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Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation.

ABSTRACT: Gemcitabine (GMZ) is a chemotherapeutic agent with well established effects on cell growth arrest and apoptosis. In this study, we investigated the potential roles of bioactive sphingolipids in mediating the growth suppressing effects of GMZ on a polyoma middle T transformed murine endothelial cell line. After 12-hour GMZ (0.6 microM) treatment, cell growth was arrested at the G(0)/G(1) phase as detected by flow cytometric cell cycle analysis and MTT cell viability analysis, and this was accompanied by dephosphorylation of the retinoblastoma protein (Rb). Furthermore, GMZ treatment resulted in increased levels of specifically the very long chain ceramides as determined by mass spectrometry. Mechanistically, GMZ did not appear to affect the activities of many enzymes of ceramide metabolism; however, GMZ caused a selective reduction in the protein levels of neutral ceramidase (NCDase), as indicated by Western blot analysis, with a concomitant decrease in NCDase activity. The significance of NCDase loss on cell cycle regulation was investigated by specific knockdown of the enzyme using small interfering RNA (siRNA). Interestingly, NCDase siRNA transfection was sufficient to induce a cell cycle arrest at G(0)/G(1) and an increase in total ceramide levels, with significant elevation in very long chain ceramides (C(24:1) and C(24:0)). NCDase siRNA also induced Rb dephosphorylation. These data provide evidence for a novel mechanism of action for GMZ and highlight downregulation of NCDase as a critical step in GMZ-mediated ceramide elevation and cell cycle arrest.

SUBMITTER: Wu BX

PROVIDER: S-EPMC2788435 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation.

Wu Bill X BX Zeidan Youssef H YH Hannun Yusuf A YA

Biochimica et biophysica acta 20090402 8

Gemcitabine (GMZ) is a chemotherapeutic agent with well established effects on cell growth arrest and apoptosis. In this study, we investigated the potential roles of bioactive sphingolipids in mediating the growth suppressing effects of GMZ on a polyoma middle T transformed murine endothelial cell line. After 12-hour GMZ (0.6 microM) treatment, cell growth was arrested at the G(0)/G(1) phase as detected by flow cytometric cell cycle analysis and MTT cell viability analysis, and this was accompa ...[more]

PMID: 19345744

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Project description:Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ?Np73?, an antagonist of p53 and p73. Inhibition of ?Np73? expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ?Np73? in this event. Chromatin immunoprecipitation experiments showed that ?Np73? is part of a negative transcriptional regulatory complex with I?B kinase beta (IKK?) that binds to a NF-?B responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1), decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation.The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

Dataset Information

Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation.

Publications

Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation.

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