|Year : 2014 | Volume
| Issue : 1 | Page : 68-72
Comparison of nucleostemin gene expression in CD133+ and CD133− cell population in colon cancer cell line HT29
Noosha Zia-Jahromi1, Seyed Hossein Hejazi2, Mojtaba Panjepour3, Kazem Parivar1, Marjan Gharagozloo4
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Biochemistry, School of Pharmacology, Isfahan University, Isfahan, Iran
4 Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||23-Apr-2014|
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran
Source of Support: This work is financially supported by Islamic Azad University, Research and Science Branch, Tehran, Iran, Conflict of Interest: None
Background: Nucleostemin has been shown to be essential for proliferation and survival of colon cancer cells. In this study, we evaluate and comparing nucleostemin expression in CD133+ and CD133- colon cancer cell line HT29.
Materials and Methods: After preparation and culturing of HT29 cell line, isolation was performed using magnetic cell separation system by CD133 MicroBeads and phycoerythrin conjugated to monoclonal anti-human CD133 monoclonal antibody and analyzed with flow cytometry. For quantitative expression of nucleostemin in HT29, CD133+ and CD133- cells used specific nucleostemin primer and glyceraldehyde 3-phosphate dehydrogenase primer as endogenous control.
Results: The results showed the percentage of CD133+ cells in HT29 colon cancer cell line ranged from 36.5% to 41.5%, whereas the percentage of CD133- cell ranged from 58.5% to 63.5%. The expression rate of nucleostemin in HT29, CD133+ and CD133- cells were 1.44 ± 0.78, 1.60 ± 0.70 and 1.00 ± 0.18 (respectively). The comparison of expression rate represents no significant difference in nucleostemin expression in CD133+, CD133- and HT29 colon cancer cells.
Conclusion: It is concluded that nucleostemin expression could not be specific in a certain type of cells in colon cancer cell line HT29 and controlling strategies in colon cancer must not be focused on one certain type of colon cancer cells as main expressing nucleostemin gene.
结果：结果表明CD133+细胞在HT29结肠癌细胞株的比例在36.5%到41.5%，而CD133−细胞占58.5%到63.5%。核干细胞因子在HT29，CD133+和CD133−细胞中的表达率分别为1.44± 0.78, 1.60± 0.70 和1.00± 0.18。表达率的比较表明核干细胞因子在CD133+、CD133−和HT29结肠癌细胞中的表达无明显差异。
Keywords: CD133, colon cancer, gene expression, HT29, nucleostemin
|How to cite this article:|
Zia-Jahromi N, Hejazi SH, Panjepour M, Parivar K, Gharagozloo M. Comparison of nucleostemin gene expression in CD133+ and CD133− cell population in colon cancer cell line HT29. J Can Res Ther 2014;10:68-72
|How to cite this URL:|
Zia-Jahromi N, Hejazi SH, Panjepour M, Parivar K, Gharagozloo M. Comparison of nucleostemin gene expression in CD133+ and CD133− cell population in colon cancer cell line HT29. J Can Res Ther [serial online] 2014 [cited 2020 May 29];10:68-72. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/68/131375
| > Introduction|| |
Colon cancer is a major public health problem world-wide and the second leading cause of cancer deaths with a world-wide cumulative incidence rate of 9.4%.  The mortality rate approaches 100% due to propensity for early metastatic spread and because the disease is highly resistant to radiation and chemotherapy. Increasing evidence of colon cancer suggests that stem cells may play a decisive role in the progression and metastasis of colon cancer.  The cancer initiating cells or cancer stem cell were first identified in hematologic malignancies and more recently in several solid tumors, including colon cancer. 
Cancer stem cells are identified by their expression of specific surface markers, which in colon cancer are most CD133. 
CD133 is a 5-transmembrane glycoprotein of 865 amino acids that is expressed on the plasma membrane of embryonical epithelial structures.  The tumorigenic potential of CD133 positive (CD133+) cells has been demonstrated  and CD133 was originally identified as a marker of tumorigenic cells acts as stem cell in colon cancer. , The percentages of CD133+ cells in the tumorigenic population ranged from 3.2% to 24.5%, whereas in the matching normal tissues, the percentage of CD133+ cells ranged from 0.4% to 2.1%. 
