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doi:10.1111/jog.12640
J. Obstet. Gynaecol. Res. Vol. 41, No. 5: 763–767, May 2015
OCT-4 and DAZL expression in precancerous lesions of the human uterine cervix Konstantinos Stefanidis1,2, Vasileios Pergialiotis1,2, Dimitrios Christakis1,2, Jessica Patta1,2, Diamanto Stefanidi1,2 and Dimitrios Loutradis1,2 1 Laboratory of Stem Cells, Division of Reproductive Medicine, Alexandra Maternity Hospital and 21st Department of Obstetrics and Gynecology, Medical School, University of Athens, Athens, Greece
Abstract Aim: To determine whether octamer-binding transcription factor 4 (OCT-4) and deleted in azoospermia like (DAZL) are expressed among cells with human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) lesions and quantify their relative expression when compared with normal cervical cultures. Methods: Cervical cells derived from normal cell cultures, HPV lesions and CIN lesions were cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% amniotic fluid and 5 ng/mL basic fibroblast growth factor at 37°C and humidified 10% CO2 in air. Real-time polymerase chain reaction (PCR) was carried out using G6PD as a reference. We used REST for statistical analysis of real-time PCR. Results: Whereas DAZL was not expressed either in normal cultures or HPV and CIN lesions, OCT-4 was expressed in all examined cell lines. Moreover its relative expression was significantly upregulated among cultures of HPV-infected cells (RE, 11.003; 95%CI: 0.054–36 704.527, P = 0.042), an observation that was also close to statistical significance among cultures of CIN lesions (P = 0.066). Conclusion: The relative expression of OCT-4 is upregulated during the early, preinvasive stages of cervical carcinogenesis. Future studies should investigate its potential as a screening marker and as a possible target of therapy. Key words: cervical, cervical intraepithelial neoplasia, DAZL, human papilloma virus, OCT-4, stemness.
Introduction Cancer cells are cells that have lost their ability to regulate their growth and to enter senescence or programmed death (apoptosis).1 In most cases this happens due to DNA defects that are triggered environmentally by carcinogens.2 The affected genes fall generally into two categories: oncogenes and tumor suppressor genes that either gain or lose function, respectively, during the cancer activation process.3 Although until recently the main theory of cancer development included only the dedifferentiation process of a somatic cell towards a progenitor state, in
recent years a revolutionary counterproposal has been introduced.4,5 This latest theory proposes the existence of stem cells in all human tissues and is supported by findings in several cancer series. It is already known that stem cells are naturally immortal until they are induced to terminally differentiate or apoptose. Stem cells have the ability to either divide symmetrically producing two equal cells with stemness, or asymmetrically, producing a cell that retains stemness and another one that has the ability to form differentiated tissue (progenitor cell).3 In both proposed theories of either dedifferentiation of somatic cells or cancerous mutation of adult stem cells it seems
Received: March 13 2014. Accepted: October 11 2014. Reprint request to: Assistant Professor Konstantinos Stefanidis, 15, Neapoleos str., Marousi 15123 – Greece. Email: [email protected]
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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K. Stefanidis et al.
that the result is the formation of cells that divide symmetrically, giving rise to cells with immortal capabilities.3 But although the first theory requires reprogramming of the somatic cell to a stem-like state, in the second one this state already exists. A number of studies have identified the presence of stemness markers in cancer cell lines derived from the human cervix.6–9 One of them examined the expression of octamer-binding transcription factor 4 (OCT-4) among human papilloma virus (HPV)-positive and HPV-negative cervical cancer cells and concluded that HPV could trigger cervical carcinogenesis by upregulation of OCT-4.10 It is well known that HPV infection is the single causative factor of cervical cancer.11 Not all women who are infected with HPV, however, ultimately develop a cervical intraepithelial neoplasia (CIN) lesion. Therefore, several distinctions among these two cell lines may exist. Although stem cell existence has been extensively studied in various cancer cell lines, studies examining the presence of stemness markers in early, pre-invasive lesions are limited. The present study examines the presence of such markers in early cervical pathology, including HPV lesions and CIN. We therefore sought to determine whether OCT-4 and deleted in azoospermia like (DAZL) are expressed among cells with HPV infection and CIN lesions, and to quantify their relative expression when compared with normal cervical cultures.
