INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling.

BACKGROUND: The gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells. RESULTS: Markedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1. CONCLUSION: These results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.

Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of fibrotic lung fibroblasts to FasL induced apoptosis.

BACKGROUND: An altered susceptibility of lung fibroblasts to Fas-induced apoptosis has been implicated in the pathogenesis of pulmonary fibrosis; however, the underlying mechanism is not completely understood. Here, we studied the susceptibility of lung fibroblasts, obtained from patients with (f-fibs) and without pulmonary fibrosis (n-fibs), to FasL- (CD95L/APO-1) induced apoptosis in relation to the expression and the amounts of membrane-bound and soluble Fas. We also analysed the effects of tumor necrosis factor-beta on FasL-induced cell death. METHODS: Apoptosis was induced with recombinant human FasL, with and without prior stimulation of the fibroblasts with tumor necrosis factor-alpha and measured by a histone fragmentation assay and flow cytometry. The expression of Fas mRNA was determined by quantitative PCR. The expression of cell surface Fas was determined by flow cytometry, and that of soluble Fas (sFas) was determined by enzyme-linked immunosorbent assay. RESULTS: When compared to n-fibs, f-fibs were resistant to FasL-induced apoptosis, despite significantly higher levels of Fas mRNA. F-fibs showed lower expression of surface-bound Fas but higher levels of sFas. While TNF-alpha increased the susceptibility to FasL-induced apoptosis in n-fibs, it had no pro-apoptotic effect in f-fibs. CONCLUSIONS: The data suggest that lower expression of surface Fas, but higher levels of apoptosis-inhibiting sFas, contribute to the resistance of fibroblasts in lung fibrosis against apoptosis, to increased cellularity and also to increased formation and deposition of extracellular matrix.

Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-beta1-signaling and upregulation of cathepsin B.

The TGF-beta signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-beta1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-beta1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-beta signaling pathways showed no alterations in TGF-beta/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-beta1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-beta1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.

Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts.

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.

Functional consequences of cathepsin L deficiency in human lung epithelial cells.

Cathepsin L is a cysteine protease of the papain family. Lung epithelial cells play an important role in host defence. The aim of the present study was to investigate the functional role of cathepsin L in the human lung carcinoma cell line A549. Cathepsin L-deficient A549 clones were generated. They showed a significant lower proliferation and secreted 5- to 8-fold more IL-8 than the control cells. The production of IL-6, IL-18, and TGF-beta1/2 was not affected significantly. It was shown that the cells upregulate IL-8 transcription and that IL-8 in the culture supernatant is necessary for the containment of cellular proliferation. In conclusion, the data show that suppression of cathepsin L expression in A549 cells leads to a growth inhibition which is partially compensated by an upregulation of IL-8 production.

Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells.

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.

Higher susceptibility to Fas ligand induced apoptosis and altered modulation of cell death by tumor necrosis factor-alpha in periarticular tenocytes from patients with knee joint osteoarthritis.

The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-alpha were studied by stimulation with TNF-alpha alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-alpha reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-alpha in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.