Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Jun;162(6):1789-806.
doi: 10.1016/S0002-9440(10)64314-3.

Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression

Geza Acs  1 , Paul J Zhang, Cindy M McGrath, Peter Acs, John McBroom, Ahmed Mohyeldin, Suzhen Liu, Huasheng Lu, Ajay Verma
Affiliations

Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression

Geza Acs et al. Am J Pathol. 2003 Jun.

Abstract

Tissue hypoxia is a characteristic property of cervical cancers that makes tumors resistant to chemo- and radiation therapy. Erythropoietin (Epo) is a hypoxia-inducible stimulator of erythropoiesis. Acting via its receptor (EpoR), Epo up-regulates bcl-2 and inhibits apoptosis of erythroid cells and rescues neurons from hypoxic damage. In addition to human papillomavirus infection, increased bcl-2 expression and decreased apoptosis are thought to play a role in the progression of cervical neoplasia. Using reverse transcriptase-polymerase chain reaction and Western blotting we showed that HeLa and SiHa cervical carcinoma cells and human cervical carcinomas express EpoR, and that hypoxia enhances EpoR expression. Exogenous Epo stimulated tyrosine phosphorylation and inhibited the cytotoxic effect of cisplatin in HeLa cervical carcinoma cells. Using immunohistochemistry, we examined the expression of Epo, EpoR, p16, hypoxia-inducible factor (HIF)-1alpha, and bcl-2 in benign and dysplastic cervical squamous epithelia and invasive squamous cell carcinomas (ISCCs). EpoR expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse EpoR expression was found in all ISCCs. Expression of Epo and HIF-1alpha was increased in dysplasias compared to benign epithelia. Focal Epo and HIF-1alpha expression was seen near necrotic areas in ISCCs, and showed correlation in their spatial distribution. Significant correlation was found between expression of EpoR, and p16 and bcl-2 in benign and dysplastic squamous epithelia. Our results suggest that increased expression of Epo and EpoR may play a significant role in cervical carcinogenesis and tumor progression. Hypoxia-inducible Epo signaling may play a significant role in the aggressive behavior and treatment resistance of hypoxic cervical cancers.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
RT-PCR analysis of Epo and EpoR expression in HeLa and SiHa cervical carcinoma cells. A: HeLa and SiHa cells cultured under normoxia (N, 21% O2) or hypoxia (H, 1% O2) were analyzed for Epo and EpoR mRNA expression. Although abundant expression of EpoR mRNA was detected in HeLa cells under both conditions, SiHa cervical carcinoma cells showed a prominent increase in EPOR mRNA expression after hypoxia treatment. Epo mRNA expression could not be detected in either cell lines. B: In contrast, human hepatoma Hep3B and glioblastoma U251 cells expressed abundant Epo mRNA.
Figure 2.
Figure 2.
HeLa cervical carcinoma cells express hypoxia-inducible and functional EpoR. A: Hypoxia activates HIF-1α and stimulates EpoR expression in cervical carcinoma cells. HeLa cells were cultured under normoxia (N, 21% O2) or hypoxia (H, 1% O2) for 3 hours and then their nuclear extracts examined by Western blotting for HIF-1α (HIF) immunoreactivity. For evaluation of EpoR expression, membranes from HeLa cells cultured under normoxia (N) or hypoxia (H) for 24 hours were used. HeLa cells cultured under normoxia (N) express a strongly immunoreactive band at Mr ∼66,000 identified with the antibody. Under normoxic culture conditions HeLa cells show very low levels of HIF-1α expression. Hypoxia (H) for 3 hours dramatically increased the level of HIF-1α protein expression and resulted in an increase in EpoR protein expression after 24 hours. B: EpoR-mediated activation of tyrosine phosphorylation in HeLa cells. HeLa cells were treated with either 10 U/ml of rHuEpo (Epo) or 1 μg/ml of Epo-mimetic peptide (Emp1) for 30 minutes and then analyzed for tyrosine phosphorylation by Western blotting. Phosphotyrosine immunoreactivity was dramatically enhanced by both Epo and Emp1 with the most prominent effect being at a molecular weight of 120 to 130 kd. C: Western blot analysis of cervical tissue samples (1, benign squamous mucosa; 2 to 8, cervical carcinoma; 9, HeLa cervical carcinoma cells) in the absence (−pep) or presence (+ pep) of EpoR-blocking peptide. In the absence of the blocking peptide a prominent immunoreactive band at Mr ∼66,000 corresponds to the EpoR protein in the tumor samples. No EpoR expression is seen in the sample of benign mucosa. Note the absence of the immunoreactive band at Mr ∼66,000 in the presence of the blocking peptide.
Figure 3.
Figure 3.
rHuEpo reverses toxicity of the chemotherapeutic drug cis-platin in cervical carcinoma cells. A: HeLa cells were treated with rHuEpo in the presence or absence of the general tyrosine kinase inhibitor genistein (10 μmol/L) for 1 hour. Cells were then exposed to cis-platin (10 μg/ml) for 20 hours. Cell viability was assessed using the MTT assay. Results show the fraction of surviving cells versus cells not treated with cis-platin. Epoetin α dose dependently inhibited cis-platin cytotoxicity and this effect was significantly diminished by genistein. B: Cis-platin-induced apoptosis in HeLa cells was examined by detection of membrane externalization of phosphatidylserine using the Annexin V assay. HeLa cells were pretreated with rHuEpo for 2 hours and then challenged with cis-platin (5 μg/ml) for 48 hours. Results are expressed as percentage of the apoptotic cell population compared to the control. Cis-platin-induced apoptosis was dose dependently reversed by rHuEpo (*, P < 0.05).