Methods Definitions In the present study we defined HPV infection as positive HC2 HPV DNA test (Digene, Gaithersburg, MD, USA) and normal cytology (no CIN). The CIN group, however, consisted of women with a combination of positive HC2 HPV DNA test (Digene) and abnormal cytology (CIN). Cytology and HPV infection detection Cervical cells were obtained after performing a routine cervical smear test with a spatula from the ectocervix and a brush from the endocervix from 30 women who had normal cervical cytology, HPV infection, or CIN lesions (10 women in each group). Conventional cytology was performed in order to detect CIN lesions. The Hybrid Capture 2 (Digene) method was used to detect the presence of HPV DNA.12 Specimens were sent for this type of analysis
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whenever a dyskaryotic phenotype was observed on cytology, without clear evidence of a CIN lesion.
Cell cultures One month after cytology analysis and HPV detection women were offered a second cervical smear. This time the brush and spatula were washed out in a tube containing 3–5 mL of Dulbecco’s Phosphate-Buffered Saline. We used our patented Stef. Medium13 (SM; US Provisional Patent No60/853420) that contains 80% basal medium (83.5% KnockOut™ Dulbecco’s modified Eagle’s medium, 12.5% of a serum-free replacement originally optimized for human ES cells, 2% l-glutamine, 1% non-essential amino acids stock [Gibco, Rockville, MD, USA], and 1% penicillinstreptomycin) and 20% amniotic fluid supplemented with 5 ng/mL basic fibroblast growth factor. We also added 90 μL of Fungizone (Invitrogen, Carlsbad, CA, USA) per 15 μL of cultured extract. RNA extraction Total RNA was extracted from the cultured medium using a commercially available kit (RNAeasy micro kit; Qiagen, Hilden, Germany), following manufacturer instructions. RNA fluorometry RNA fluorometry was performed using the commercially available Quant-iT™ RNA BR Assay Kit (Invitrogen™) in order to measure the quantity of the extracted RNA. Reverse transcription Total RNA extracted from CIN lesions was used to obtain complimentary DNA (cDNA) with reverse transcription. Superscript® First-Strand Synthesis System for real-time polymerase chain reaction (RT-PCR) was used during this part of the experiment (Invitrogen™). The kit provides two alternative methods of obtaining cDNA, one involving random primers (first-strand synthesis using random primers) and one involving oligonucleotides (first-strand synthesis using oligo[dT]). Both of them were used in the current experiment. Real-time PCR Real-time PCR was performed with the Light Cycler (Roche, Basel, Switzerland). The primers and hybrid probes used for this experiment were provided by TIB MOLBIOL (Berlin, Germany) and are indexed in Table 1. Oligonucleotides used during DAZL RT-PCR
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
OCT-4 and DAZL in CIN and HPV infection
Table 1 Oligonucleotides used during RT-PCR Deleted in azoospermia-like (DAZL) Primers DAZL S DAZL A Probes DAZL FL DAZL LC Primers OCT-4 S OCT-4 A Probes OCT-4 FL OCT-4 LC
Sequence gCTATgTTgTACCTCCggTTA gCCCgACTTCTTCTAAAgATg Sequence TTTCAgAgggTggAgTAgCTTCATg-FL 640-ACTgAACATTCATTTggACAACTTCAgCT P OCT-4 Sequence AAgCAgAAACCCTCgTg ACTCggACCACATCCCT Sequence AACAAATTCTCCAggTTgCCTC-FL 640-CACTCggTTCTCgATACTggTTCgC P
FL, upstream oligonucleotide labeled at the 3′ end with fluorescein; LC, downstream oligonucleotide labeled at the 5′ end with LightCycler Red 640; RT-PCR, real-time polymerase chain reaction.
