Abstract
The CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate physiological processes such as energy metabolism, inflammation, cell cycle, and the development and differentiation of several tissues including skin. Recently, a role for C/EBPs in tumor cell proliferation and differentiation has been proposed, but the incomplete characterization in the literature of multiple translational isoforms of these proteins has made interpretation of these roles difficult. Therefore, we have carefully reexamined C/EBP isoform expression in human non-melanoma skin cancers. C/EBPα, C/EBPβ, and C/EBPδ were analyzed histologically in squamous cell carcinomas (SCC). The individual isoforms of C/EBPα and C/EBPβ were examined by immunofluorescent digital imaging, western blotting and DNA binding activity (electrophoretic mobility shift analysis). Expression of all C/EBP family proteins was decreased in SCC tumors. Suppression was greatest for C/EBPα, less for C/EBPβ, and least for C/EBPδ. Western analyses confirmed that C/EBPα p42 and p30 isoforms were decreased. For C/EBPβ, only the abundant full-length isoform (C/EBPβ−1, LAP*, 55 kD) was reduced, whereas the smaller isoforms, C/EBPβ−2 (LAP, 48 kD) and C/EBPβ−3 (LIP, 20 kD), which are predominantly nuclear, were significantly increased in well- and moderately-differentiated SCC (up to 14-fold for C/EBPβ−3). These elevations correlated with increases in PCNA, a marker of proliferation. Although C/EBPβ displayed increased post-translational modifications in SCC, phosphorylation of C/EBPβ−1 (Thr 235) was not altered. C/EBP-specific DNA binding activity in nuclear and whole-cell extracts of cultured cells and tumors was predominantly attributable to C/EBPβ. In summary, two short C/EBPβ isoforms, C/EBPβ−2 and C/EBPβ−3, represent strong candidate markers for epithelial skin malignancy, due to their preferential expression in carcinoma versus normal skin, and their strong correlation with tumor proliferation.
Highlights
C/EBP transcription factors (C/EBPs), a family of six gene members in which C/EBPa, C/EBPb, and C/EBPd have been the most intensively studied, were originally identified as regulators of growth and differentiation in normal tissues [1,2,3]
C/EBPd gene has been reported to be methylated in acute myelomoncytic leukemia (AML), cervical, breast and hepatocellular carcinoma and reduced expression associated with progression of breast tumors [22]
To obtain qualitative information about expression levels and intracellular localization of C/EBP transcription factors in skin cancers in vivo, we used primary antibodies to C/EBPa, C/ EBPb, and C/EBPd to perform immunostaining on frozen squamous cell carcinomas (SCC) specimens and adjacent normal skin (Fig. 1)
Summary
C/EBP transcription factors (C/EBPs), a family of six gene members in which C/EBPa, C/EBPb, and C/EBPd have been the most intensively studied, were originally identified as regulators of growth and differentiation in normal tissues [1,2,3]. C/EBPb expression, in contrast to C/EBPa, was reportedly increased in advanced cases of breast, ovarian, colorectal, renal, and gastric carcinoma [15,16,17,18,19,20,21]. Interpretation of those reports is complicated by the existence of three different isoforms of C/EBPb, a major topic of the current study (see below). Other C/EBP family genes have received relatively less attention, but potential involvement of dysregulated C/EBPe [23], and C/EBPf/gadd153 [24] in leukemia and melanoma, respectively, has been suggested
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