Sphere-forming cell subpopulations with cancer ste

Background

Hepatocellular carcinoma(HCC)is the fifth most com-mon cancer and the third leading cause of cancer death worldwide[1,2].Its overall incidence remains alarmingly high in the developing countries and is steadily rising across most of the developed countries[1-3].Although the cytologic pathogenesis of HCC remains unclear,it has been proposed that only a small fraction of cancer cells with stem cell properties,named cancer stem cells (CSCs),is responsible for the initiation,progression, local and distant recurrence/metastasis of HCC,also for the failure of chemo-and radiotherapy.The clinical cor-ollary of this hypothesis has been extended to proposals to treat cancer by targeting the putative liver CSCs.

Numerous attempts have been made to identify cells with stem cell properties in established HCC cell lines. Different research groups have reported that liver CSC fractions could be successfully enriched by some cell sur-face phenotypes,specifically

CD133,CD90,CD44,

epithelial cell adhesion molecule(EpCAM),OV6and

CD13[4-9].Nevertheless,these proteins,which are

involved in embryonic and somatic stem cell function,

embryonic development,hepatocyte membrane transport

and growth control,have been demonstrated a relative

lack of sensitivity and specificity for identifying liver

CSCs[10-12].So far,no markers for putative liver CSCs

have yet been generally accepted,and further study is

needed to explore the isolation method for liver CSCs.

A major advance in adult stem cell research was

achieved in1996when it was discovered that the undif-

ferentiated multipotent neural cells could be grown and

maintained in suspension using the neurosphere assay *Correspondence:yinzfk@yahoo.com.cn

1Department of Molecular Oncology,Eastern Hepatobiliary Surgery Hospital,

Second Military Medical University,Shanghai,China

Full list of author information is available at the end of the article

Cao et al.BMC Gastroenterology2011,11:71

http://www.biomedcentral.com/1471-230X/11/71

©2011Cao et al;licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons

Attribution License(http://creativecommons.org/licenses/by/2.0),which permits unrestricted use,distribution,and reproduction in

any medium,provided the original work is properly cited.[13].Then anchorage-independent sphere culture of stem cells was instrumental in the study of adult stem cells including the nerve,prostate and mammary stem cells[14-17].Recently,as a functional approach,sphere formation is particularly useful to enrich the potential CSC subpopulations when the specific CSC makers have not been defined as the case for most CSCs[18-25]. However,there have been few reports for sphere culture in liver cancer.Therefore,the present study intends to establish an alternate approach to isolate,identify and characterize liver cancer cell subsets with CSC properties. Methods

Cell lines and sphere culture

Human hepatoma cell lines,PLC/PRF/5and HepG2,were obtained from the Cell Bank of Chinese Academy of Sciences(Shanghai,China).MHCC97H was obtained from the Liver Cancer Institute,Zhongshan Hospital, Fudan University(Shanghai,China).All of the cells were maintained as a monolayer in high glucose DMEM with 10%fetal bovine serum(FBS),100IU/ml penicillin G and 100μg/ml streptomycin at37°C in a humidified5%CO2 incubator.Cells were collected and washed to remove serum,then suspended in serum-free DMEM/F12supple-mented with100IU/ml penicillin,100μg/ml streptomy-cin,20ng/ml human recombinant epidermal growth factor(hrEGF),10ng/ml human recombinant basic fibro-blast growth factor(hrbFGF),2%B27supplement without vitamin A,1%N2supplement(Invitrogen,Carlsbad,CA, USA).The cells were subsequently cultured in ultra low attachment6-well plates(Corning Inc.,Corning,NY, USA)at a density of no more than5,000cells/well. Sphere passage and sphere formation assay

The spheres were collected by gentle centrifugation,then dissociated with trypsin-EDTA and mechanically dis-rupted with a pipette.The resulting single cells were then centrifuged to remove the enzyme and re-suspended in serum-free medium allowed to re-form spheres.The spheres should be passaged every5-8days before they reached a diameter of100μm.The dissociated single sphere-forming cells were also diluted to a density of 500cells/ml.Then,the2μl/well diluted cell suspension was plated to ultra low attachment96-well plate(Corning Inc.,Corning,NY,USA),and150μl of serum-free med-ium was added.The wells with only one cell were marked and observed everyday.

