Cell Cycle Regulators Cyclin D3 Research Articles

IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation.

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa.1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized withPalbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thusidentifies USP10 as the first deubiquitinase of CCND3and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells.

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by "inertia." We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.

Restoration of microRNA-212 causes a G0/G1 cell cycle arrest and apoptosis in adult T-cell leukemia/lymphoma cells by repressing CCND3 expression

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive T-cell malignancy. This study was designed to explore the expression and functional significance of microRNA (miR)-212 in ATL. The expression of miR-212...

Cell Cycle Differences in Vitro between Primitive Hematopoietic Cell Populations from Adult and Umbilical Cord Blood

Lineage-negative (Lin(-)) cell populations, obtained by negative selection from umbilical cord blood (UCB) and adult mobilized peripheral blood (aMPB), were cultured in serum-free liquid cultures supplemented with a mixture of seven stimulatory cytokines. On specific days, proliferation potential was assessed and cell cycle status was determined by DNA content. Expression of the cell cycle regulators cyclin D3 (cD3), cyclin-dependent kinase 4 (cdk4), p21(cip1/waf1) (p21), and p27(kip1) (p27) was also determined. As expected, UCB cells showed significantly higher proliferation potentials than aMPB cells, particularly during the first 7 days of culture. During this period of time, higher numbers of cell cycles were observed in UCB cells (7-9 cycles), as compared to aMPB cells (5-6 cycles). Higher levels of cD3, cdk4, and p27 were also detected in UCB cells. Our results confirm that UCB cells possess an intrinsically higher proliferation potential, as compared to aMPB cells, and suggest that such a biological difference is due, at least in part, to differences in cell cycle status. This, in turn, seems to result from the differential expression of cell cycle regulatory molecules.

Nuclear ??-Catenin Expression Is Rare and its Potential Association With Short Survival in Colorectal Signet-Ring Cell Carcinoma

Colorectal signet-ring cell carcinoma (SRCC) is a rare cancer and the prognosis is usually very poor. The biologic pathways involved in its oncogenesis are unknown. beta-catenin, a key target in the Wnt-signaling pathway, is recognized to play an important role in the carcinogenesis in conventional colorectal carcinoma. This study explores the involvement of Wnt-signaling molecules beta-catenin and cyclin D1, cell cycle regulators cyclin D3, proliferative index Ki-67, apoptotic index, and angiogenic indicator CD31 in 20 colorectal SRCC paraffin-embedded specimens. Results showed that there were 2 specimens with nuclear beta-catenin and higher expression of cyclin D1 than the remaining 18 specimens. Surprisingly, those 2 patients had a much shorter survival of 6 months than the remaining 15 patients, who had around 24 months. Moreover, all colorectal SRCC specimens had an overexpression of cyclin D1, cyclin D3, and Ki-67, as well as much more angiogenesis and apoptosis than adjacent normal epithelial tissues. The authors make the preliminary comment that nuclear beta-catenin is a rare phenomenon in colorectal SRCC, but the involvement of it may indicate a worse prognosis with shorter survival than colorectal SRCC without nuclear beta-catenin expression. Besides, overexpression of cyclin D1, cyclin D3, Ki-67, and increased angiogenesis and apoptosis may play a vital role in promoting colorectal SRCC development.

Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death.

Precancerous cells that enter S phase without appropriate growth and viability factors undergo programmed cell death, suggesting that apoptosis may help guarantee organismic integrity [Evan, G. & Littlewood, T. (1998) Science 281, 1317-1322]. However, the connection between proliferation and cell death has remained unclear. Here, we show that the positive cell cycle regulator cyclin D3 [Matsushime H., Roussel M. F., Ashmun, R. A. & Sherr, C. J. (1991) Cell 65, 701-713] interacts with the death enzyme Caspase 2 [Wang, L., Miura, M., Bergeron, L., Zhu, H. & Yuan, J. (1994) Cell 78, 739-750]. Directed expression of cyclin D3 and Caspase 2 in human cells potentiated apoptosis compared with expression of Caspase 2 alone. Cyclin D3 expression increased the amount of cleaved (active) Caspase 2. We describe a PCR mutagenesis/ligation/two-hybrid/green fluorescent protein approach that facilitates the isolation of missense mutant proteins defective in interaction with particular partners absent other phenotypes or knowledge of the system. We used this approach to isolate Caspase 2 mutants that did not bind cyclin D3 (noninteractors). Noninteractors were sensitive to apoptosis-dependent proteolysis, but did not potentiate apoptosis. Noninteractors did not block apoptosis caused by wild-type Caspase 2. Our results are consistent with the idea that an interaction with cyclin D3 may stabilize Caspase 2, and suggest that a physical interaction between cyclin D3 and Caspase 2 connects the genetic networks that govern cell-cycle progression with those that govern cell death.

Developmentally regulated expression of cyclin D3 and its potential in vivo interacting proteins during murine gametogenesis.

To begin to assess the function of the cell cycle regulator cyclin D3 during gametogenesis, the present study examined its expression, interacting partners, and associated kinase activity in the murine testis and ovary. In the early stages of postnatal testicular development, cyclin D3 protein was detected in spermatogonia and Leydig cells. In the adult testis, cyclin D3 was also expressed in terminally differentiating spermatids. In the embryonic ovary, detection of cyclin D3 was limited to somatic cells. In the postnatal ovary, its localization was predominantly in the nuclei of oocytes in primordial and small follicles, a localization that diminished with oocyte growth. Cdk4 and p27 were expressed in a similar subset of testicular and ovarian cells, suggesting that they may regulate cyclin D3 function during testicular and ovarian development in a cell type-specific manner. Cyclin D3-associated kinase activity was detected in immature, but not adult, testes and ovaries. These observations suggest unique roles for cyclin D3 in the control of cell division and differentiation in the germ line and the differential regulation of mitotic and meiotic cell cycles during male and female gametogenesis.

Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.

The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0. (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.