Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment

Cell lines and reagents

Human acute monocytic leukemia cell line THP-1, human nasopharyngeal cancer cell line CNE-2 and human neuroblastoma cell line SK-N-CH were obtained from American Tissue Culture Collection (Rockville, MD, USA).

Antibodies including CD133 and CD133-PE were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). All the other reagents were purchased from Sigma (St Louis, MO, USA) unless otherwise noted.

Single clone selection

Cells were seeded in a 24-well plate with 1 cell/ml and single cell clones were isolated from wells with only a solitary colony. Selecting 50 single cell clones were subjected to expansion culture until sufficient amounts of cells were obtained. The 50 single cell clones were detected by the flow cytometric analysis of SP and non-SP distribution respectively. One CNE-2 single clone is named CNE-2-2 with little SP phenotype.

Macrophage-conditioned medium

THP-1 cells at a density of 0.5 × 10⁶/ml were cultured in RPMI 1640 (Invitrogen, San Diego, CA, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA) and 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h at 37°C in a humidified atmosphere of 5% CO₂ incubator. After nonadherent cells were discarded, remaining cells were washed twice in PBS and once in RPMI 1640 with 15-min incubation at 37°C. These PMA induced macrophage-like cells were cultivated for 24 h with fresh RPMI 1640 containing 10% FBS (8 ml per 75-cm³ flask). The supernatant after culture was subsequently filtered and used as the macrophage-conditioned medium.

Cell culture

NPC Cells were cultured in a 25-cm² tissue culture flask (1.2 × 10⁴cells/cm²) in RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin for 24 h. Cells were washed with PBS and the conditioned medium produced by PMA-activated THP-1 cells was added. After indicated cultivation days, nonadherent cells were gently washed away with PBS and adherent cells were harvested with 0.05% trypsin-EDTA for test.

Flow cytometry

To identify the SP cells in the cancer cells, we selected the single clone cells as described above. The cells were removed from the culture dish with trypsin/EDTA (Invitrogen) and resuspended at 10 cells per ml RPMI 1640 supplemented with 2% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were preincubated in a 1.5-ml Eppendorf tube for 90 min at 37°C with 2.5 μg/ml Hoechst 33342 dye (Invitrogen), either alone or in combination with 50 M verapamil, an inhibitor of some verapamil-sensitive ABC transporters [29]. Finally, the cells were counterstained with 1 μg/ml propidium iodide to label dead cells. Cells were analyzed in a FACSVantage fluorescence-activated cell sorter (Becton Dickinson). Propidium iodide-positive dead cells (<15%) were excluded from the analysis.

The non-SP Cells from the single clone detected by FACS were seeded into a 6-well plate at 1 × 10⁵ cells/well and incubated with conditioned medium at 37°C. After 24 h of incubation, cells were collected with trypsin/EDTA. SK-N-CH cells were incubated with CD133-PE antibody for 10 minutes at 4°C before FACS analysis. CNE-2-2 single-cell suspension was fixed in ice-cold 70% ethanol at -20°C for 16 h, labeled with propidium iodide (50 μg/ml) for at least 15 min in dark and analyzed directly on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Immunofluorescence staining

Cells grown on cover slips were washed in cold PBS and fixed in 2% paraformaldehyde-PBS for 20 min and permeabilized in 0.5% Triton X-100 in PBS for 10 min at 4°C. The cells were incubated with 1% BSA for 30 min at room temperature (RT) to block nonspecific binding before the primary antibody reaction. Cells were incubated with the primary antibodies to CD133 for 1 h at RT, and then a specific FITC conjugated secondary antibody for 1 h. Cells were counterstained with DAPI (1 μg/μl), and examined using a fluorescence microscope (Olympus BX51).