Colony formation assay

The PLC/PRF/5spheres were enzymatically dissociated as described above.Trypan blue staining was used to determine cell viability,and more than95%of cells with viability were acceptable for the following experiments.The single cells were seeded in DMEM with10%FBS at a density of2000cells/well on6-well plates that were pre-coated with Matrigel(BD Biosciences,San Jose,CA, USA).After7days,the colony formation ability was assessed by counting the number of colonies(>70cells) under a microscope after crystal violet staining(Sigma-Aldrich,St.Louis,MO,USA).Representative views were photographed.The parental cells were plated at the same density as the control.

Chemotherapy sensitivity assays

The sensitivity of the PLC/PRF/5parental and sphere-forming cells to chemotherapeutic drugs was measured by MTT assay.Briefly,cells were seeded in96-well plates that were precoated with Matrigel,and various concentrations of cisplatin(Sigma-Aldrich)were added at the beginning,co-incubated for12h or24h.After changing to fresh medium without cisplatin,cells were cultured for another72h.The MTT reagent(Sigma-Aldrich)was then added to each well according to the manufacturer’s instructions.Absorbance was measured at490nm.

Immunofluorescent staining

Cells were fixed in4%paraformaldehyde and blocked with normal goat serum.The primary antibodies, including mouse anti-human OV6(R&D Systems Inc., Minneapolis,MN,USA),mouse anti-human CD133and rabbit anti-human CD44(Santa Cruz Biotechnology, Inc.Santa Cruz,CA.USA)were added and incubated overnight at4°C.After washing3times with PBS,the goat anti-mouse IgG and goat anti-rabbit secondary antibodies conjugated with Cy3,FITC or TRITC(Jack-son ImmunoResearch Laboratories Inc.,West Grove, PA,USA)were added and incubated at room tempera-ture for1h.Cells were then counterstained with DAPI (Sigma-Aldrich)and the images were captured using an Olympus-IX71fluorescent microscope(Olympus Inc., Center Valley,PA,USA).

In vivo tumorigenicity experiments

All mice were cared for in accordance with institutional guidelines.The PLC/PRF/5parental and the third,sixth and ninth passages of sphere-forming cells were used in tumorigenicity experiments.Trypan blue staining was used to assess cell viability,and various numbers of viable single cells were subcutaneously injected into5-week-old NOD/SCID male mice(Shanghai Laboratory Animal Center,Chinese Academy of Sciences,Shanghai, China)in serum-free DMEM/Matrigel(1:1)using100μl microsyringe.Mice were killed at8weeks after cell injection,then the tumors were harvested for further examination.Real-time PCR microarray analysis

Human Stem Cell RT2Profiler™PCR Array(PAHS-405A,SABioscience,USA)was done according to the manufacturer’s instructions.Briefly,RNA samples from both the PLC/PRF/5parental cells(as control)and the tertiary passage spheres were prepared.After removal of contaminating DNA from RNA preparations,total RNA samples were cleaned up and the resulting RNA were assessed for both yield and quality.After the first strand cDNA was synthesized,real-time PCR was performed. The data were analyzed using theΔΔCt method. Western blotting analysis

Quantified protein lysates were resolved on SDS-PAGE gels,transferred onto a polyvinylidene difluoride mem-brane(Millipore,Billerica,MA,USA),and immuno-blotted with the primary antibodies against EpCAM, CD133,activated Notch1,Ep300(Abcam,Cambridge, MA,USA),DTX1,Oct3/4(Santa Cruz,CA,USA),or OV6,CD44(R&D Systems Inc.,MN,USA),followed by incubation with the horseradish peroxidase-conjugated secondary antibody.The blots were visualized using a supersignal west femto maximum sensitivity substrate kit(Pierce,Waltham,MA,USA).GAPDH was used as a loading control.