are also indexed in the same label. The 96-well plates were used during RT-PCR and the total volume of each reaction was 20 μL. The reference solution for DAZL and OCT-4 contained 4 μL of Light Cycler 480 Genotyping Master Mix, 0.5 μL Primer S, 0.5 μL Primer A, 0.2 μL Hyprobe LC, 0.2 μL Hyprobe LC and 9.6 μL LightCycler 480 Genotyping Master: H2O, PCR grade (Roche). The G6PD control gene was amplified using a solution that contained 2 μL of Light Cycler 480 Genotyping Master Mix, 4 μL of Primer mix for G6PD, 2.4 μL LightCycler 480 Genotyping Master: MgCl2 solution and 6.6 μL of LightCycler 480 Genotyping Master: H2O, PCR grade (Roche). Preincubation of the solution was performed at 95°C for 10 min. The next step included 40 cycles of amplification, each one repeating the next four steps: (i) DNA denaturation at 95°C for 10 min; (ii) primer-DNA annealing for 20 s; (iii) DNA extension at 72°C for 10 s; and (iv) termination of reaction with a cooling stage at 40°C for 30 s.
Statistical analysis Analysis of results was carried out using the Relative Expression Software Tool (REST) developed by Pfaffl et al.14 Relative quantification analysis among groups of normal cultures, HPV-infected cultures and CIN lesions used the pair-wise fixed reallocation randomization test in REST. REST interprets downregulation or upregulation of the studied (targeted) gene among different groups of cultures. It also provides 95%CI, and results of statistical significance (in the present study, defined as P < 0.05).
Results G6PD expression G6PD was expressed in all 30 women, thus we were able to evaluate all of them for the expression of DAZL and OCT-4. DAZL expression DAZL was expressed neither in normal cultures nor in CIN lesions, with the exception of one sample that gave a crossing point value of 33.15 at a concentration of 2.64 × 104 cells/ml. This result, however, can be explained according to the theory of Sottile, who proposed that stem cells pass from the bone marrow to the vasculature.15 We must state at this point that the current specimen was contaminated with an abundance of red cells, among which we only can speculate that certain stem cells could be present. OCT-4 expression Using REST we observed that OCT-4 was upregulated 11-fold among women with HPV infection. This result was statistically significant (P = 0.042; Table 2). Women with CIN lesions, however, were not significantly different to healthy controls, although the results were close to statistical significance (P = 0.066; Table 2).
Discussion According to a recent theory, stem-cells seem to contribute to cancer development.16 Therefore, we find it imperative that future studies examine whether
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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K. Stefanidis et al.
Table 2 Relative expression Gene
Type
Reaction efficiency
Expression
SE
95%CI
P
HPV OCT-4 G6PD
TRG REF
0.97 0.99
11.003 1.00
0.619–192.922
0.054–36 704.527
0.042
CIN lesions OCT-4 G6PD
TRG REF
0.97 0.99
5.088 1.00
0.488–62.490
0.073–922.560
0.066
REF, reference gene; TRG, target gene.
markers of stemness are present in HPV-infected cells and in situ lesions. In the present study we studied the expression of two known genes that are expressed in human pluripotent cells. DAZL is a protein that is naturally localized to the nucleus and cytoplasm of fetal germ cells and to the cytoplasm of developing oocytes.17 It seems to participate during early proliferation and differentiation of these germ cells. Collier et al. showed that it specifically triggers the initiation of the process of translation.18 OCT-4 is a protein that is also known as POU5F1, which is encoded by POU5F1.19 OCT-4 seems to either suppress or induce the function of the Rex1 promoter, a known factor of pluripotency that is found in undifferentiated embryonic stem cells.20,21 Previously it has been used in experimental models along with Klf4 in order to produce pluripotent stem cells with the process of cell reprogramming.22 Several studies have proposed the existence of OCT-4 in several cancer cell lines as a marker of the existence of cancer stem cells.23–25 We have previously demonstrated that both DAZL and OCT-4 are expressed in HeLa cell lines.9 Liu et al. have confirmed that OCT-4 is overexpressed in HPVinfected cervical cancer cell lines.10 In two, almost synchronous studies, they also reported that OCT-4 expression was higher among HPV-16 lesions, compared to HPV-negative lesions.16,26 They proposed that, in normal states, a repressor complex binds to the hyper-methylated regulatory regions of OCT-4 and suppress its expression in HPV-negative cervical cells. When cervical cancer is triggered by HPV-16, however, its oncoproteins destroy the repressor complex, thus leading to upregulation of OCT-4. Due to these observations we studied the expression of both DAZL and OCT-4 in normal cell cultures and precancerours lesions, in order to determine whether these pathways are triggered at early, precancerous stages or late, after the invasion of the basal membrane of the cervical epithelium. In the present study we