Transwell migration assay

CNE-2-2 cells were starved overnight in assay media (RPMI 1640 with 1% FBS). The top chamber of a 24-well transwell plate (Corning Inc.) was pretreated with 1% Matrigel™ (BD Biosciences, San Jose, CA, USA) in PBS and incubated 1 h at RT. Cells (10⁵/ml) were added to the top chambers of the plates. Media with or without activated macrophage-like cells was added to both chambers. After overnight incubation, top cells were removed and bottom cells were fixed and stained with DAPI (5 μg/ml) to visualize nuclei. The number of migrating cells in five fields was counted under fluorescence microscope, and the mean for each chamber was determined.

Nucleic acid preparation

All of the cells were snap-frozen in liquid nitrogen and stored at -80°C. The tested cells were lysed in Trizol Reagent (Invitrogen, CA, USA), and total RNA was isolated according to the manufacturer's instructions. mRNA of the conditioned culture non-SP single cell clone and non-conditioned culture cells was purified from total RNA using the Oligotex mRNA Spin-Column kit (Qiagen, Hilden, Germany).

Suppression subtractive hybridization (SSH)

SSH was performed between conditioned culture non-SP single cell clone and non-conditioned culture cells using the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer's recommendations. In the subtraction, conditioned culture non-SP single clone cells were used as a tester and non-conditioned culture single clone cells were used as a driver. Double-strand cDNA was synthesized from poly (A)+ RNA and blunted according to the kit manual. Two tester populations were created with different adaptors, but no adaptors with the driver.

Differential Screening

120 individual recombinant clones were picked and used as templates for PCR amplification using vector primers M13F and M13R. Each PCR product (about 1 μl) was transferred to a nylon membrane (Hybond-N, Amersham Pharamacia Biotech, Uppsala, Sweden). For denaturing purposes, the membrane was submerged for 2 - 3 min in turn on the three pieces of Whatman 3 MM paper saturated with 1.5 M NaCl and 0.5 M NaOH, 1.5 M NaCl and 0.5 M Tris-HCl (pH 8.0), 0.2 M Tris-HCl (pH 7.5) and 2 × SSC buffer, respectively. Subtracted and unsubtracted PCR products were used as probes labeling with digoxigenin-dUTP at 37°C overnight according to the DIG High Prime Labeling Kit protocol (Roche Molecular Biochemicals, Mannheim, Germany). The dot blot hybridization was performed overnight at 42°C using the DIG Easy Hyb system (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's instructions. The blots were washed at RT twice at low stringency (2 × SSC, 0.1% SDS) for 5 min and then at 68°C twice at high stringency (0.1 × SSC, 0.1% SDS) for 15 min. Hybridized probes were subsequently detected by alkaline phosphatase-conjugated anti-DIG antibody and the CSPD chemiluminescent detection system (Roche Molecular Biochemicals, Mannheim, Germany).

Sequencing and Sequence analysis

Nucleotide sequences were compared with the GenBank using the BLASTn programs from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST/ and the BLASTx for the protein. Protein patterns were then identified by searching the PROSITE database http://www.expasy.ch/tools/ and Pfam database http://pfam.sanger.ac.uk/.

The DNA was sequenced from both strands on an ABI PRISM 377 Genetic Analyzer (PE Biosystems, Foster City, CA, USA).

Northern blotting analysis

Digoxigenin-labelled DNA probes were applied for Northern blotting analysis. Oligonucleotide primers for the DNA probes were designed based on the known sequences (Table 1). The programs were as follows: 94°C for 5 min, followed by 35 cycles of 30 sec at 94°C, 30 sec at 56°C, and 90 sec at 72°C, and a final polymerization step at 72°C for 10 min, and then the PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The fragments were labeled with digoxigenin-dUTP as above.

Each set of total RNA was extracted from conditioned culture non-SP single clone cells and non-conditioned culture single clone cells described above. Total RNA was quantified on a spectrophotometer at 260 nm. Aliquots containing 10 μg of total RNA corresponding to each tissue were mixed with glyoxal and DMSO and heated at 50°C for 30 min and then placed on ice over 1 min. The treated RNAs were then separated through a 1% agarose (Seakem) gel. The intensity of ethidium bromide-stained rRNA bands was examined visually to ensure that equal amounts of total RNA were loaded onto the gels. The RNA was then transferred to a nylon membrane (Hybond-N, Amersham Pharamacia Biotech, Uppsala, Sweden), and UV cross-linked. Hybridization was performed overnight at 50°C, washed at RT twice at low stringency for 5 min and then at 55°C twice at high stringency for 15 min and were detected as above.