Statistical analysis

All values in the figures and text were showed as means±SD.Statistical analyses were performed using the SPSS statistical software package(SPSS/PC+,SPSS Inc.,Chi-cago,IL,USA).Any significant differences among mean values were evaluated by the student’s t test.A two-sided P<0.05was accepted as significant.

Results

Hepatoma cells could form anchorage-independent,self-renewing spheres

The hepatoma cells were plated in stem cell conditioned culture medium in6-well plates at a density of5,000 cells/well which allowed for the formation of colonies separated from each other.In this condition,cells grew as nonadherent,three-dimensional sphere clusters,called spheres.Figure1A showed anchorage-independent spheres formed by the HepG2,MHCC97H and PLC/ PRF/5cells.After5to8days,when the spheres grew to 70to100μm in diameter,they were passaged and the

Hepatoma cells formed the anchorage-independent,self-renewing spheres.(A)Hepatoma cell lines,including could form the anchorage-independent3-D spheres in stem cell conditioned culture medium(200×).(B)Generation PLC/PRF/5cell.The propagation of a single cell cultured in a96-well dish was recorded at day1,3,5,7,9andsingle cell from spheres could propagate to form new spheres again.A key property of all normal and cancer stem cells is their unique ability to self-renew.One of the methods to determine the self-renewal capacity of sphere-forming cells is to test their capability of serial passage.The PLC/PRF/5,HepG2and MHCC97H spheres had been serially passaged for more than12gen-erations,indicating their self-renewal capability in vitro. To corroborate the finding that a sphere could be gen-erated from a single cell,one PLC/PRF/5cell per well was plated to a96-well plate and the wells with one cell were visualized everyday.Figure1B showed the process of single PLC/PRF/5cell forming a sphere.

Sphere-forming cells proliferate extensively in vitro

We compared the proliferative ability of the tertiary pas-sage PLC/PRF/5spheres and its parental cell line using clonogenicity assay.Both of them were adherently plated and were alive,but were unable to form comparable colonies.The sphere-forming cells proliferated signifi-cantly faster and induced bigger and greater numbers of tumor colonies than the parental cells.Based on count-ing the number of colonies per2,000seeded cells,it was 175.67±30.07/381.00±61.02(P=0.006)for the paren-tal/sphere-forming PLC/PRF/5cells(Figure2A).The sphere-forming cells were capable of extensive prolifera-tion,indicating that the sphere-forming cells could play an important role in the maintenance of tumor growth. Sphere-forming cells possess the ability of resistance to conventional chemotherapy in vitro

HCC cells are commonly resilient to chemotherapy.It is speculated that cancer stem or progenitor cells in HCC are more resistant to conventional chemotherapy contri-buting to recurrence.To examine whether the self-renewing sphere-forming cells possess a hypothesized CSC chemoresistant property,the sensitivity of the PLC/PRF/5parental cells versus the tertiary passage sphere-forming cells to cisplatin was assessed.The sur-vival rates of sphere-forming cells were higher under the treatment of3μg/ml,5μg/ml and9μg/ml cisplatin for 12h,being1.4-fold,1.9-fold,1.8-fold,respectively,com-pared with the parental PLC/PRF/5cells(P<0.01), whereas,under the treatment for24h,the relative sur-vival rates were increased to1.5-fold,2.1-fold and2.3-fold respectively(Figure2B,P<0.01).

We also tested the sensitivity of sphere-forming cells to other4drugs in addition to cisplatin.The PLC/PRF/ 5sphere-forming cells exhibited general resistance to5-Fu,gemcitabine,mitomycin and sorafenib in the treat-ment of36h.Compared with the PLC/PRF/5parental cells,the survival rates of PLC/PRF/5sphere-forming cells were higher under200μmol/L,400μmol/L of5-Fu(1.60-fold,1.98-fold respectively,P<0.01);5mmol/L,10mmol/L of gemcitabine(1.99-fold,2.49-fold respectively,P<0.01);0.5μmol/L,1.0μmol/L of mito-mycin(1.24-fold,2.33-fold respectively,P<0.01);and6μmol/L,12μmol/L of serafenib(2.07-fold,15.21-fold respectively,P<0.01).The results support a role for these sphere-forming cells in HCC chemoresistance, which may explain why current therapies fail to eradi-cate progenitors and prevent tumor re-growth.

