Tumor suppressor genes are often silenced in human cancer; this can occur by transcriptional repression by deacetylation in the promoter regions, mediated by histone deacetylase (HDAC). HDAC inhibitors can block cancer cell growth by restoring expression of tumor suppressor genes. In this study, we investigated the effects of a HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) on pancreatic cancer cells. SAHA inhibited the growth of 6 pancreatic cancer cell lines in a dose-dependent manner as measured by MTT and clonogenic assays (ED 50 10 26 M) associated with induction of apoptosis, G2 cell cycle arrest and also induced differentiation as indicated by morphology and increased expression of cytokeratin 7. It increased expression of p21 WAF1 (independent of the mutational status of p53), C/EBPa, RARa and E-cadherin; these genes have been associated with decreased proliferation in other cancers. SAHA decreased cyclin B1 expression; this cyclin normally promotes progression through G2 of the cell cycle. SAHA mediated acetylation of histone H3 globally, as well as, associated with the p21 WAF1 promoter, as measured by chromatin immunoprecipitation. SAHA also decreased levels of c-myc and cyclin D1, independent of an active b-catenin pathway. In further studies, the combination of SAHA and an inhibitor of DNA methylation, 5-Aza-2 0 -deoxycytidine, had an enhanced antiproliferative effect on pancreatic cancer cells. In summary, SAHA inhibited the growth of human pancreatic cancer cells by inducing apoptosis, differentiation and cell cycle arrest, as well as increase in the expression of several tumor suppressor genes. SAHA is a novel, promising therapeutic agent for human pancreatic cancers. ' 2007 Wiley-Liss, Inc.

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Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPα, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPα was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPα was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPα markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPα and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPα localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPα causes dysregulation of its function, suggesting that C/EBPα is a novel candidate tumor suppressor gene in pancreatic cancer cells.

TSC-22 (Transforming growth factor-beta stimulated clone-22) was originally isolated as a TGF-beta-inducible gene in mouse osteoblastic cells. TSC-22 encodes a putative transcriptional regulator containing a leucine zipper-like structure. Several differentiation-inducing stimuli up-regulate the TSC-22 gene. Furthermore, TSC-22 acts as an effector that integrates multiple extracellular signals during embryogenesis of Drosophila and mouse. Separately, we identified TSC-22 cDNA as an anti-cancer drug (vesnarinone)-inducible gene in a human salivary gland cancer cell line, TYS. Vesnarinone is known to have a differentiation-inducing activity in several cell types. We showed that TSC-22 negatively regulated the growth of TYS cells, and that down-regulation of TSC-22 played a major role in the salivary gland tumorigenesis. Subsequently, we found that artificial overexpression of TSC-22 enhanced chemosensitivity and radiation-sensitivity by inducing apoptosis in TYS cells. Recently, we isolated TSC-22 genomic DNA and analyzed the transcriptional and post-transcriptional regulation of the TSC-22 gene. Then, we confirmed by the luciferase reporter assay that several differentiation-inducing stimuli directly activated the promoter region of TSC-22 gene. Now we are investigating the chemical compounds, which could enhance the transcription of the TSC-22 gene. Thus, because TSC-22 is a key molecule for differentiation of several cells, it can be used as a molecular target for cancer differentiation therapy in salivary gland cancer.

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IntroductionAdvancing industrial-scale manufacture of cells as therapeutic products is an example of the wide applications of regenerative medicine. However, one bottleneck in establishing stable and efficient cell manufacture is quality control. Owing to the lack of effective in-process measurement technology, analyzing the time-consuming and complex cell culture process that essentially determines cellular quality is difficult and only performed by manual microscopic observation. Our group has been applying advanced image-processing and machine-learning modeling techniques to construct prediction models that support quality evaluations during cell culture. In this study, as a model of errors during the cell culture process, intentional errors were compared to the standard culture and analyzed based only on the time-course morphological information of the cells.MethodsTwenty-one lots of human mesenchymal stem cells (MSCs), including both bone-marrow-derived MSCs and adipose-derived MSCs, were cultured under 5 conditions (one standard and 4 types of intentional errors, such as clear failure of handlings and machinery malfunctions). Using time-course microscopic images, cell morphological profiles were quantitatively measured and utilized for visualization and prediction modeling. For visualization, modified principal component analysis (PCA) was used. For prediction modeling, linear regression analysis and the MT method were applied.ResultsBy modified PCA visualization, the differences in cellular lots and culture conditions were illustrated as traits on a morphological transition line plot and found to be effective descriptors for discriminating the deviated samples in a real-time manner. In prediction modeling, both the cell growth rate and error condition discrimination showed high accuracy (>80%), which required only 2 days of culture. Moreover, we demonstrated the applicability of different concepts of machine learning using the MT method, which is effective for manufacture processes that mostly collect standard data but not a large amount of failure data.ConclusionsMorphological information that can be quantitatively acquired during cell culture has great potential as an in-process measurement tool for quality control in cell manufacturing processes.

The number of bone marrow granulocyte-macrophage progenitor cells (CFU-C) and fibroblastoid colony-forming units (CFU-F) was significantly smaller in acute leukemia patients. To analyze the mechanism by which CFU-C and CFU-F were reduced, the study on murine myeloid leukemia was carried out. In murine leukemia, bone marrow CFU-C and CFU-F decreased in number in correlation with the proliferation of leukemic cells. Murine leukemic cells suppressed the growth of CFU-C and CFU-F in the mixed culture of leukemic cells and normal murine marrow cells. Furthermore, leukemic cell-conditioned medium inhibited the growth of CFU-F. The results show that murine leukemic cells themselves and/or humoral factors produced by them give inhibitory effects on the growth of not only CFU-C but also CFU-F.

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