技术资料

CD147 (alias emmprin or basigin),an integral plasma membrane glycoprotein and a member of the Ig superfamily,is widespread in normal tissues,but highly up-regulated in many types of malignant cancer cells. CD147 is multifunctional,with numerous binding partners. Recent studies suggest that complexes of CD147 with the hyaluronan receptor CD44 and associated transporters and receptor tyrosine kinases are enriched in the plasma membrane of cancer stem-like cells. Here,we show that subpopulations of tumor cell lines constitutively expressing high levels of cell-surface CD147 exhibit cancer stem-like cell properties; that is,they exhibit much greater invasiveness,anchorage-independent growth,spheroid formation,and drug resistance in vitro and higher tumorigenicity in vivo than those constitutively expressing low levels of cell-surface CD147. Primary CD147-rich cell subpopulations derived from mouse mammary adenocarcinomas also exhibit high levels of invasiveness and spheroid-forming capacity,whereas CD147-low cells do not. Moreover,localization at the plasma membrane of CD44,the EGF receptor,the ABCB1 and ABCG2 drug transporters,and the MCT4 monocarboxylate transporter is elevated in cells constitutively expressing high levels of cell-surface CD147. These results show that CD147 is associated with assembly of numerous pro-oncogenic proteins in the plasma membrane and may play a fundamental role in properties characteristic of cancer stem-like cells. View Publication

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death,but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis,or of its underlying transcriptome network. Therefore,the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes,whereas MeHg altered fewer transcripts. To attenuate batch effects,analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (backslashtextless20 % overlap). Moreover,within one test system,little overlap between the PS changed by the two compounds has been observed. However,using TFBS enrichment,a relatively large ‘common response' to VPA and MeHg could be distinguished from ‘compound-specific' responses. In conclusion,the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. View Publication

Progestins play a deleterious role in the onset of breast cancer,yet their influence on existing breast cancer and tumor progression is not well understood. In luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancer,progestins induce a fraction of cells to express cytokeratin 5 (CK5),a marker of basal epithelial and progenitor cells in the normal breast. CK5(+) cells lose expression of ER and PR and are relatively quiescent,increasing their resistance to endocrine and chemotherapy compared to intratumoral CK5(-)ER(+)PR(+) cells. Characterization of live CK5(+) cells has been hampered by a lack of means for their direct isolation. Here,we describe optical (GFP) and bioluminescent (luciferase) reporter models to quantitate and isolate CK5(+) cells in luminal breast cancer cell lines utilizing the human KRT5 gene promoter and a viral vector approach. Using this system,we confirmed that the induction of GFP(+)/CK5(+) cells is specific to progestins,is dependent on PR,can be blocked by antiprogestins,and does not occur with other steroid hormones. Progestin-induced,fluorescence-activated cell sorting-isolated CK5(+) cells had lower ER and PR mRNA,were slower cycling,and were relatively more invasive and sphere forming than their CK5(-) counterparts in vitro. Repeated progestin treatment and selection of GFP(+) cells enriched for a persistent population of CK5(+) cells,suggesting that this transition can be semi-permanent. These data support that in PR(+) breast cancers,progestins induce a subpopulation of CK5(+)ER(-)PR(-) cells with enhanced progenitor properties and have implications for treatment resistance and recurrence in luminal breast cancer. View Publication

Regenerative medicine is in need of solid,large animal models as a link between rodents and humans to evaluate the functionality,immunogenicity,and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model,genetically close to humans and readily used worldwide in clinical research. Until now,only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore,we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV,SB431542,PD0325901,and ascorbic acid via a novel,excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4,KLF4,SOX2,C-MYC). Endogenous pluripotency markers like OCT3/4,KLF4,SOX2,C-MYC,LIN28,NANOG,and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors,megakaryocytes,adipocytes,chondrocytes,and osteogenic cells. Thus,all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations. View Publication

Green tea is a popular drink consumed daily by millions of people around the world. Previous studies have shown that some polyphenol compounds from green tea possess anticancer activities. However,systemic evaluation was limited. In this study,we determined the cancer chemopreventive potentials of 10 representative polyphenols (caffeic acid,CA; gallic acid,GA; catechin,C; epicatechin,EC; gallocatechin,GC; catechin gallate,CG; gallocatechin gallate,GCG; epicatechin gallate,ECG; epigallocatechin,EGC; and epigallocatechin gallate,EGCG),and explored their structure-activity relationship. The effect of the 10 polyphenol compounds on the proliferation of HCT-116 and SW-480 human colorectal cancer cells was evaluated using an MTS assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with propidium iodide (PI)/RNase or annexin V/PI. Among the 10 polyphenols,EGCG showed the most potent antiproliferative effects,and significantly induced cell cycle arrest in the G1 phase and cell apoptosis. When the relationship between chemical structure and anticancer activity was examined,C and EC did not show antiproliferative effects,and GA showed some antiproliferative effects. When C and EC esterified with GA to produce CG and ECG,the antiproliferative effects were increased significantly. A similar relationship was found between EGC and EGCG. The gallic acid group significantly enhanced catechin's anticancer potential. This property could be utilized in future semi-synthesis of flavonoid derivatives to develop novel anticancer agents. View Publication