Nucleostemin (guanine nucleotide binding protein-like 3) encoded a nuclear GTP-binding protein highly enriched in stem cells and cancer cells, including embryonic stem cells, neural stem cells and cancer stem cells, but not in most terminally differentiated cells.  Nucleostemin has been shown to be essential for stem cell and cancer cell proliferation and survival. 
In this study, we evaluated the expression of nucleostemin as a possible marker in different cell fraction of colon cancer cells. To address the issue, we examined the expression of nucleostemin in the human colon cancer cell line HT29 and compared expression patterns of nucleostemin in CD133+ and CD133− colon cancer cells.
| > Materials and Methods|| |
The human colon cancer cell line HT29 was obtained from Pasture Institute (Iran). HT29 cells were cultured in RPMI1640 medium supplemented with 5% (vol/vol) fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 1% (vol/vol) penicillin (100 U/ml), streptomycin (100 U/ml) and 1% (vol/vol) L-glutamine (GIBCO-BRI, Grand Island, NY). Cells were maintained at 37°C and 5% CO 2 . Media and supplement exchange were performed when 90% confluence was obtained. 
Cells were removed from culture dishes by trypsinization (0.25% trypsin-ethylenediaminetetraacetic acid [EDTA]) and after washing twice in isolation buffer (phosphate buffered saline, pH: 7.2, supplemented with 0.5% bovine serum albumin and 2 mmol/L EDTA), cells were resuspended in 300 μL of the same buffer per 10 8 total cells. Then FcR blocking reagent (100 μL/10 8 cells) and CD133 MicroBeads conjugated to monoclonal anti-human CD133 antibodies (100 μL/10 8 cells) were added (Miltenyi Biotec. Auburn, CA, USA) and incubated for 30 min in the 2-8°C.
Staining phycoerythrin (PE)-conjugated anti-human CD133 monoclonal antibody (50 μL) were added (CD133/2 (293C3)-PE, Cat. No. 130-090-853, Miltenyi Biotec. Auburn, CA, USA) and incubated in dark and cool place (2-8°C) for 5 min. After washing, cells were resuspend in 500 μL of the above buffer and subjected to the cell isolation process. The cell isolation procedure was performed according to the instruction of manufacture, using the magnetic cell separation system ([MACS], Miltenyi Biotec. Bergisch Gladbach, Germany). The magnetically labeled cells were subjected to a magnetic separation column placed on the magnetic field of MACS and the labeled cells were obtained by positive selection, as the population of CD133+ cells. The non-labeled cells were obtained by negative selection, as the CD133− population. The purity of the obtained CD133+ and CD133− cells was confirmed by flow-cytometry. Furthermore, unseparated HT29 cells were prepared and used as control. The unseparated HT29 cells exposed to staining antibody, used as a positive control and the unseparated HT29 cells were not exposed to staining antibody, used as negative control in flow cytometry analysis.
All samples (HT29 unseparated and separated to subpopulation cells) were analyzed by flow cytometer (BD Biosciences FACS Calibur; Becton Dickinson, San Jose, CA, USA), using the CellQuest software. Results were expressed as the mean fluorescence intensity. Total ribonucleic acid (RNA) was extracted from CD133+, CD133− and HT29 cells using a RNeasy kit (Qiagen, Valencia, CA, USA) and complementary deoxyribonucleic acid was synthesized using a QantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA) according to instruction of manufacture.
Real-time reverse transcription polymerase chain reaction (RT-PCR) for Nucleostemin gene was performed using specific QuantiTect primer (GNL3, Cat no. QT00038703, Qiagen, Valencia, CA, USA) using the QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA, USA).
The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer as endogenous control, were synthesized by Bioneer (South Korea). The primer sequences for GAPDH were as follows: Forward primer, 5'-AAGCTCATTTCCTGGTATG-3'; reverse primer, 5'- CTTCCTCTTGTGCTCTTG-3' with product size 125 bp.
Quantitative real-time RT-PCR was performed in a 20 μL reaction volume containing 10 μL of the SYBR Green PCR master mix (Fermentas, Germany), 1.5 μL of the RT reaction mixture, 1 μL primers and 7.5 μL dH 2 O using the Applied Biosystems StepOnePlus TM Real-time PCR system (Foster City, CA, USA).
