Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix - PubMed (original) (raw)

Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix

S Nakagawa et al. Br J Cancer. 2004.

Abstract

Recently, a LAP protein, scribble, was identified in Drosophila epithelia as a basolateral protein that controls the apical-basolateral polarity. Loss of scribble causes disorganisation and overgrowth of the epithelia. Scribble has a human homologue, human scribble (hScrib), which is a substrate of ubiquitin-mediated degradation by human papillomavirus E6 and the E6AP ubiquitin-protein ligase. In the present study, we revealed that hScrib localised to the basolateral regions of the epithelial cell line MDCK and human uterine cervical epithelial tissues by immunofluorescence. Human scribble colocalised rather with the adherens junction protein E-cadherin, but not with the tight junction protein ZO-1. Histochemical analysis showed a dramatic decrease in the expression of hScrib with the progression of disease from normal uterine cervical tissues to invasive cervical cancers through the precursor lesions. In contrast, the expression of hScrib was retained in the throughout epithelial layer of the HPV-negative cervical high-grade squamous intraepithelial lesions (H-SIL). Although quantitative RT-PCR revealed no significant downregulation of hScrib mRNA expression in the H-SIL, it revealed a clear downregulation in the invasive cancers. These results suggest the possibility that degradation by HPV E6 is one of the causal roles for the progressive decrease of hScrib expression during the disease progression from low-grade squamous intraepithelial lesions to H-SIL, and a cooperative role of downregulation of hScrib mRNA expression and ubiquitin-mediated degradation of hScrib by E6 and E6AP led to the complete decrease of hScrib expression during the process of carcinogenesis from H-SIL to invasive cancer. These data underscore the importance of hScrib in the construction of tissue architecture and prevention of cancer development.

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Figures

Figure 1

Figure 1

Construction of the anti-hScrib antibody using its C-terminus. (A) Structure of hScrib consists of 16 canonical LRRs, followed by LAPSD (LAP-specific domain) and four PDZ domains. We generated an anti-hScrib antibody (anti-hScrib C-terminus) using C-terminus (AA 1208–1630) as antigens. (B) Anti-hScrib C-terminus antibody detected the endogenous hScrib protein with 220 kDa in molecular weight in 293 T cells (lane 1) and _in vitro_-translated hScrib (lane 2), but not _in vitro_-translated hDlg by Western blotting (lane 3).

Figure 2

Figure 2

Confocal images of immunofluorescence staining of hScrib, E-cadherin, and ZO-1 in the MDCK epithelial cell line. (A) Partial colocalisation of hScrib and E-cadherin at the basolateral membrane. (B) Localisation of hScrib and ZO-1. ZO-1 localised at the top of the basolateral membrane, where hScrib did not localise.

Figure 3

Figure 3

Immunofluorescence staining of hScrib and E-cadherin in uterine endocervical tissues. (A) Basolateral localisation of hScrib. (B) Localisation of E-cadherin, which shows the area of adherens junction. (C) Colocalisation of hScrib with E-cadherin, an adherens junctional protein.

Figure 4

Figure 4

Immunohistochemical detection of hScrib in normal uterine cervical epithelia and neoplasm. There was a dramatic decrease in the expression of hScrib with disease progression. (A) Normal squamous epithelia. (B) Endocervical epithelia. (C) Cervical condyloma, an HPV-related wart. (D) L-SIL (CIN1). (E) H-SIL (CIN3). (F) Invasive cervical cancer.

Figure 5

Figure 5

Western blot analysis of hScrib protein levels in cervical neoplasm and the normal counterparts using the anti-hScrib C-terminus antibody. (A) The representative result of three independent experiments. (B) Analysis of hScrib protein expression relative to _α_-tubulin in the cervical neoplasm. The results are expressed as the mean percentage of the normal counterpart. Lanes 1, 3, and 5: the normal counterpart; Lane 2: L-SIL; Lane 4: H-SIL; Lane 6: invasive cancer.

Figure 6

Figure 6

Immunohistochemical detection of hScrib in a case with H-SIL without HPV infection. The normal hScrib expression is seen throughout the epithelial layer.

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