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relied on pluripotent properties of DAZL and OCT-4 in order to evaluate whether they are expressed in cultures from cervices without pathology and from cervices with HPV and CIN lesions. It seems that DAZL expression does not participate in cultures of normal cervices and those with early preinvasive lesions. It may be that its role is detected in the subsequent dedifferentiation of cancerous cells. This hypothesis still needs further investigation in future studies. The present study, however, has shown that both normal cervices and early precancerous lesions (including HPV infection and CIN) express OCT-4. Furthermore OCT-4 was significantly upregulated in HPV-infected lesions; an effect that is, however, lost in lesions with CIN. It is possible that OCT-4 may interfere during the precancerous cervical activity, thus having significant participation in early cervical pathology. A weakness of the present study was that CIN cells could be mixed with HPV-only infected cells during specimen collection. Therefore, future studies should investigate the present findings among isolated strains of cell cultures in order to investigate whether OCT-4 truly represents an early marker of cervical carcinogenesis and whether it could serve as a target for the treatment of cervical cancer. OCT-4 is expressed both in cells of normal cervical cultures and among those derived from HPV and CIN lesions. What is more important to denote is that its expression is significantly upregulated during the early stages of cervical pathology. Future studies should clarify whether OCT-4 is an important factor that could be used in the future for early screening and targeted therapy of HPV and CIN cervical lesions.
Disclosure The authors report no declarations of interest.
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
OCT-4 and DAZL in CIN and HPV infection
References 1. Dixon SC, Soriano BJ, Lush RM, Borner MM, Figg WD. Apoptosis: Its role in the development of malignancies and its potential as a novel therapeutic target. Ann Pharmacother 1997; 31: 76–82. 2. Li S, Hursting SD, Davis BJ, McLachlan JA, Barrett JC. Environmental exposure, DNA methylation, and gene regulation: Lessons from diethylstilbesterol-induced cancers. Ann N Y Acad Sci 2003; 983: 161–169. 3. Trosko JE. Review paper: Cancer stem cells and cancer nonstem cells: From adult stem cells or from reprogramming of differentiated somatic cells. Vet Pathol 2009; 46: 176–193. 4. Sell S. On the stem cell origin of cancer. Am J Pathol 2010; 176: 2584–2494. 5. Wu XZ. Origin of cancer stem cells: The role of self-renewal and differentiation. Ann Surg Oncol 2008; 15: 407–414. 6. Feng D, Peng C, Li C et al. Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri. Oncol Rep 2009; 22: 1129–1134. 7. Carlson JW, Nucci MR, Brodsky J, Crum CP, Hirsch MS. Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: The role of TTF-1, WT-1 and HPV analysis. Histopathology 2007; 51: 305–312. 8. Guenin S, Mouallif M, Deplus R et al. Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis. PLoS ONE 2012; 7: e42704. 9. Stefanidis K, Loutradis D, Vassiliou LV et al. Nevirapine induces growth arrest and premature senescence in human cervical carcinoma cells. Gynecol Oncol 2008; 111: 344–349. 10. Liu D, Zhou P, Zhang L, Wu G, Zheng Y, He F. Differential expression of Oct4 in HPV-positive and HPV-negative cervical cancer cells is not regulated by DNA methyltransferase 3A. Tumour Biol 2011; 32: 941–950. 11. Wright TC. Pathology of HPV infection at the cytologic and histologic levels: Basis for a 2-tiered morphologic classification system. Int J Gynaecol Obstet 2006; 94: S22–S31. 12. Clavel C, Masure M, Putaud I et al. Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions. J Clin Pathol 1998; 51: 737–740. 13. Stefanidis K, Loutradis D, Anastasiadou V et al. Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL. Biosystems 2009; 98: 122–126.
14. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002; 30: e36. 15. Sottile V. Bone marrow as a source of stem cells and germ cells? Perspectives for transplantation. Cell Tissue Res 2007; 328: 1–5. 16. Miller SJ, Lavker RM, Sun TT. Interpreting epithelial cancer biology in the context of stem cells: Tumor properties and therapeutic implications. Biochim Biophys Acta 2005; 1756: 25–52. 17. Brook M, Smith JW, Gray NK. The DAZL and PABP families: RNA-binding proteins with interrelated roles in translational control in oocytes. Reproduction 2009; 137: 595–617. 18. Collier B, Gorgoni B, Loveridge C, Cooke HJ, Gray NK. The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells. EMBO J 2005; 24: 2656– 2666. 19. Takeda J, Seino S, Bell GI. Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic Acids Res 1992; 20: 4613–4620. 20. Ben-Shushan E, Thompson JR, Gudas LJ, Bergman Y. Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol Cell Biol 1998; 18: 1866–1878. 21. Shi W, Wang H, Pan G, Geng Y, Guo Y, Pei D. Regulation of the pluripotency marker Rex-1 by Nanog and Sox2. J Biol Chem 2006; 281: 23319–23325. 22. Kim JB, Zaehres H, Wu G et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 2008; 454: 646–650. 23. Kim SY, Kang JW, Song X et al. Role of the IL-6-JAK1-STAT3Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells. Cell Signal 2013; 25: 961–969. 24. Ghisolfi L, Keates AC, Hu X, Lee DK, Li CJ. Ionizing radiation induces stemness in cancer cells. PLoS ONE 2012; 7: e43628. 25. Hu X, Ghisolfi L, Keates AC et al. Induction of cancer cell stemness by chemotherapy. Cell Cycle 2012; 11: 2691–2698. 26. Mine H, Sakurai T, Kashida H et al. Association of gankyrin and stemness factor expression in human colorectal cancer. Dig Dis Sci 2013; 58: 2337–2344.
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doi:10.1111/jog.12640
J. Obstet. Gynaecol. Res. Vol. 41, No. 5: 763–767, May 2015
OCT-4 and DAZL expression in precancerous lesions of the human uterine cervix Konstantinos Stefanidis1,2, Vasileios Pergialiotis1,2, Dimitrios Christakis1,2, Jessica Patta1,2, Diamanto Stefanidi1,2 and Dimitrios Loutradis1,2 1 Laboratory of Stem Cells, Division of Reproductive Medicine, Alexandra Maternity Hospital and 21st Department of Obstetrics and Gynecology, Medical School, University of Athens, Athens, Greece
Abstract Aim: To determine whether octamer-binding transcription factor 4 (OCT-4) and deleted in azoospermia like (DAZL) are expressed among cells with human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) lesions and quantify their relative expression when compared with normal cervical cultures. Methods: Cervical cells derived from normal cell cultures, HPV lesions and CIN lesions were cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% amniotic fluid and 5 ng/mL basic fibroblast growth factor at 37°C and humidified 10% CO2 in air. Real-time polymerase chain reaction (PCR) was carried out using G6PD as a reference. We used REST for statistical analysis of real-time PCR. Results: Whereas DAZL was not expressed either in normal cultures or HPV and CIN lesions, OCT-4 was expressed in all examined cell lines. Moreover its relative expression was significantly upregulated among cultures of HPV-infected cells (RE, 11.003; 95%CI: 0.054–36 704.527, P = 0.042), an observation that was also close to statistical significance among cultures of CIN lesions (P = 0.066). Conclusion: The relative expression of OCT-4 is upregulated during the early, preinvasive stages of cervical carcinogenesis. Future studies should investigate its potential as a screening marker and as a possible target of therapy. Key words: cervical, cervical intraepithelial neoplasia, DAZL, human papilloma virus, OCT-4, stemness.