Sphere-forming cells exhibit high tumorigenicity in vivo To confirm that the sphere-forming cells exhibit greater tumor initiating capability,NOD/SCID mice were trans-planted with varying amounts of the PLC/PRF/5sphere-forming cells ranging from the amount that is unable to initiate tumor growth to the amount that always initiates tumor formation.The PLC/PRF/5parental cells were operated as controls.As few as500sphere-forming cells were sufficient for tumor development,whereas,at least 2×105parental cells were necessary to consistently generate a tumor in the same model,and not to men-tion,requiring a longer period of time(Table1Figure 3).The tumor nodules formed by the PLC/PRF/5 sphere-forming cells displayed similar histology to that by the parental cells.To inspect whether the tumor initiating capability could be decreased as the spheres were passaged,we also compared the tumorigenicity of different generations of spheres.The results showed that both the tumor initiating capability and phenotypic appearance were similar for the3th,6th and9th genera-tions of sphere-forming cells(Table1).The tumorigenic efficacies of three cell lines HepG2,PLC/PRF/5and MHCC97H were also compared in nude mice(Addi-tional file1Figure S1).The results suggested that the tumorigenic efficacies of sphere-forming cancer cells were enhanced compared with the parental cells,and the volumes of tumors were positively correlated with malignant grade of the cell lines(malignant grade HepG2Sphere-forming cells overexpress liver CSC related proteins and the CSL-independent Notch signaling pathway might play a role in liver CSCs

To date,anti-OV6,a monoclonal antibody raised against cells isolated from carcinogen treated rat liver[26], remains the best available marker of hepatic stem cells [27],even though it also reacts with bile duct epithelium in rats and humans[28].Besides,Yang et al reported that the OV6+liver cancer cells may represent a potential stem/progenitor-like cell population[8].Immunofluores-cent staining for OV6showed that most of the PLC/PRF/2Sphere-forming cells proliferated extensively and possessed resistance ability to conventional chemotherapeutics Comparison of in vitro clonogenecity of the PLC/PRF/5parental cells and sphere-forming cells.Cells were plated at a density of2,000 6-well plates and cultured for1week.At the end,cells were stained with crystal violet,photographed,and analyzed for their proliferation efficiency.Each experiment was performed in triplicate,and the representative examples were shown(Columns,mean;bars,SD;*,P<0.01).

PLC/PRF/5sphere-forming cells showed a drug resistance phenotype.The parental cells and sphere-forming cells were treated with at the beginning of plating for12h or24h and5-Fu,gemcitabine,mitomycin and sorafenib for36h.Cell survival was determined assay(**,P<0.01).5sphere-forming cells are OV6positive(Figure4A). Then,liver CSCs related proteins,including Oct3/4, OV6,EpCAM,CD133and CD44were examined by Western blotting.The results displayed that all of them were significantly increased as compared to the parental cells(Figure4B),denoting that the PLC/PRF/5spheres possess stem cell-like properties.We also compared the expression of candidate CSC markers CD133and CD44 between the parental and sphere-forming cells of HepG2 and MHCC97H by immunofluorescent labelling.It was found that CD44expression was obviously enriched in HepG2and MHCC97H sphere-forming cells compared with their parental cells(Additional file2,Figure S2).

