Decreased synthesis and expression of TGF-?1, ?2, and ?3 in epithelium of HPV 16-positive cervical precancer: a study by microdissection, quantitative RT-PCR, and immunocytochemistry

Abstract

Cervical carcinogenesis is a multistep process initiated by 'high-risk' human papillomaviruses (HR-HPVs), most commonly HPV 16. Transforming growth factor-beta (TGF-beta) inhibits epithelial proliferation and down-regulates transcription of E6/E7 genes of HPV. Altered TGF-beta expression may be important in carcinogenesis. Quantitative RT-PCR was used to investigate TGF-beta1, beta2, and beta3 mRNA levels in nine specimens of normal cervix and 15 cervical precancers (eight HPV-positive, including five HPV 16-positive). Immunocytochemical expression of TGF-beta1, beta2, and beta3 was examined in cervical intraepithelial neoplasia (CIN) positive for HPV 16 (26), and in HPV-negative, normal ectocervical epithelium (9); reserve cell hyperplasia (12); and immature (7) and mature (15) squamous metaplasia. The intensity of staining for TGF-beta1 was measured using grey-scale image analysis. Microdissection was used to investigate epithelial and stromal (excluding crypts) levels of TGF-beta1 mRNA in HPV 16-positive cervical precancer. Normal cervix, including reserve cells and immature and mature metaplasia, showed strong immunocytochemical expression of all TGF-beta isoforms. Expression was decreased in the basal third of the epithelium in CIN 1, in the basal and middle thirds in CIN 2, and in all layers in CIN 3. Quantitative analysis of TGF-beta1 expression showed that the changes in CIN compared with normal ectocervix and mature metaplasia were statistically highly significant (p<0.001, ANOVA). TGF-beta1, beta2, and beta3 mRNA levels showed a significant decrease only in the five HPV 16-positive CIN samples when compared with normal (p=0. 0034, 0.0033, and 0.029, respectively). TGF-beta mRNA levels in HPV 16-positive epithelium also decreased from normal through low-grade to high-grade precancer. Stromal TGF-beta1 was absent or very low compared with epithelial production and was not altered in HPV 16 precancer. Progressive loss of epithelial TGF-beta expression and synthesis may be important in HPV 16-associated human cervical carcinogenesis.

Depletion of Langerhans Cells in Human Papillomavirus Type 16-Infected Skin Is Associated with E6-Mediated Down Regulation of E-Cadherin

“…TGF-␤1 Ϫ/Ϫ SCID mice are deficient of epidermal LC but LC infiltration can be induced at localized sites in these mice after injection with TGF-␤1-expressing cells (42). HPV16 ϩ cervical precancerous lesions display reduced expression of TGF-␤1, and this is may impact on the infiltration and maturation state of LC at the site of the infection (16). These data suggest that in addition to the loss of E-cadherin in HPV16-infected tissue and its effect on LC retention, signals for LC immigration and maturation may also be impaired.…”

Quantitative analysis of IL-10 and IFN-? mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer

“…The mimic was slightly different in size (five bases shorter) from the target gene fragment for definite Genescan detection, but was close enough in size to preserve minimal difference in amplification efficiency. To localize the cellular source of cytokines, microdissection from archival histological material was combined successfully with accurate mRNA quantitation, as in our previous study of TGF‐β34. Both frozen sections and paraffin‐embedded tissues have been used to study mRNAs, with variable results35–37.…”