Identification of SP cells in the CNE-2 single clones

To determine whether different percentages of SP cells were contained in CNE-2 cells, we screened the CNE-2 cells by single clone selection. We stained the single clone cells respectively with Hoechst 33342, which is actively extruded by verapamil-sensitive ABC transporters. The SP cells from representative single clone CNE-2 ranged from 0.1% to 73.7% as estimated by FACS analysis (Fig. 1A, B and 1C). In each case, by definition, the number of SP cells was decreased greatly by treatment with verapamil (Fig. 1 bottom panel). We could detect little SP cells in just a single clone cell (Fig. 1C, clone 2). Thus, most of the CNE-2 cancer cell clones contained a small or big component of SP cells. Our study is focus on whether microenvironment might have an effect on the percentage of SP cell in CNE-2, i.e, whether it play a role in the cancer stem cell pool, so the further experiments were mainly performed the human CNE-2 single clone (namely CNE-2-2) with little SP phenotype.

We cultured the CNE-2-2 clone with conditioned medium and carried out FACS analysis for the presence of SP. As shown in Fig. 1D, the CNE-2-2 cell clone contained 9.8% SP cells after cultured with macrophage-like cells, whereas the control CNE-2-2 cells only contained 0.1% SP. The data therefore suggested that macrophage might be responsible for maintaining the CNE-2 cancer stem cell pool.

Validation SP alteration in neuroblastoma SK-N-CH cells

To further confirm the above phenomena, we selected another cell line, the human neuroblastoma cell line SK-N-CH in which we could barely detect the presence of SP and the expression of CD133, one of the markers of neuroblastoma cancer stem cells. After cultured with activated macrophage medium, SK-N-CH cells were stained with CD133 antibody for FACS analyses. As shown in Fig. 2A, the condition cultured SK-N-CH cells contained 4.5% cancer stem cells, whereas the control SK-N-CH cells only contained 0.6%.

Furthermore, we examined the expression of CD133 on SK-N-CH cells in response to the conditioned culture induced by macrophage-like cells with immunofluorescence. The fluorescence density of CD133 positive cells was evidently stronger than that in control (Fig. 2B). These results allowed us to conclude that human macrophages did play a role in maintaining cancer stem cell pool at least in several solid tumors.

G1-S cell cycle and migration of CNE-2 single clone cell affected by conditioned culture

Next, we analyzed cell cycle distribution in CNE-2-2 cells with conditioned medium. Cell cycle profiling of CNE-2-2 cells revealed that the conditioned culture induced reduction of the G1-S transition at 24 hours (Fig. 3A and 3B). By contrast, Control experiments showed no cell growth arrest features at 24 hours.

Following culture with activated macrophage-like cells, derived from human THP-1 cells, in a Transwell Boyden chamber system without cell-to-cell contact, CNE-2-2 cells resulted in a robust increase (>4 folds) in cell motility after 18 hours, whereas in control the normal culture conditions did not enhance the migration ability of CNE-2-2 cells (Fig. 3C and 3D). Taken together, these results clearly indicated that the functional components in macrophage-like cells responsible for cancer stem cell were complicated.

Differential gene expression profile of conditioned culture CNE-2 single clone cell

The SSH data was further validated by Northern blotting analysis. Results revealed that mRNAs of ADP-ribosylation factor-like 6 interacting protein (ARMER), poly(rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-β subunit (PDHB) were only expressed in conditioned culture CNE-2-2 (Fig. 4A, B and 4C), and the level of ubiquitin-like protein fubi and ribosomal protein S30 precursor (FUBI) mRNA transcript was higher in conditioned cultured cells compared with control (Fig. 4D).