Anat Cell Biol.  2014 Mar;47(1):1-11. 10.5115/acb.2014.47.1.1.

The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines

• Amini S
• Fathi F
• Mobalegi J
• Sofimajidpour H
• Ghadimi T

• KMID: 2168851
• DOI: http://doi.org/10.5115/acb.2014.47.1.1

Abstract

Uncontrolled self-renewal plays a direct function in the progression of different types of carcinomas. The same molecular pathway that manages self-renewal in normal stem cells also seems to manage cancer stem cells. Here, we examine the expressions of self-renewal regulatory factors Oct4, Nanog, Sox2, nucleostemin, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 in tissue samples of colon, prostate, and bladder carcinomas as well as cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2. We used reverse transcriptase polymerase chain reaction to examine expressions of the above mentioned regulatory factors in cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2 and in 20 tumor tissue samples. Total RNA was isolated by the ISOGEN method. RNA integrity was checked by agarose gel electrophoresis and spectrophotometry. Expressions of Oct4 and nucleostemin at the protein level were determined by immunocytochemistry. A significant relationship was found between tumor grade and self-renewal gene expression. Expressions of stem cell specific marker genes were detected in all examined cancer cell lines, in 40% to 100% of bladder cancer samples, and in 60% to 100% of colon and prostate cancer samples. Oct4 expressed in 100% of tumor tissue samples. Our data show that stem cell markers Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 significantly express in cancer cell lines and cancer tissues. Hence, these markers might be useful as potential tumor markers in the diagnosis and/or prognosis of tumors.

Keyword

Neoplastic stem cells; Self-renewal gene; Colonic neoplasms; Prostatic neoplasms; Urinary bladder neoplasms

MeSH Terms

Cell Line*
Colon*
Colonic Neoplasms
Diagnosis
Electrophoresis, Agar Gel
Gene Expression
Immunohistochemistry
Neoplastic Stem Cells
Prognosis
Prostate*
Prostatic Neoplasms*
Reverse Transcriptase Polymerase Chain Reaction
RNA
Spectrophotometry
Stem Cells*
Biomarkers, Tumor
Urinary Bladder Neoplasms
Urinary Bladder*
RNA

Figure

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  Fig. 1 Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of the expressions of Oct4, Nanog, Sox2, nucleostemin, Esrrb, Tbx3, Tcl1, Dppa4, Zfx, and Bmi in HT-1376 (bladder cancer cell line), LNCaP (pro state cancer cell line), HepG2 (hepa tocarcinoma cell line), HT-29, and Caco-2 (colon cancer cell lines). RT-PCR analysis was performed using carefully designated primers for specific amplification of each gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the internal control. U87, a glioblastoma cell line, was the positive control for Sox2, nucle ostemin, and Zfx. NT2, a human embryonal carcinoma cell line, was the positive control for Oct4, Nanog, Bmi, Tcl1, Tbx3, Dppa4, Zfx, and Esrrb. Note: All genes were expressed in the cancer cell lines.

• 

  Fig. 2 Reverse transcriptase polymerase chain reaction analysis shows the expressions of Oct4, Nanog, Sox2, nucleostemin, Esrrb, Tbx3, Tcl1, Dppa4, Zfx, and Bmi in bladder cancer tissues. Note: Some samples did not express some of genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.

• 

  Fig. 3 Reverse transcriptase polymerase chain reaction analysis shows expressions of Oct4, Nanog, Sox2, nucleostemin, Esrrb, Tbx3, Tcl1, Dppa4, Zfx, and Bmi in prostate cancer tissues. Although all genes were expressed in some of the samples, some samples did not express some of genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

• 

  Fig. 4 Reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows the expressions of Oct4, Nanog, Sox2, nucleostemin, Esrrb, Tbx3, Tcl1, Dppa4, Zfx, and Bmi in the colon cancer tissues. RT-PCR analysis was performed using carefully designated primers for specific amplification of each gene. Note: All genes were expressed in some of the samples, however other samples did not express some of genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

• 

  Fig. 5 Reverse transcriptase polymerase chain reaction analysis shows expression of Oct4, Sox2, Nanog, Tcl1, Tbx3, Esrrb, Dppa4, and nucleostemin in normal colon tissues. Low expressions of Oct4 and nucleostemin were observed in some normal colon tissues.

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  Fig. 6 Histogram of polymerase chain reaction results in colon, bladder and prostate cancer samples, as well as HT-29, Caco-2, HT-1376, LNCaP, and HepG2 cancer cell lines. Oct4 was expressed in 100% of bladder, colon, and prostate tumor samples. Expression of Nanog was detected in 100% of the colon cancer samples, 90% of the bladder cancer samples and 80% of the prostate cancer samples. Nucleostemin was detected in 100% of the prostate cancer samples, 80% of the bladder cancer samples, and 60% of the colon cancer samples.

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  Fig. 7 Immunocytochemistry (ICC) of cancer cell lines stained with anti-Oct4 antibody. (A) NT2 cells were used as positive controls. HT-1376 (C), HepG2 (E), and LNCaP (G) cell lines were positively stained for Oct4. Panels (B), (D), (F), and (H) show the phase contrast counterparts of panels (A), (C), (E), and (G), respectively. Result is representative of three independent experiments. Scale bars=50 µm (A-H).

• 

  Fig. 8 Immunocytochemistry (ICC) of the cancer cell lines stained with anti-nucleostemin antibody. (A) NT2 cells were used as positive controls. HT-1376 (C), HepG2 (E), and LNCaP (G) cell lines were positively stained for nucleostemin. Panels (B), (D), (F), and (H) show the phase contrast counterparts of panels (A), (C), (E), and (G), respectively. Result is representative of three independent experiments. Scale bar=50 µm (A-H).

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