Figure 4.
Figure 4.
Expression of p16INK4a (p16) (A), EpoR (B), Epo (C), and HIF-1α (D) in benign cervical squamous epithelium, cervical dysplasia (CIN, grades I to III), and ISCC, determined by immunohistochemistry. Bars indicate median immunostaining score values.
Figure 5.
Figure 5.
Immunohistochemical expression of p16INK4a (p16), EpoR, Epo, HIF-1α, and bcl-2 in benign cervical squamous epithelium (benign), low-grade squamous dysplasia (CIN I), high-grade squamous dysplasia (CIN III), and ISCC (immunohistochemical stains with hematoxylin counterstain; H&E).
Figure 6.
Figure 6.
Expression of EpoR increases with increasing severity of dysplasia in cervical squamous epithelia. A: Immunohistochemical expression of EpoR in benign squamous epithelium and CIN grades I, II and III. In benign squamous epithelium EpoR expression is seen only in the basal one or two cell layers. With increasing severity of dysplasia EpoR immunostaining appears in the more superficial cell layers, and in CIN III, EpoR immunostaining is present in full thickness of the epithelium (immunohistochemical stains for EpoR with hematoxylin counterstain; H&E). B: EpoR expression increases with increasing severity of dysplasia in cervical squamous epithelia. Data are mean values ± SEM of percent epithelium thickness showing EpoR expression. C: Strong EpoR expression in endothelial and smooth muscle cells of a small artery and a lymphatic channel (arrowhead) (immunofluorescence staining for EpoR, tetramethyl-rhodamine isothiocyanate, red; nuclei are stained with 4,6-diamidino-2-phenylindole, blue; original magnification, ×400).
Figure 7.
Figure 7.
Expression of Epo and HIF-1α in cervical squamous cell carcinomas show correlation in spatial distribution. Prominent Epo (A) and HIF-1α (C) expression are present in tumor cells adjacent to necrotic areas (asterisk). Epo (B) and HIF-1α (D) expression is also present adjacent to tumor areas showing tumor cell keratinization. Double-immunohistochemical stains (E and F) show co-expression of Epo (cytoplasmic staining, red) and HIF-1α (nuclear staining, brown) in cervical carcinomas adjacent to necrotic areas (asterisk) (immunohistochemical stains with hematoxylin counterstain).
Figure 8.
Figure 8.
Distribution of HIF-1α immunostaining in benign and dysplastic cervical squamous epithelia. Basal, mid, and upper designate the basal, mid, and upper thirds of the squamous epithelium. Each bar represents one case and indicates the presence of immunostaining in the respective portion of the squamous epithelium. Note that in benign squamous epithelia HIF-1α immunostaining is primarily restricted to the suprabasal, middle portion of the epithelium. In contrast, in most cases of dysplasia HIF-1α immunostaining showed an abnormal distribution and was present in the basal and/or more superficial epithelial cells as well.
Figure 9.
Figure 9.
Correlation of immunohistochemical expression of EpoR with p16INK4a (p16) and bcl-2 in benign and dysplasic cervical squamous epithelia. A: The level of EpoR expression shows positive correlation with the expression of p16 (r = 0.8644, P < 0.0001, Spearman’s test). The line represents the calculated regression line. B: The level of EpoR expression shows positive correlation with the expression of bcl-2 (r = 0.4632, P < 0.0001, Spearman’s test). The line represents the calculated regression line.
Figure 10.
Figure 10.
Cells of cervical squamous dysplasia (CIN III) and ISCC show co-expression of EpoR (red) and p16INK4a (p16, green). Note that benign squamous epithelium (asterisk) shows weak EpoR expression in the basal cell layers, but no expression of p16INK4a. (Double-immunofluorescence stain for EpoR, tetramethyl-rhodamine isothiocyanate, red, and p16INK4a, fluorescein isothiocyanate, green; nuclei are stained with 4,6-diamidino-2-phenylindole, blue; H&E.)
Figure 11.
Figure 11.
Expression of EpoR and p16INK4a in dysplastic cervical squamous cells in SurePath liquid-based cervicovaginal cytological preparations. A, B, and E: Papanicolaou-stained preparations showing high-grade squamous intraepithelial lesion and background benign squamous cells. C and D: Cells of high-grade squamous intraepithelial lesion show prominent EpoR expression, whereas no immunostaining is seen in benign squamous cells (immunohistochemical stain for EpoR with hematoxylin counterstain). F to J: Cells of high-grade squamous intraepithelial lesion co-express p16INK4a (F and I, green), and EpoR (G and J, red). H: Triple band pass filter image showing the co-expression of both proteins in the same cells (yellow). (Double-immunofluorescence stain for EpoR, tetramethyl-rhodamine isothiocyanate, red, and p16INK4a, fluorescein isothiocyanate, green; nuclei are stained with 4,6-diamidino-2-phenylindole, blue.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rose PG: Chemoradiotherapy for cervical cancer. Eur J Cancer 2002, 38:270-278 - PubMed
    1. Advances in the treatment of cervical cancer. 20April2001. International Network for Cancer Treatment and Research, Brussels, Belgium Meeting
    1. Parkin DM, Pisani P, Ferlay J: Global cancer statistics. CA Cancer J Clin 1999, 49:31-64 - PubMed
    1. Parkin DM, Pisani P, Ferlay J: Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999, 80:827-841 - PubMed
    1. Greenlee RT, Murray T, Bolden S, Wingo PA: Cancer statistics, 2000. CA Cancer J Clin 2000, 50:7-33 - PubMed

Publication types

MeSH terms