Cyclin D1‐Specific Single‐Chain Variable Fragment Antibody that Inhibits HepG2 Cell Growth and Proliferation

Cyclin D1 is a key regulatory factor of the G1 to S transition during cell cycle progression. Aberrant cyclin D gene amplification and abnormal protein expression have been linked to hepatocellular carcinoma (HCC) tumorigenesis. Intrabodies, effective anticancer therapies that specifically inhibit target protein function within all intracellular compartments, may block cyclin D1 function. Here, a single‐chain variable fragment (scFv) antibody against cyclin D1 (ADκ) selected from a human semi‐synthetic phage display scFv library is expressed in Escherichia coli as soluble ADκ. Purified ADκ specifically binds to recombinant and endogenous cyclin D1 with high affinity. To enable blocking of intracellular cyclin D1 activity, an endoplasmic reticulum (ER) retention signal sequence is added to the ADκ sequence to encode anti‐cyclin D1 intrabody ER‐ADκ. Transfection of HepG2 cells with expression vector encoding ER‐ADκ elicited intracellular ER‐ADκ expression leading to cyclin D1 binding, significant G1 phase arrest, and apoptosis that are mechanistically tied to decreased intracellular phosphorylated retinoblastoma protein (Rb) levels. Meanwhile, ER‐ADκ dramatically inhibited subcutaneous human HCC xenografts growth in nude mice in vivo after injection of tumors with expression vector encoding ER‐ADκ. These results demonstrate the potential of intrabody‐based cyclin D1 targeting therapy as a promising treatment for HCC.     

DICER1 regulated let‐7 expression levels in p53‐induced cancer repression requires cyclin D1

Let‐7 miRNAs act as tumour suppressors by directly binding to the 3′UTRs of downstream gene products. The regulatory role of let‐7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let‐7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let‐7. Down‐regulated let‐7 has been identified in many types of tumours, suggesting a feedback loop may exist between let‐7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence‐specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1‐induced let‐7 expression. In addition, we found that let‐7 miRNAs expression decreased because of the p53‐induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin‐3 and TAX‐induced let‐7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let‐7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let‐7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.     

Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.

Cyclin D1 is the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle. Deregulated cyclin D1 expression has been implicated in several human neoplasms, most consistently in centrocytic B lymphoma, where the cyclin D1 gene usually has been translocated to an immunoglobulin locus. To determine directly whether constitutive cyclin D1 expression is lymphomagenic, transgenic mice were generated having the cyclin D1 gene linked to an immunoglobulin enhancer. Despite abundant transgene expression, their lymphocytes were normal in cell cycle activity, size and mitogen responsiveness, but young transgenic animals contained fewer mature B- and T-cells. Although spontaneous tumours were infrequent, lymphomagenesis was much more rapid in mice that co-expressed the cyclin D1 transgene and a myc transgene than in mice expressing either transgene alone. Moreover, the spontaneous lymphomas of myc transgenic animals often ectopically expressed the endogenous cyclin D1 gene. These findings indicate that this G1 cyclin can modulate differentiation and collaborate with myc-like genes in oncogenesis.     

Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.

Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.     

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Background

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G₁ phase.

Methodology/Principal Findings

In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G₀-G₁ phase arrest, but also reduced the number of cells at S and G₂-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G₀-G₁ phase despite the reduction of G₀-G₁ regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G₀-G₁ regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.

Conclusions/Significance

This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G₀-G₁ phase.     

Cyclin D1 and molecular chaperones: implications for tumorigenesis

We recently investigated the mechanisms of cyclin D1 action in human cancer using global analyses of gene expression. With an experimentally-determined expression signature for cyclin D1 overexpression, gene expression data from human tumors, and a novel data-mining method, we were able to reveal a previously unappreciated and apparently predominant functional interdependency between cyclin D1 and C/EBPbeta. Many of the genes we found to be affected by cyclin D1 overexpression are recognized as molecular chaperones or their regulators. Might this provide insights to the role of the cyclin D1-C/EBPbeta axis in carcinogenesis?     

Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.     

Genomic organization and evolutionary conservation of plant D-type cyclins

Plants contain more genes encoding core cell cycle regulators than other organisms but it is unclear whether these represent distinct functions. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition, and Arabidopsis (Arabidopsis thaliana) contains 10 CYCD genes in seven defined subgroups, six of which are conserved in rice (Oryza sativa). Here, we identify 22 CYCD genes in the poplar (Populus trichocarpa) genome and confirm that these six CYCD subgroups are conserved across higher plants, suggesting subgroup-specific functions. Different subgroups show gene number increases, with CYCD3 having three members in Arabidopsis, six in poplar, and a single representative in rice. All three species contain a single CYCD7 gene. Despite low overall sequence homology, we find remarkable conservation of intron/exon boundaries, because in most CYCD genes of plants and mammals, the first exon ends in the conserved cyclin signature. Only CYCD3 genes contain the complete cyclin box in a single exon, and this structure is conserved across angiosperms, again suggesting an early origin for the subgroup. The single CYCD gene of moss has a gene structure closely related to those of higher plants, sharing an identical exon/intron structure with several higher plant subgroups. However, green algae have CYCD genes structurally unrelated to higher plants. Conservation is also observed in the location of potential cyclin-dependent kinase phosphorylation sites within CYCD proteins. Subgroup structure is supported by conserved regulatory elements, particularly in the eudicot species, including conserved E2F regulatory sites within CYCD3 promoters. Global expression correlation analysis further supports distinct expression patterns for CYCD subgroups.     

Homocysteine induces smooth muscle cell proliferation through differential regulation of cyclins A and D1 expression

The mechanism of homocysteine‐induced cell proliferation in human vascular smooth muscle cells (SMCs) remains unclear. We investigated the molecular mechanisms by which homocysteine affects the expression of cyclins A and D1 in human umbilical artery SMCs (HUASMCs). Homocysteine treatment induced proliferation of HUASMCs and increased the expression levels of cyclins A and D1. Knocking down either cyclin A or cyclin D1 by small interfering RNA (siRNA) inhibited homocysteine‐induced cell proliferation. Furthermore, treatment with extracellular signal‐related kinase (ERK) inhibitor (PD98059) and dominant negative Ras (RasN17) abolished homocysteine‐induced cyclin A expression; and treatment with phosphatidylinositol 3‐kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine‐induced cyclin D1 expression. Homocysteine also induced transient phosphorylation of ERK, Akt, and p70 ribosomal S6 kinase (p70S6K). Neutralizing antibody and siRNA for β1 integrin blocked cell proliferation, expression of cyclins A and D1, and phosphorylation of ERK and Akt. In conclusion, homocysteine‐induced differential activation of Ras/ERK and PI3K/Akt/p70S6K signaling pathways and consequent expression of cyclins A and D1 are dependent on β1 integrin. Homocysteine may accelerate progression of atherosclerotic lesions by promoting SMC proliferation. J. Cell. Physiol. 226: 1017–1026, 2011. © 2010 Wiley‐Liss, Inc.     

Cyclin D1 and Molecular Chaperones: Implications for Tumorigenesis

We recently investigated the mechanisms of cyclin D1 action in human cancer using global analyses of gene expression. With an experimentally-determined expression signature for cyclin D1 overexpression, gene expression data from human tumors, and a novel data-mining method, we were able to reveal a previously unappreciated and apparently predominant functional interdependency between cyclin D1 and C/EBPb. Many of the genes we found to be affected by cyclin D1 overexpression are recognized as molecular chaperones or their regulators. Might this provide insights to the role of the cyclin D1-C/EBPb axis in carcinogenesis?