Introduction Cancer cells are cells that have lost their ability to regulate their growth and to enter senescence or programmed death (apoptosis).1 In most cases this happens due to DNA defects that are triggered environmentally by carcinogens.2 The affected genes fall generally into two categories: oncogenes and tumor suppressor genes that either gain or lose function, respectively, during the cancer activation process.3 Although until recently the main theory of cancer development included only the dedifferentiation process of a somatic cell towards a progenitor state, in
recent years a revolutionary counterproposal has been introduced.4,5 This latest theory proposes the existence of stem cells in all human tissues and is supported by findings in several cancer series. It is already known that stem cells are naturally immortal until they are induced to terminally differentiate or apoptose. Stem cells have the ability to either divide symmetrically producing two equal cells with stemness, or asymmetrically, producing a cell that retains stemness and another one that has the ability to form differentiated tissue (progenitor cell).3 In both proposed theories of either dedifferentiation of somatic cells or cancerous mutation of adult stem cells it seems
Received: March 13 2014. Accepted: October 11 2014. Reprint request to: Assistant Professor Konstantinos Stefanidis, 15, Neapoleos str., Marousi 15123 – Greece. Email: [email protected]
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
763
K. Stefanidis et al.
that the result is the formation of cells that divide symmetrically, giving rise to cells with immortal capabilities.3 But although the first theory requires reprogramming of the somatic cell to a stem-like state, in the second one this state already exists. A number of studies have identified the presence of stemness markers in cancer cell lines derived from the human cervix.6–9 One of them examined the expression of octamer-binding transcription factor 4 (OCT-4) among human papilloma virus (HPV)-positive and HPV-negative cervical cancer cells and concluded that HPV could trigger cervical carcinogenesis by upregulation of OCT-4.10 It is well known that HPV infection is the single causative factor of cervical cancer.11 Not all women who are infected with HPV, however, ultimately develop a cervical intraepithelial neoplasia (CIN) lesion. Therefore, several distinctions among these two cell lines may exist. Although stem cell existence has been extensively studied in various cancer cell lines, studies examining the presence of stemness markers in early, pre-invasive lesions are limited. The present study examines the presence of such markers in early cervical pathology, including HPV lesions and CIN. We therefore sought to determine whether OCT-4 and deleted in azoospermia like (DAZL) are expressed among cells with HPV infection and CIN lesions, and to quantify their relative expression when compared with normal cervical cultures.
Methods Definitions In the present study we defined HPV infection as positive HC2 HPV DNA test (Digene, Gaithersburg, MD, USA) and normal cytology (no CIN). The CIN group, however, consisted of women with a combination of positive HC2 HPV DNA test (Digene) and abnormal cytology (CIN). Cytology and HPV infection detection Cervical cells were obtained after performing a routine cervical smear test with a spatula from the ectocervix and a brush from the endocervix from 30 women who had normal cervical cytology, HPV infection, or CIN lesions (10 women in each group). Conventional cytology was performed in order to detect CIN lesions. The Hybrid Capture 2 (Digene) method was used to detect the presence of HPV DNA.12 Specimens were sent for this type of analysis
764
whenever a dyskaryotic phenotype was observed on cytology, without clear evidence of a CIN lesion.
