Limits...
Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection.

Shukla S, Shishodia G, Mahata S, Hedau S, Pandey A, Bhambhani S, Batra S, Basir SF, Das BC, Bharti AC - Mol. Cancer (2010)

Bottom Line: Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells.In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades.We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, I-7, Sector-39, NOIDA, U.P., India.

ABSTRACT

Background: Recent observations indicate potential role of transcription factor STAT3 in cervical cancer development but its role specifically with respect to HPV infection is not known. Present study has been designed to investigate expression and activation of STAT3 in cervical precancer and cancer in relation to HPV infection during cervical carcinogenesis. Established cervical cancer cell lines and prospectively-collected cervical precancer and cancer tissues were analyzed for the HPV positivity and evaluated for STAT3 expression and its phosphorylation by immunoblotting and immunohistochemistry whereas STAT3-specific DNA binding activity was examined by gel-shift assays.

Results: Analysis of 120 tissues from cervical precancer and cancer lesions or from normal cervix revealed differentially high levels of constitutively active STAT3 in cervical precancer and cancer lesions, whereas it was absent in normal controls. Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells. Expression and activity of STAT3 were found to change as a function of severity of cervical lesions from precancer to cancer. Expression of active pSTAT3 was specifically high in cervical precancer and cancer lesions found positive for HPV16. Interestingly, site-specific accumulation of STAT3 was observed in basal and suprabasal layers of HPV16-positive early precancer lesions which is indicative of possible involvement of STAT3 in establishment of HPV infection. In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades.

Conclusion: We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.

Show MeSH

Related in: MedlinePlus

Cervical cancer cells over-express STAT3 with phosphorylation on both tyrosine and serine residues. (A) Cervical cancer cell lines C33a (HPV-), SiHa & CaSki (HPV16+) and HeLa (HPV18+) (2 × 106 cells) were lysed, and 50 μg of total cellular proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a PVDF membrane, and probed for STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin expression by respective antibodies. (B) STAT3 expression and phosphorylation increases as a function of severity of cervical lesions. Representative immunoblot of STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin indicating expression of respective protein in total cellular proteins (50 μg) isolated from normal (N), low grade squamous intra-epithelial lesion (LSIL1 & 2), high grade SIL (HSIL) and cervical cancer biopsy tissues (CaCx1, CaCx2) as described in Methods. (C) STAT3 RT-PCR analysis of cDNA derived from cervical cancer cell lines and cervical tissues. Representative ethidium bromide-stained agarose gel (3%) showing specific amplification STAT3 transcripts (318 bp) in the cDNA derived from total RNA of indicated samples (middle panel). Quality and quantity of RNA used for cDNA preparation was examined and confirmed by 1% agarose gel electrophoresis (upper panel). GAPDH RT-PCR (amplicon size 520 bp) was used as internal control (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2984472&req=5

Figure 1: Cervical cancer cells over-express STAT3 with phosphorylation on both tyrosine and serine residues. (A) Cervical cancer cell lines C33a (HPV-), SiHa & CaSki (HPV16+) and HeLa (HPV18+) (2 × 106 cells) were lysed, and 50 μg of total cellular proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a PVDF membrane, and probed for STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin expression by respective antibodies. (B) STAT3 expression and phosphorylation increases as a function of severity of cervical lesions. Representative immunoblot of STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin indicating expression of respective protein in total cellular proteins (50 μg) isolated from normal (N), low grade squamous intra-epithelial lesion (LSIL1 & 2), high grade SIL (HSIL) and cervical cancer biopsy tissues (CaCx1, CaCx2) as described in Methods. (C) STAT3 RT-PCR analysis of cDNA derived from cervical cancer cell lines and cervical tissues. Representative ethidium bromide-stained agarose gel (3%) showing specific amplification STAT3 transcripts (318 bp) in the cDNA derived from total RNA of indicated samples (middle panel). Quality and quantity of RNA used for cDNA preparation was examined and confirmed by 1% agarose gel electrophoresis (upper panel). GAPDH RT-PCR (amplicon size 520 bp) was used as internal control (lower panel).

Mentions: To determine STAT3 expression and its phosphorylation at Y705 and S727 residues in cervical cancer, HPV positive [SiHa & CaSki (HPV16+) and HeLa (HPV18+)] and HPV- cervical cancer cell lines (C33a) were tested for expression of STAT3 and pSTAT3 (Y705 & S727) by immunoblotting. As shown in Figure 1A, immunoblot analysis of cellular proteins demonstrated constitutive STAT3 expression in all cervical cancer cell lines although the degree of expression was variable. In contrast to HPV- C33a cells, all HPV positive cells had higher expression of STAT3. These cellular proteins were simultaneously tested for expression of phosphorylated STAT3 at Y705 & S727 by monoclonal antibodies that could distinguish between the two phosphorylated forms of STAT3 from each other. Interestingly, all four cell lines irrespective of HPV infection expressed STAT3 phosphorylated at Y705 & S727 residues, though HPV16+ cells expressed a comparatively higher level of phosphorylated forms in comparison to HPV- (C33a) or HPV18+ (HeLa) cells.


Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection.

Shukla S, Shishodia G, Mahata S, Hedau S, Pandey A, Bhambhani S, Batra S, Basir SF, Das BC, Bharti AC - Mol. Cancer (2010)

Cervical cancer cells over-express STAT3 with phosphorylation on both tyrosine and serine residues. (A) Cervical cancer cell lines C33a (HPV-), SiHa & CaSki (HPV16+) and HeLa (HPV18+) (2 × 106 cells) were lysed, and 50 μg of total cellular proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a PVDF membrane, and probed for STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin expression by respective antibodies. (B) STAT3 expression and phosphorylation increases as a function of severity of cervical lesions. Representative immunoblot of STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin indicating expression of respective protein in total cellular proteins (50 μg) isolated from normal (N), low grade squamous intra-epithelial lesion (LSIL1 & 2), high grade SIL (HSIL) and cervical cancer biopsy tissues (CaCx1, CaCx2) as described in Methods. (C) STAT3 RT-PCR analysis of cDNA derived from cervical cancer cell lines and cervical tissues. Representative ethidium bromide-stained agarose gel (3%) showing specific amplification STAT3 transcripts (318 bp) in the cDNA derived from total RNA of indicated samples (middle panel). Quality and quantity of RNA used for cDNA preparation was examined and confirmed by 1% agarose gel electrophoresis (upper panel). GAPDH RT-PCR (amplicon size 520 bp) was used as internal control (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984472&req=5

Figure 1: Cervical cancer cells over-express STAT3 with phosphorylation on both tyrosine and serine residues. (A) Cervical cancer cell lines C33a (HPV-), SiHa & CaSki (HPV16+) and HeLa (HPV18+) (2 × 106 cells) were lysed, and 50 μg of total cellular proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a PVDF membrane, and probed for STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin expression by respective antibodies. (B) STAT3 expression and phosphorylation increases as a function of severity of cervical lesions. Representative immunoblot of STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin indicating expression of respective protein in total cellular proteins (50 μg) isolated from normal (N), low grade squamous intra-epithelial lesion (LSIL1 & 2), high grade SIL (HSIL) and cervical cancer biopsy tissues (CaCx1, CaCx2) as described in Methods. (C) STAT3 RT-PCR analysis of cDNA derived from cervical cancer cell lines and cervical tissues. Representative ethidium bromide-stained agarose gel (3%) showing specific amplification STAT3 transcripts (318 bp) in the cDNA derived from total RNA of indicated samples (middle panel). Quality and quantity of RNA used for cDNA preparation was examined and confirmed by 1% agarose gel electrophoresis (upper panel). GAPDH RT-PCR (amplicon size 520 bp) was used as internal control (lower panel).
Mentions: To determine STAT3 expression and its phosphorylation at Y705 and S727 residues in cervical cancer, HPV positive [SiHa & CaSki (HPV16+) and HeLa (HPV18+)] and HPV- cervical cancer cell lines (C33a) were tested for expression of STAT3 and pSTAT3 (Y705 & S727) by immunoblotting. As shown in Figure 1A, immunoblot analysis of cellular proteins demonstrated constitutive STAT3 expression in all cervical cancer cell lines although the degree of expression was variable. In contrast to HPV- C33a cells, all HPV positive cells had higher expression of STAT3. These cellular proteins were simultaneously tested for expression of phosphorylated STAT3 at Y705 & S727 by monoclonal antibodies that could distinguish between the two phosphorylated forms of STAT3 from each other. Interestingly, all four cell lines irrespective of HPV infection expressed STAT3 phosphorylated at Y705 & S727 residues, though HPV16+ cells expressed a comparatively higher level of phosphorylated forms in comparison to HPV- (C33a) or HPV18+ (HeLa) cells.

Bottom Line: Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells.In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades.We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, I-7, Sector-39, NOIDA, U.P., India.

ABSTRACT

Background: Recent observations indicate potential role of transcription factor STAT3 in cervical cancer development but its role specifically with respect to HPV infection is not known. Present study has been designed to investigate expression and activation of STAT3 in cervical precancer and cancer in relation to HPV infection during cervical carcinogenesis. Established cervical cancer cell lines and prospectively-collected cervical precancer and cancer tissues were analyzed for the HPV positivity and evaluated for STAT3 expression and its phosphorylation by immunoblotting and immunohistochemistry whereas STAT3-specific DNA binding activity was examined by gel-shift assays.

Results: Analysis of 120 tissues from cervical precancer and cancer lesions or from normal cervix revealed differentially high levels of constitutively active STAT3 in cervical precancer and cancer lesions, whereas it was absent in normal controls. Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells. Expression and activity of STAT3 were found to change as a function of severity of cervical lesions from precancer to cancer. Expression of active pSTAT3 was specifically high in cervical precancer and cancer lesions found positive for HPV16. Interestingly, site-specific accumulation of STAT3 was observed in basal and suprabasal layers of HPV16-positive early precancer lesions which is indicative of possible involvement of STAT3 in establishment of HPV infection. In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades.

Conclusion: We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.

Show MeSH
Related in: MedlinePlus