The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood,limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest,differentiation and cell death. Conversely,inactivation of the entire RB family (RB,p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells,mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally,loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells,uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells. View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

Heterogeneity is an often unappreciated characteristic of stem cell populations yet its importance in fate determination is becoming increasingly evident. Although gene expression noise has received greater attention as a source of non-genetic heterogeneity,the effects of stochastic partitioning of cellular material during mitosis on population variability have not been researched to date. We examined self-renewing human embryonic stem cells (hESCs),which typically exhibit a dispersed distribution of the pluripotency marker NANOG. In conjunction with our experiments,a multiscale cell population balance equation (PBE) model was constructed accounting for transcriptional noise and stochastic partitioning at division as sources of population heterogeneity. Cultured hESCs maintained time-invariant profiles of size and NANOG expression and the data were utilized for parameter estimation. Contributions from both sources considered in this study were significant on the NANOG profile,although elimination of the gene expression noise resulted in greater changes in the dispersion of the NANOG distribution. Moreover,blocking of division by treating hESCs with nocodazole or colcemid led to a 39% increase in the average NANOG content and over 68% of the cells had higher NANOG level than the mean NANOG expression of untreated cells. Model predictions,which were in excellent agreement with these findings,revealed that stochastic partitioning accounted for 17% of the total noise in the NANOG profile of self-renewing hESCs. The computational framework developed in this study will aid in gaining a deeper understanding of how pluripotent stem/progenitor cells orchestrate processes such as gene expression and proliferation for maintaining their pluripotency or differentiating along particular lineages. Such models will be essential in designing and optimizing efficient differentiation strategies and bioprocesses for the production of therapeutically suitable stem cell progeny. View Publication

A small molecule compound,JTP-74057/GSK1120212/trametinib,had been discovered as a very potent antiproliferative agent able to induce the accumulation of CDK inhibitor p15INK4b. To conduct its drug development rationally as an anticancer agent,molecular targets of this compound were identified as MEK1/2 using compound-affinity chromatography. It was shown that JTP-74057 directly bound to MEK1 and MEK2 and allosterically inhibited their kinase activities,and that its inhibitory characteristics were similar to those of the known and different chemotype of MEK inhibitors PD0325901 and U0126. It was further shown that JTP-74057 induced rapid and sustained dephosphorylation of phosphorylated MEK in HT-29 colon and other cancer cell lines,while this decrease in phosphorylated MEK was not observed in PD0325901-treated cancer cells. Physicochemical analyses revealed that JTP-74057 preferentially binds to unphosphorylated MEK (u-MEK) in unique characteristics of both high affinity based on extremely low dissociation rates and ability stabilizing u-MEK with high thermal shift,which were markedly different from PD0325901. These findings indicate that JTP-74057 is a novel MEK inhibitor able to sustain MEK to be an unphosphorylated form resulting in pronounced suppression of the downstream signaling pathways involved in cellular proliferation. View Publication

Aldehyde dehydrogenases (ALDHs),enzymes responsible for detoxification and retinoic acid biosynthesis,are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date,however,there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay,and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor,diethylaminobenzaldehyde (DEAB),together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition,the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore,a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation,suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity. View Publication

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus,many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study,we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells,which are shown by multiple marker or phenotypes of CSCs. Thus,these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus,further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer. View Publication

Metformin,the first-line drug for treating diabetes,inhibits cellular transformation and selectively kills cancer stem cells in breast cancer cell lines. In a Src-inducible model of cellular transformation,metformin inhibits the earliest known step in the process,activation of the inflammatory transcription factor NF-κB. Metformin strongly delays cellular transformation in a manner similar to that occurring upon a weaker inflammatory stimulus. Conversely,inhibition of transformation does not occur if metformin is added after the initial inflammatory stimulus. The antitransformation effect of metformin can be bypassed by overexpression of Lin28B or IL1β,downstream targets of NF-κB. Metformin preferentially inhibits nuclear translocation of NF-κB and phosphorylation of STAT3 in cancer stem cells compared with non-stem cancer cells in the same population. The ability of metformin to block tumor growth and prolong remission in xenografts in combination with doxorubicin is associated with decreased function of the inflammatory feedback loop. Lastly,metformin-based combinatorial therapy is effective in xenografts involving inflammatory prostate and melanoma cell lines,whereas it is ineffective in noninflammatory cell lines from these lineages. Taken together,our observations suggest that metformin inhibits a signal transduction pathway that results in an inflammatory response. As metformin alters energy metabolism in diabetics,we speculate that metformin may block a metabolic stress response that stimulates the inflammatory pathway associated with a wide variety of cancers. View Publication