Cell cultures One month after cytology analysis and HPV detection women were offered a second cervical smear. This time the brush and spatula were washed out in a tube containing 3–5 mL of Dulbecco’s Phosphate-Buffered Saline. We used our patented Stef. Medium13 (SM; US Provisional Patent No60/853420) that contains 80% basal medium (83.5% KnockOut™ Dulbecco’s modified Eagle’s medium, 12.5% of a serum-free replacement originally optimized for human ES cells, 2% l-glutamine, 1% non-essential amino acids stock [Gibco, Rockville, MD, USA], and 1% penicillinstreptomycin) and 20% amniotic fluid supplemented with 5 ng/mL basic fibroblast growth factor. We also added 90 μL of Fungizone (Invitrogen, Carlsbad, CA, USA) per 15 μL of cultured extract. RNA extraction Total RNA was extracted from the cultured medium using a commercially available kit (RNAeasy micro kit; Qiagen, Hilden, Germany), following manufacturer instructions. RNA fluorometry RNA fluorometry was performed using the commercially available Quant-iT™ RNA BR Assay Kit (Invitrogen™) in order to measure the quantity of the extracted RNA. Reverse transcription Total RNA extracted from CIN lesions was used to obtain complimentary DNA (cDNA) with reverse transcription. Superscript® First-Strand Synthesis System for real-time polymerase chain reaction (RT-PCR) was used during this part of the experiment (Invitrogen™). The kit provides two alternative methods of obtaining cDNA, one involving random primers (first-strand synthesis using random primers) and one involving oligonucleotides (first-strand synthesis using oligo[dT]). Both of them were used in the current experiment. Real-time PCR Real-time PCR was performed with the Light Cycler (Roche, Basel, Switzerland). The primers and hybrid probes used for this experiment were provided by TIB MOLBIOL (Berlin, Germany) and are indexed in Table 1. Oligonucleotides used during DAZL RT-PCR
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
OCT-4 and DAZL in CIN and HPV infection
Table 1 Oligonucleotides used during RT-PCR Deleted in azoospermia-like (DAZL) Primers DAZL S DAZL A Probes DAZL FL DAZL LC Primers OCT-4 S OCT-4 A Probes OCT-4 FL OCT-4 LC
Sequence gCTATgTTgTACCTCCggTTA gCCCgACTTCTTCTAAAgATg Sequence TTTCAgAgggTggAgTAgCTTCATg-FL 640-ACTgAACATTCATTTggACAACTTCAgCT P OCT-4 Sequence AAgCAgAAACCCTCgTg ACTCggACCACATCCCT Sequence AACAAATTCTCCAggTTgCCTC-FL 640-CACTCggTTCTCgATACTggTTCgC P
FL, upstream oligonucleotide labeled at the 3′ end with fluorescein; LC, downstream oligonucleotide labeled at the 5′ end with LightCycler Red 640; RT-PCR, real-time polymerase chain reaction.
are also indexed in the same label. The 96-well plates were used during RT-PCR and the total volume of each reaction was 20 μL. The reference solution for DAZL and OCT-4 contained 4 μL of Light Cycler 480 Genotyping Master Mix, 0.5 μL Primer S, 0.5 μL Primer A, 0.2 μL Hyprobe LC, 0.2 μL Hyprobe LC and 9.6 μL LightCycler 480 Genotyping Master: H2O, PCR grade (Roche). The G6PD control gene was amplified using a solution that contained 2 μL of Light Cycler 480 Genotyping Master Mix, 4 μL of Primer mix for G6PD, 2.4 μL LightCycler 480 Genotyping Master: MgCl2 solution and 6.6 μL of LightCycler 480 Genotyping Master: H2O, PCR grade (Roche). Preincubation of the solution was performed at 95°C for 10 min. The next step included 40 cycles of amplification, each one repeating the next four steps: (i) DNA denaturation at 95°C for 10 min; (ii) primer-DNA annealing for 20 s; (iii) DNA extension at 72°C for 10 s; and (iv) termination of reaction with a cooling stage at 40°C for 30 s.
Statistical analysis Analysis of results was carried out using the Relative Expression Software Tool (REST) developed by Pfaffl et al.14 Relative quantification analysis among groups of normal cultures, HPV-infected cultures and CIN lesions used the pair-wise fixed reallocation randomization test in REST. REST interprets downregulation or upregulation of the studied (targeted) gene among different groups of cultures. It also provides 95%CI, and results of statistical significance (in the present study, defined as P < 0.05).
Results G6PD expression G6PD was expressed in all 30 women, thus we were able to evaluate all of them for the expression of DAZL and OCT-4. DAZL expression DAZL was expressed neither in normal cultures nor in CIN lesions, with the exception of one sample that gave a crossing point value of 33.15 at a concentration of 2.64 × 104 cells/ml. This result, however, can be explained according to the theory of Sottile, who proposed that stem cells pass from the bone marrow to the vasculature.15 We must state at this point that the current specimen was contaminated with an abundance of red cells, among which we only can speculate that certain stem cells could be present. OCT-4 expression Using REST we observed that OCT-4 was upregulated 11-fold among women with HPV infection. This result was statistically significant (P = 0.042; Table 2). Women with CIN lesions, however, were not significantly different to healthy controls, although the results were close to statistical significance (P = 0.066; Table 2).
