The expression of tumor suppressor gene Cyld is upregulated by histone deacetylace inhibitors in human hepatocellular carcinoma cell lines.

CYLD is a deubiquitinating enzyme that exerts a tumor suppressive function. Its downregulation or inactivation has been associated with the development of several types of malignancies including hepatocellular carcinoma (HCC). HCC cells display significantly lower Cyld expression compared to primary human hepatocytes, and Cyld downregulation can contribute to apoptotic resistance of HCC cells. Little is known about the mechanism of Cyld downregulation in human HCC cells. In the present study we explored the possible regulation of Cyld expression by histone deacetylases (HDACs) in human HCC cell lines. We demonstrated that the HDAC inhibitors suberoylanilide hydroxamic acid, sodium butyrate, and trichostatin A induced the upregulation of both mRNA and protein levels of CYLD in two different HCC cell lines, HepG2 and Huh7. Our results demonstrate the involvement of HDACs in the downregulation of Cyld expression in HCC cells and support and may improve the use of HDAC inhibitors for the treatment for HCC.

T cell proliferation and homeostasis: an emerging role for the cell cycle inhibitor geminin.

Thymic T cell differentiation to peripheral T cells aims to assist the generation of effector cells mediating adaptive immune responses. During this process, which takes place during embryogenesis and in adulthood, proliferation is coupled with changes in chromatin organization and transcription. Moreover, B and T lymphocytes start to proliferate and rapidly expand their numbers when activated following an encounter with an antigen. This expansion phase is accompanied by differentiation of naïve T cells and is followed by a period of population contraction, resulting in only a small fraction of the expanded population surviving and entering the memory cell pool. The kinetics of the expansion and contraction affect the speed of antigen clearance and the clinical course of disease. Molecules that are involved in the coordination of proliferation, chromatin reorganization, and transcriptional regulation are likely to play an important role in T cell generation, homeostasis, and disease. Here we review how cell cycle regulators affect lymphoid system development and homeostasis and discuss recent evidence implicating the cell cycle inhibitor Geminin in this process. Geminin has been shown to coordinate proliferation and differentiation by regulating cell cycle progression, chromatin organization, and transcription in the nervous system. In the immune system, progenitor T cell commitment and differentiation progresses normally in the absence of Geminin. However, Geminin is required for TCR response in vitro and T cell proliferation upon lymphopenia-induced proliferation, suggesting that Geminin might be an essential factor for T cell expansion during the immune response.

Differential geminin requirement for proliferation of thymocytes and mature T cells.

Stem/progenitor cells coordinate proliferation and differentiation, giving rise to appropriate cell numbers of functionally specialized cells during organogenesis. In different experimental systems, Geminin was shown to maintain progenitor cells and participate in fate determination decisions and organogenesis. Although the exact mechanisms are unclear, Geminin has been postulated to influence proliferation versus differentiation decisions. To gain insight into the in vivo role of Geminin in progenitor cell division and differentiation, we have generated mice that specifically lack Geminin in cells of lymphoid lineage through Cre-mediated recombination. T cells lacking Geminin expression upregulate early activation markers efficiently upon TCR stimulation in vitro and are able to enter the S phase of cell cycle, but show a marked defect in completing the cycle, leading to a large proportion of T cells accumulating in S/G2/M phases. Accordingly, T cells deficient in Geminin show a reduced ability to repopulate lymphopenic hosts in vivo. Contrary to expectations, Geminin deficiency does not alter development and differentiation of T cells in vivo. Our data suggest that Geminin is required for the proliferation events taking place either in vitro upon TCR receptor activation or during homeostatic expansion, but appears to be redundant for the proliferation and differentiation of the majority of progenitor T cell populations.

Cdt1 and Geminin in cancer: markers or triggers of malignant transformation?