Amplification program included of initial denaturation step at 95°C for 10 min, flowed by 40 amplification cycles consisting of denaturation at 95°C for 15 s, annealing at 60°C for 40 s and extension at 72°C for 20 s. Real-time PCR assays were performed in triplicate and repeated three times. Relative gene expression was calculated using the standard curve method.
All data were expression as mean ± standard deviation and statistically analysis by t-test. P <0.05 was considered to be significant. Confidence interval of 95% was considered for all tests. All statistical analysis was performed using the statistical package for social sciences (SPSS) version 18.0 software (Chicago, Inc, USA).
| > Results|| |
By cell sorting, the CD133+ and CD133− subpopulation of HT29, with purities of 91.5 ± 1.5% and 97 ± 2% were obtained, respectively. The purity of CD133+ and CD133− in unseparated HT29 cells that stained with PE-conjugate anti-human CD133 monoclonal antibody was 38.80 ± 2.52% and 61.20 ± 2.52%, respectively. The purity of subpopulation cells in unsepareted and stained cell represent the percentage of CD133+ cells in HT29 colon cancer cell line that ranged from 36.5 to 41.5%, whereas the percentage of CD133− cell ranged from 58.5 to 63.5% [Figure 1].
The purity of 99.7% of CD133− in unsepareted-unstained HT29 cells showed the validity of cell separation system.
|Figure 1: Flow cytometric analysis of CD133+ and CD133− population in colon cancer cell line HT29|
Click here to view
We compared expression of nucleostemin in HT29 cell line and derive subpopulation cells of HT29 cell line comprising CD133+ and CD133−. The expression rate of nucleostemin in HT29, CD133+ and CD133− cells were 1.44 ± 0.78, 1.60 ± 0.70 and 1.00 ± 0.18 respectively. Interestingly, the comparison of expression rate represents no significant difference in nucleostemin expression in CD133+, CD133− and HT29 colon cancer cells [Figure 2]. Therefore, the expression of nucleostemin in HT29 may be related to both CD133+ and CD133− cells in HT29 cell line.
|Figure 2: The expression of nucleostemin gene in HT29, CD133+ and CD133− colon cancer cells|
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| > Discussion|| |
Cancer stem cells in colon cancer tissues have been shown to be isolated by means of expression of specific cell surface markers. Several molecules have been proposed as cancer stem cell markers, including CD133, CD44, Musashi-1, integri, superfamily of transmembrane protein subunits α2 and β1 and aldehyde dehydrogenase-1.  Until now, CD133 appears to be the main colon cancer stem cells marker and subset of CD133+ colon cancer cells have demonstrated increased tumorigenic potential in transplantation studies both in vivo and in vitro. ,,,
Nucleostemin, a novel p53 - bonding protein, is abundantly present in the stem and cancer cells, but silenced in differentiated cells, has been shown to be essential for stem and cancer cell proliferation and survival.  The expression of nucleostemin has been reported in cancer cells in various organs including gastric liver,  kidney  and epithelial cell.  At present, no data concerning nucleostemin expression in the subpopulation of CD133+ and CD133− colon cancer cells are available.
For this, in this study the messenger RNA (mRNA) expression of nucleostemin was studied in subsets of CD133+ and CD133− colon cancer cells in HT29 cell line. Our finding showed there is no significant difference in nucleostemin mRNA expression in CD133+ and CD133− colon cancer cells in HT29 cell line.
Nucleostemin has a functional role in proliferating and maintaining cell cycle progression and it may be important for proliferation of cancer cells, since its over-expression has been reported in a number of human cancer cell lines a several malignant tissues. ,,, Therefore, the expression of nucleostemin in CD133+ and CD133− colon cancer cells revealed the proliferate capacity of CD133+ and CD133− colon cancer cells in HT29 cell line. However, its exact mechanism of action and whether it has a similar role in CD133+ and CD133− colon cancer cells are not clear. Recently, nucleostemin expression has been observed in normal and malignant renal tissues  and differentiated heart cells following pathological stresses.  These findings challenge the concept of nucleostemin being a specific player for stem and cancer cell proliferation. Furthermore, Nikpour et al.  were found over-expression of nucleostemin in the most uroepithelial carcinoma cell lines. They suggest involvement of nucleostemin differs even between tumors of the same type, like invasive bladder cancers. Interestingly, we also, observed expression of nucleostemin in CD133+ and CD133− colon cancer cell line HT29. These findings revealed nucleostemin not only could not be specific in a certain type of cells in normal and cancer cell, but may not to be specific in different fractions of colon cancer, e.g. cell line HT29 comprising CD133+ and CD133− cells.