Discussion According to a recent theory, stem-cells seem to contribute to cancer development.16 Therefore, we find it imperative that future studies examine whether
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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K. Stefanidis et al.
Table 2 Relative expression Gene
Type
Reaction efficiency
Expression
SE
95%CI
P
HPV OCT-4 G6PD
TRG REF
0.97 0.99
11.003 1.00
0.619–192.922
0.054–36 704.527
0.042
CIN lesions OCT-4 G6PD
TRG REF
0.97 0.99
5.088 1.00
0.488–62.490
0.073–922.560
0.066
REF, reference gene; TRG, target gene.
markers of stemness are present in HPV-infected cells and in situ lesions. In the present study we studied the expression of two known genes that are expressed in human pluripotent cells. DAZL is a protein that is naturally localized to the nucleus and cytoplasm of fetal germ cells and to the cytoplasm of developing oocytes.17 It seems to participate during early proliferation and differentiation of these germ cells. Collier et al. showed that it specifically triggers the initiation of the process of translation.18 OCT-4 is a protein that is also known as POU5F1, which is encoded by POU5F1.19 OCT-4 seems to either suppress or induce the function of the Rex1 promoter, a known factor of pluripotency that is found in undifferentiated embryonic stem cells.20,21 Previously it has been used in experimental models along with Klf4 in order to produce pluripotent stem cells with the process of cell reprogramming.22 Several studies have proposed the existence of OCT-4 in several cancer cell lines as a marker of the existence of cancer stem cells.23–25 We have previously demonstrated that both DAZL and OCT-4 are expressed in HeLa cell lines.9 Liu et al. have confirmed that OCT-4 is overexpressed in HPVinfected cervical cancer cell lines.10 In two, almost synchronous studies, they also reported that OCT-4 expression was higher among HPV-16 lesions, compared to HPV-negative lesions.16,26 They proposed that, in normal states, a repressor complex binds to the hyper-methylated regulatory regions of OCT-4 and suppress its expression in HPV-negative cervical cells. When cervical cancer is triggered by HPV-16, however, its oncoproteins destroy the repressor complex, thus leading to upregulation of OCT-4. Due to these observations we studied the expression of both DAZL and OCT-4 in normal cell cultures and precancerours lesions, in order to determine whether these pathways are triggered at early, precancerous stages or late, after the invasion of the basal membrane of the cervical epithelium. In the present study we
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relied on pluripotent properties of DAZL and OCT-4 in order to evaluate whether they are expressed in cultures from cervices without pathology and from cervices with HPV and CIN lesions. It seems that DAZL expression does not participate in cultures of normal cervices and those with early preinvasive lesions. It may be that its role is detected in the subsequent dedifferentiation of cancerous cells. This hypothesis still needs further investigation in future studies. The present study, however, has shown that both normal cervices and early precancerous lesions (including HPV infection and CIN) express OCT-4. Furthermore OCT-4 was significantly upregulated in HPV-infected lesions; an effect that is, however, lost in lesions with CIN. It is possible that OCT-4 may interfere during the precancerous cervical activity, thus having significant participation in early cervical pathology. A weakness of the present study was that CIN cells could be mixed with HPV-only infected cells during specimen collection. Therefore, future studies should investigate the present findings among isolated strains of cell cultures in order to investigate whether OCT-4 truly represents an early marker of cervical carcinogenesis and whether it could serve as a target for the treatment of cervical cancer. OCT-4 is expressed both in cells of normal cervical cultures and among those derived from HPV and CIN lesions. What is more important to denote is that its expression is significantly upregulated during the early stages of cervical pathology. Future studies should clarify whether OCT-4 is an important factor that could be used in the future for early screening and targeted therapy of HPV and CIN cervical lesions.
Disclosure The authors report no declarations of interest.
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
OCT-4 and DAZL in CIN and HPV infection
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