To further explore the biological properties of liver CSCs,the Stem Cell RT2Profiler™PCR array was per-formed.This array profiles the expression of84genes related to the identification,growth and differentiation of stem cells.Among the significantly distinguishing genes, we noted the DTX1and Ep300,CSL-independent Notch signaling pathway related genes,were4.24-fold and2.36-fold,respectively,more abundant in the spheres than those in the control.Consistent results were confirmed by Western blotting(Figure5A).To determine the role of CSL-independent Notch signals,blocking Notch path-way was performed by a gamma-secretase inhibitor MRK003.The secondary dissociated PLC/PRF/5sphere-forming cells were treated with10μM MRK003or DMSO control for7days.The inactivation of Notch1and down-regulations of downstream target genes DTX1 and Ep300were confirmed by Western blotting(Figure 5B).The sphere formation ability of the MRK003-treated groups was significantly inhibited in comparison to the DMSO-treated controls(Figure5C).The results indi-cated that the CSL-independent Notch signaling pathway might play an important role in liver CSCs and MRK003 could partly eliminate the stem-like cells.

Discussion

There are two classical models of carcinogenesis[29,30]. One is the stochastic model,which is based on the belief that most of tumor cells are capable of extensive prolifera-tion and contribute substantially to tumor maintenance; carcinogenesis could results from the random mutations and the subsequent clonal selections.The other is the hierarchical model,which is based on the belief that there are hierarchical differences among tumor cells,and only a small number of specific cells capable of extensive prolif-eration can contribute to carcinogenesis.The discovery of CSCs in solid tumors strongly supports the hierarchical model.The CSC hypothesis considers that CSCs subsets are located in the top of the hierarchical structure of tumor cells and directly affect the organization and con-struction of lower hierarchical cells.Therefore,the identi-fication of tumorigenic liver CSCs could provide new insight into the HCC tumorigenic process and possibly bear great therapeutic implications.

Table1Tumorigenicity experiments of PLC/PRF/5sphere-forming cells and parental cells in NOD/SCID mice Cell type Cell numbers injected Tumor incidence†Latency(days)‡Sphere-forming cells of the3rd generation2×1020/3-

5×1021/335

1×1033/323

2.5×1033/323

5×1033/320

1×1043/316

5×1042/29

1×1052/27

2×1052/27 Sphere-forming cells of the6th generation2×1020/3-

5×1022/349

1×1033/328 Sphere-forming cells of the9th generation2×1020/3-

5×1021/340

1×1033/330 PLC/PRF/5parental cells5×1040/3-

1×1050/3-

2×1052/330

5×1053/316

1×1062/29

†The number of tumors detected/number of injections.

‡Approximate number of days from tumor cell injection to appearance of a tumor.

Usually,the isolation and identification of liver CSCs involve two types of methods:one is based on the sorting of side population(SP)cells that can exclude the hoechst 33342dye[31].However,Hoechst33342is cytotoxic; consequently,SP cells are protected by their membrane transport properties,whereas unprotected non-SP cells suffer toxicity and are unable to grow.Thus the differing tumor-initiation abilities of SP and non-SP cells are most likely due to an artifact of Hoechst33342toxicity,rather than due to intrinsic stem-cell properties[32].The other type includes the fluorescence activated cell sorting (FACS)and the magnetic activated cell sorting(MACS), which are based on cell surface markers.The proposed markers for liver CSCs include CD133,CD90,CD44, CD13,EpCAM and OV6,on the basis of the hypothesis that CSCs are originated from somatic stem cells and

Sphere-forming cells exhibited high tumorigenicity in vivo.(A)The representative examples of xenograft tumors injection with the PLC/PRF/5parental cells and sphere-forming cells.(B)The top row shows the nodules formed by PLC/PRF/5parental cells,separately.The bottom row shows the nodules formed by injecting5×102,1×103

PLC/PRF/5sphere-forming cells,separately.(C)H&E staining revealed that the histological features of xenograft sphere-forming cells were similar to those induced by the parental cells.accordingly express the same surface markers[33,34]. Although they have been reported to be used to enrich CSC fraction,their sensitivity and specificity for identify-ing liver CSCs are being challenged.For example,Kimura et al reported CD133+fraction in Hep3B and Huh7were 16.8%and2.7%,respectively[7],whereas some other groups reported more than90%in Hep3B and60%in Huh7[5,6].In Huh7the CD13-positive cells typically existed in a CD133strong fraction,but in PLC/PRF/5the CD13-positive cells were CD133-negative[9].Some researchers indicated that different culture conditions and differentiated degree of the cells,especially the latter, were important factors.The roles of these phenotypes in defining functionally distinct populations of cells from progenitor to differentiated hepatocytes need to be sys-temically studied.