Cdt1 and its inhibitor Geminin are important regulators of replication licensing. In normal cells, a critical balance between these two proteins ensures that firing of each origin along the genome will take place only once per cell cycle. Cdt1 overexpression in cell lines and animals leads to aberrant replication, activates DNA damage checkpoints and predisposes for malignant transformation. Geminin inactivation mimics the effects of Cdt1 overexpression in cells and generates mitotic defects and abnormal chromosome segregation. Aberrant expression of Cdt1 and Geminin is thus linked to DNA replication defects, aneuploidy and genomic instability. These traits are considered integral to precancerous states and essential elements for malignant transformation. Moreover, Cdt1 and Geminin expression is deregulated in human tumor specimens and Cdt1 and Geminin may represent novel markers useful for cancer diagnosis and prognosis.

Inactivation of CYLD in intestinal epithelial cells exacerbates colitis-associated colorectal carcinogenesis - a short report.

PURPOSE: CYLD is a tumor suppressor that has been linked to the development of various human malignancies, including colon cancer. The tumor-suppressing function of CYLD is associated with its deubiquitinating activity, which maps to the carboxyl-terminal region of the protein. In the present study we evaluated the role of intestinal epithelial CYLD in colitis-associated cancer using a conditional mouse CYLD inactivation model. METHODS: In order to evaluate the role of CYLD in intestinal epithelial carcinogenesis, mice (IEC-Cyld (Δ9) mice) that carry a mutation that eliminates the deubiquitinating domain of CYLD in intestinal epithelial cells (IEC) were generated by crossing Villin-Cre transgenic mice to previously generated mice carrying a loxP-flanked Cyld exon 9 (Cyld (flx9) mice). RESULTS: We found that IEC-Cyld (Δ9) mice did not present spontaneous intestinal abnormalities up to one year of age. However, upon challenge with a combination of genotoxic (AOM) and pro-inflammatory (DSS) agents we found that the number of adenomas in the IEC-Cyld (Δ9) mice was dramatically increased compared to the control mice. Inactivation of CYLD in intestinal epithelial cells did not affect the classical nuclear factor-kappaB (NF-κB) and c-Jun kinase (JNK) activation pathways under physiological conditions, suggesting that these pathways do not predispose CYLD-deficient intestinal epithelia to colorectal cancer development before the onset of genotoxic and/or pro-inflammatory stress. CONCLUSIONS: Our findings underscore a critical tumor-suppressing role for functional intestinal epithelial CYLD in colitis-associated carcinogenesis. CYLD expression and its associated pathways in intestinal tumors may be exploited for future prognostic and therapeutic purposes.

Reduced Geminin levels promote cellular senescence.

Cellular senescence is a permanent out-of-cycle state regulated by molecular circuits acting during the G1 phase of the cell cycle. Cdt1 is a central regulator of DNA replication licensing acting during the G1 phase and it is negatively controlled by Geminin. Here, we characterize the cell cycle expression pattern of Cdt1 and Geminin during successive passages of primary fibroblasts and compare it to tumour-derived cell lines. Cdt1 and Geminin are strictly expressed in distinct subpopulations of young fibroblasts, similarly to cancer cells, with Geminin accumulating shortly after the onset of S phase. Cdt1 and Geminin are down-regulated when primary human and mouse fibroblasts undergo replicative or stress-induced senescence. RNAi-mediated Geminin knock-down in human cells enhances the appearance of phenotypic and molecular features of senescence. Mouse embryonic fibroblasts heterozygous for Geminin exhibit accelerated senescence compared to control fibroblasts. In contrast, ectopic expression of Geminin in mouse embryonic fibroblasts delays the appearance of the senescent phenotype. Taken together, our data suggest that changes in Geminin expression levels affect the establishment of senescence pathways.

Life without geminin.

The interplay of proliferation and differentiation is essential for normal development and organogenesis. Geminin is a cell cycle regulator which controls licensing of origins for DNA replication, safeguarding genomic stability. Geminin has also been shown to regulate cellular decisions of self-renewal versus commitment of neuronal progenitor cells. We discuss here our recent analysis of mice with conditional inactivation of the Geminin gene in the immune system. Our data indicate that Geminin is not indispensable for every cell division: in the absence of Geminin, development of progenitor T cells appears largely unaffected. In contrast, rapid cell divisions, taking place in vitro upon TCR receptor activation or in vivo during homeostatic proliferation, are defective.