However, the no differences in nucleostemin expression in CD133+ and CD133− colon cancer cells may be related to the method of CD133 detection, since most CD133 antibody recognize glycosylation-dependent epitope that may change with differential and transformation status the cell and complicating the use of this reagents to fractionate CD133+ and CD133− colon cancer cells.  However, we conclude that nucleostemin expression is not unique feature for CD133+ colon cancer cells, neither for CD133− colon cancer cells in HT29 colon cancer cell line. Although several evidences in major human cancer types, including brain,  prostate,  kidney,  hepatocellular, , colon , and pancreatic tumor , revealed much higher tumorigenic potential of CD133+ compared with CD133−, the result of our study regard to the same expression of nucleostemin in CD133+ and CD133− colon cancer cells, demonstrated that the difference in tumorigenicity may not be for nucleostemin expression. Hence the nucleostemin expression is an indicator of the proliferative capacity of cells,  therefore the nucleostemin expression in CD133+ and CD133− colon cancer cells represent the proliferative potential of D133 - same as CD133+ colon cancer cells or the hypothesis that proliferative potential of cancer cells may not be related to nucleostemin, exclusively.
In this study by cell sorting, the CD133+ and CD133− subpopulation of HT29, with purities over 90%, were obtained. The percentage of CD133+ and CD133− cancer cells among HT29 cell were 38.80 ± 2.52 and 61.20 ± 2.52, respectively. Previously, classified different colorectal carcinomas cell lines into three groups according to their CD133 surface presentation and inserted HT29 to cell lines that contain two distinct populations of CD133+ and CD133− appearing cells.  In the present study, we detect CD133+ and CD133− cells in HT29 and our finding was in agreement by Dittfeld et al.
Huang and Wicha  stated the percentage of CD133+ and CD133− cells in the tumorigenic populations ranged from 3.2% to 24.5%, whereas in the machine normal tissues, the percentage of CD133+ cells ranged from 0.04% to 2.1%. In colon cancer samples, Puglisi et al.  analyzed the CD133+ populations in colon cancer samples and compared with a healthy colon tissues. The percentage of CD133+ cells was higher in tumor samples and ranged from 0.1% to 20.44% while the percentage of CD133+ in normal colon tissues were lower and ranged from 0% to 3.3%.  Our Result is disagreement with study by Elsaba et al.  who reported HT29 has a high level of CD133 expression (>95%) in the cell line HT29. However, the percentage of CD133+ and CD133− can be change by many factors. So that, the cell line HT29 showed that highly variable CD133+ fraction of 2-90% in different laboratories. , It seems that the expression of surface markers can be influenced by physiological and pathophysiobgical parameters.  Previously showed that oxygen availability in vitro and in vivo condition impact the distribution of CD133+ and CD133− fractions in glioblastoma cell culture and brain tumor. , Furthermore, Hongo et al.  represent the method of cell sorting can influence the purity of CD133+ and the percentage of CD133+ could be changed with time.
| > Conclusion|| |
In overall, we conclude HT29 colon cancer line contain the CD133+ and CD133− population that nucleostemin expression is not unique in CD133+ or CD133− cells in HT29. It is concluded that nucleostemin expression could not be specific in a certain type of cells in colon cancer cell line HT29 and controlling strategies in colon cancer must not be focused on one certain type of colon cancer cells as main expressing nucleostemin gene. However, extended studies with more cell line and colon cancer samples are needed to approve the hypothesis that nucleostemin expression in CD133+ is same as CD133−.
| > Acknowledgments|| |
author gratefully acknowledgement the excellent technical assistance of Mrs. Mehrafarin Fesharaki, Ms Mohammad Kazemi, School of Medicine, Isfahan University of Medicine Science, Isfahan, Iran.
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[Figure 1], [Figure 2]