Recently,sphere culture has been increasingly used as a method for enriching stem cells which relies on their property of anchorage independent growth.Researchers have reported the application of sphere culture to iso-late,enrich,maintain or expand potential CSC subpopu-lations from various types of cancers[18-25].The sphere-forming cells from primary tumors,such as breast cancer and ovarian cancer,showed stem-like properties and expressed their CSC markers[19,23].It is generally agreed that,like all stem cells,the tumor sphere-forming cells are capable of proliferation,self-renewal and possess higher tumorigenicity.Using neural crest stem cell conditions and sphere formation system, Hansford et al[35]for the first time successfully expanded tumor cells both from low-risk neuroblasto-mas and from the bone marrow metastases of high-risk tumors.The latter formed metastatic tumors in a mur-ine xenograft model with as few as10cells and could also be serially passaged[35].To our knowledge,there have been few reports on the isolation and long-term propagation of liver CSCs by the method of sphere culture.

Figure4Sphere-forming cells overexpressed liver CSC related proteins and downstream proteins of the CSL-independent Notch pathway.(A)The PLC/PRF/5sphere-forming cells expressed the hepatic stem cell maker OV6,as observed under fluorescence microscopy. Nuclei were stained with DAPI(400×).(B)Western blotting assay showed the liver CSC related proteins(Oct3/4,OV6,EpCAM,CD133and CD44) and the relative band intensities were calculated by densitometry and normalized to the loading control GAPDH.In the present study,using stem cell conditioned culture system,we tested three human hepatoma cell lines,PLC/ PRF/5,MHCC97H and HepG2.Cells are plated at a low density(<5000cells/well in6-well plate)to avoid sponta-neous cell aggregation.The three cell lines could form clo-nal nonadherent3-D spheres,and without exceptions, could also be serially passaged.We evaluated the PLC/ PRF/5sphere-forming cells for their stemness characteris-tics.It has been showed that they were capable of self-renewal,proliferation,drug resistance,and overexpressing liver CSC related proteins.Xenotransplantation is the gold standard for evaluating tumorigenicity of tumor cells.We tested the third,sixth and ninth generations of the PLC/ PRF/5sphere-forming cells for their tumor initiating cap-ability.It was demonstrated that as few as500cells from the PLC/PRF/5spheres were able to form a tumor when subcutaneously injected into NOD/SCID mice,while2×105parental cells were needed.This was400times higher than that of sphere-forming cells.Moreover,the tumor initiating capability was not decreased as the spheres were passaged.Similar CSC properties of self-renewal,strongly proliferation,drug resistance and tumorigenicity are also found in the MHCC97H and HepG2spheres.Indeed,the HepG2parental cells at106cells/mouse could not form

CSL-independent Notch signaling pathway might play a role in liver CSCs and MRK003could partly eliminate downstream proteins levels of the CSL-independent Notch pathway(DTX1and Ep300)were significantly increased compared with the parental cell line.GAPDH was used as a loading control.(B)10μM MRK003could reduce downstream components of CSL-independent Notch signaling pathway DTX1and Ep300were consequently decreased. dissociated PLC/PRF/5sphere-forming cells were treated with10μM MRK003or DMSO control for7days.The sphere MRK003-treated groups were significantly inhibited in comparison to DMSO-treated controls.

visible xenografts in nude mice,but its sphere-forming cells at the same amount of cells could form xenograft tumors,suggesting the tumorigenic efficacies of sphere-forming cells were enhanced compared with the parental cancer cells.

