Heterogeneic nature of adult cardiac side population cells

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, α-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2⁺ cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin⁻ cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin⁻ cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.     

Functional heterogeneity of side population cells in skeletal muscle

Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31(-)CD45(-) SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also some mesenchymal lineage markers. CD31(-)CD45(-) SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31(-)CD45(-) SP cells participate in muscle regeneration.     

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

Stem cell properties and the side population cells as a target for interferon-alpha in adult T-cell leukemia/lymphoma

The cancer stem cell theory suggests that chemoresistance and recurrence of tumors are often due to the similarity of stem cell properties between normal and cancer cells. Adult T-cell leukemia/lymphoma (ATLL) has poor prognosis, suggesting that ATLL cells possess common stem cell properties. We analyzed side population (SP), a characteristic stem cell phenotype, and CD markers in ATLL cell lines. We found that several lines contained SP with expressions of some hematopoietic stem cell markers. On the other hand, treatment with interferon (IFN)-α is sometimes effective in ATLL, particularly combined with other drugs. We examined its effect on ATLL cells and found that IFN-α significantly reduced the SP proportion. Moreover, CD25-positive cells and phosphorylation of STAT1/5 and ERK were upregulated during this process. These data suggest that their stem cell properties render ATLL cells therapy-resistant, and IFN-α exerts its clinical effect through a reduction of the SP cell population.     

Radioresistance mechanisms of side population cells in mouse melanoma cell line B16

As we showed earlier, side population (SP) cells are more resistant to low-LET radiation than the rest of mouse melanoma B16 cells (Matchuk et al., 2012). The goal of this study was elucidation of some mechanisms of radioresistance; therefore, we analyzed the SP and non-SP cell-cycle distribution, spontaneous and radiation-induced DNA double-strand breaks (the number of γ-H2AX foci), and intracellular NO concentration. The obtained results indicate that SP cells have a significantly lower number of double-strand DNA breaks after irradiation at a dose of 3 Gy than do non-SP cells (24.4 vs. 40.3, respectively, p < 0.05, Mann-Whitney U-test). The SP cells are more quiescent than are non-SP ones (the G₁/G₀-fraction is 85 vs. 39%, respectively, p < 0.01). Most non-SP cells are in the S or G₂/M phases (61%), which are believed to be rather radiosensitive. Thus, the difference between the SP and non-SP cell radiosensitivity can be partly explained by peculiarities of the cell cycle distribution. The NO concentration is 1.5 times higher in the SP than in the non-SP cells (p < 0.05); since it is known that NO inhibits apoptosis, being one of the mechanisms of genetic stability maintenance, that there is a higher number of the spontaneous double-strand DNA breaks in the SP cells is not surprising (p < 0.05). The above-given results to a certain extent explain the higher resistance of the SP cells to low-LET radiation in comparison with the non-SP cells. Further study of this problem may become the basis for development of tools to target SP cells and, eventually, for more effective treatment of oncological diseases.     

Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line

CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population ) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell-like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self-renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem-like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.     

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip^® data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.     

SNAIL- and SLUG-induced side population phenotype of HCT116 human colorectal cancer cells and its regulation by BET inhibitors

Epithelial-mesenchymal transition (EMT) is associated with cancer malignancies such as invasion, metastasis, and drug resistance. In this study, HCT116 human colorectal cancer cells were transduced with SLUG or SNAIL retroviruses, and EMT cells with mesenchymal morphology were established. The EMT cells showed a high invasive activity and resistance to several anticancer agents such as methotrexate, SN-38, and cisplatin. Furthermore, they contained about 1–10% side population (SP) cells that were not stained by Hoechst 33342. This SP phenotype was not stable; the isolated SP cells generated both SP and non-SP cells, suggesting a potential for differentiation. Gene expression analysis of SP cells suggested the alteration of genes that are involved in epigenetic changes. Therefore, we examined the effect of 74 epigenetic inhibitors, and found that two inhibitors, namely I-BET151 and bromosporine, targeting the bromodomain and extra-terminal motif (BET) proteins, decreased the ratio of SP cells to <50% compared with the control, without affecting the immediate efflux of Hoechst 33342 by transporters. In addition, compared with the parental cells, the EMT cells showed a higher sensitivity to I-BET151 and bromosporine. This study suggests that EMT development and SP phenotype can be independent events but both are regulated by BET inhibitors in SLUG- or SNAIL-transducted HCT116 cells.     

Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K(2)CO(3) just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C(18) 3.5 micrometer (150x4.6 mm) column (Waters) equipped with a NovaPak C(18) Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 degrees C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min-->(40:60) at 30 min-->(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH(2)PO(4) and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH(2)PO(4) and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r>0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.     

Cancer stem cell-like population is preferentially suppressed by EGFR-TKIs in EGFR-mutated PC-9 tumor models

Aims

Although the epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling systems synergistically regulate many essential developmental and regenerative processes in lung cancer, the mechanisms of their crosstalk remain poorly defined. Our study aimed to investigate an interaction between EGFR and the β-catenin signal.

Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods

The cancer stem cell (CSC) hypothesis has provided insights into the initiation and recurrence of brain tumor. Specific identification and targeted elimination of these CSCs within the tumor mass represents a promising therapeutic strategy for refractory brain tumors. In this study, we attempted to identify CSCs in the rat C6 glioma cell line by three different identification methods. It is interesting to note that single-cell clonal analysis showed most C6 cells are cancer stem-like cells with characteristics of self-renewal, multilineage differentiation potentials in vitro, and tumorigenic capacity in vivo. It is surprising to note that CD133 failed to identify the total cancer stem-like cell population in the C6 line, since both CD133 (+) and CD133 (-) C6 cells have cancer stem-like cell fractions. Moreover, Hoechst 33342 staining, which is used in flow cytometry to isolate the side population (SP), was found to be harmful to C6 cells. Therefore, CD133 (-) and non-SP C6 cells may also harbor cancer stem-like cells. These results imply the limitation of using current identification methods in C6 line and underscore the importance of defining the genetic and molecular basis of CSCs.     

The presence of a side population and its marker ABCG2 in human deciduous dental pulp cells

Stem cells have been identified using the DNA-binding dye Hoechst 33342 and flow cytometry (FCM) in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population cells in human deciduous dental pulp cells (DPCs). Flow cytometric identification revealed that 2% of human deciduous DPCs were SP cells and that this SP profile disappeared in the presence of verapamil. The SP marker ABCG2 protein was localized to DPCs in the cell membrane by immunofluorescence staining, and flow cytometric analysis demonstrated that 3.6% of DPCs were ABCG2-positive. Furthermore, quantitative real-time PCR proved that ABCG2 mRNA expression in DPCs isolated from human exfoliated deciduous teeth was higher than in DPCs from permanent teeth. Our findings demonstrate that DPCs from human exfoliated deciduous teeth contain a higher proportion of the SP phenotype than permanent teeth and that they may constitute a stem cell population.     

Kinetic analyses as a critical parameter in defining the side population (SP) phenotype

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.     

小鼠骨髓瘤中肿瘤干细胞的初步分析

探讨小鼠骨髓瘤(SP2/0细胞)中肿瘤干细胞存在与否。以克隆形成试验检测SP2/0细胞中具有形成克隆能力细胞的大体比例;采用BrdU标记滞留试验检测SP2/0细胞中含有DNA永生化链的细胞,即具有干细胞特性的细胞;检测SP2/0细胞中具有干细胞特性的SP细胞存在情况及其比例。结果显示,SP2/0细胞中有一部分细胞具有形成克隆的能力;SP2/0细胞中含有DNA永生化链的细胞;SP2/0细胞中存在SP细胞,其比例约为0.7%。而且SP2/0细胞中存在肿瘤干细胞。     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation. Margarita Salcedo and Nadège Bercovici both contributed equally to this work