Immunocytological and preliminary immunohistochemical studies of prothymosin alpha, a human cancer-associated polypeptide, with a well-characterized polyclonal antibody

Abstract

Prothymosin alpha (ProTalpha) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTalpha immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTalpha or the synthetic fragments ProTalpha[1-28], ProTalpha[87-109], and ProTalpha[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTalpha[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTalpha in tumorigenesis.

Figure 1

ELISA displacement curves obtained with the anti-prothymosin α (ProTα)[101-109]/keyhole limpet hemocyanin (KLH) antibody G in the presence of increasing concentrations (100 ng/ml to 100 μg/ml) of ProTα, ProTα[1-28], ProTα[87-109], and ProTα[101-109]. Symbols corresponding to the intact polypeptide and the different synthetic fragments are shown as an inset.

Figure 2

Western blot analysis of cell and nuclear lysates from HeLa cells, PANC-1 cells, and human fetal fibroblasts: PANC-1 cells, cell lysate, 60 μg total protein (Lane 1); PANC-1 cells, cell lysate, 30 μg total protein (Lane 2); HeLa cells, cell lysate, 60 μg total protein (Lane 3); HeLa cells, cell lysate, 30 μg total protein (Lane 4); human fetal fibroblasts, cell lysate, 30 μg total protein (Lane 5); ProTα, 10 ng (Lane 6); PANC-1 cells, nuclear lysate, 30 μg total protein (Lane 7); HeLa cells, nuclear lysate, 60 μg total protein (Lane 8).

Figure 3

Immunolocalization of ProTα in HeLa (A) and PANC-1 (B) cells. Dual-labeling (A1,B1), labeling for DNA (A2,B2), and specific immunolabeling for ProTα with the anti-ProTα[101-109] antibody G (A3,B3) are presented. As clearly observed, ProTα-like immunoreactivity is localized almost exclusively in the cell nucleus.

Figure 4

(A) ProTα-negative normal prostatic gland adjacent to strongly positive adenocarcinoma cells. (B) Diffuse strong ProTα positivity of both adenocarcinoma and prostatic intraepithelial neoplasia lesions. (C) Prostatic tissue stained with anti-ProTα[101-109]/KLH antibody G. (D) Negative control, i.e., prostatic tissue stained with anti-ProTα[101-109]/KLH antibody G preincubated with ProTα.