To further explore the CSC properties of sphere-form-ing cells,we examined the sensitivity of sphere-forming cells to chemotherapeutics and the expression of candidate CSC markers.The PLC/PRF/5sphere-forming cells exhib-ited general resistance to cisplatin,5-Fu,gemcitabine,mitomycin and sorafenib,and showed higher survival per-centages compared with its parental cells.Synchronously,we found that CD44expression was obviously enriched in HepG2and MHCC97H sphere-forming cells compared with their parental cells.CD44is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins,normally takes part in cell-cell and cell-matrix adhesion interactions,which is involved in cancer cell migration,proliferation and metastasis.Accordingly,CD44expression enrichment in sphere-forming cells may account for their increased survival ability and tumorigeni-city.Therefore,we propose that the nonadherent tumor spheres cultured in serum-free condition possess liver CSC properties.This long-term culture system may also provide the means of further purifying and functionally characterizing the biological properties of the liver CSC fraction,with the goal of developing new therapeutic stra-tegies directed specifically against liver CSCs.

Accumulating evidence has been established that the Notch signaling pathway plays vital and universal roles not only in cell differentiation,embryonic development and tissue self-renewal,but also in pathogenesis of some types of human cancers and genetic disorders.Recent advance-ments have further revealed that the Notch signals pro-duce a marked effect either in stem cells or CSCs.The activated Notch signals can inhibit hematopoietic stem cell differentiation and maintain their pluripotency [36,37],and maintaining the stem cell population in several solid tissue types,including several neuroectodermal tissues [38].Only when the Notch signals are activated the cancer stem cell activity could be enhanced to promote intestinal tumor formation [39].Generally,the Notch signaling path-way is mediated in two different pathways.One is through CSL-DNA binding proteins;the other is the CSL-indepen-dent pathway.DTX1(Deltex-1)is an important transcrip-tional regulator that is downstream of the Notch receptor in the CSL-independent Notch signaling pathway [40].Ep300,also known as p300,is a transcriptional co-activa-tor protein.It functions as a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differ-entiation.It has been reported that Ep300can work as a transcriptional co-activator of DTX1.Yamamoto et al [41]reported that DTX1inhibited the transcriptional

activation of the neural-specific helix-loop-helix type tran-scription factor MASH1by binding to Ep300.This mechanism is likely responsible for the differentiation inhibition of neural progenitor cells.In our study,the result of stem cell microarrays showed that DTX1and Ep300were highly expressed in liver cancer stem-like cells.This was further confirmed by Western blotting.Although the molecular mechanism and function of the CSL-independent Notch signaling pathway have not been elucidated,and little has been known about its involve-ment in HCC,we suppose that the CSL-independent Notch signaling pathway play an important role in the dif-ferentiation and propagation of liver CSCs.

Conclusions

In summary,the study demonstrated that the nonadher-ent tumor spheres from human hepatoma cell lines which are cultured in stem cell conditioned medium possess liver cancer stem cell properties,and the CSL-independent Notch signaling pathway may play a role in the differentiation and propagation of liver CSCs.Additional material

Acknowledgements

This study was supported by National High-Tech Research and Development Program of China (No.2007AA02Z461),China National Key Projects for Infectious Disease (2008ZX10002-021)and National Natural Science Foundation of China (No.30672002,30801342).

Author details

1

Department of Molecular Oncology,Eastern Hepatobiliary Surgery Hospital,Second Military Medical University,Shanghai,China.2Department of Hepato-Biliary-Pancreato-Vascular Surgery,the First affiliated Hospital of Xiamen University,Xiamen,China.

Authors ’contributions

Lu C conceived the study and participated in the study design,performance,coordination and manuscript writing.YMZ participated in the study performance and writing.MCW participated in the study design and

coordination.ZFY participated in the study design,coordination and writing.BBZ,Jian L,WX,RXZ,Jing L,YZ,Lei C and HHQ performed the research.All authors have read and approved the final manuscript.Competing interests

The authors declare that they have no competing interersts.Received:21October 2010Accepted:14June 2011Published:14June 2011

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Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-230X/11/71/prepub