Recently, Yang et al (1998) produced a polyclonal antibody that proved to be specific for an actin fragment generated by caspase 3 and possibly other caspases during apoptosis. That antibody, named fractin, did not react with intact actin filaments. Fractin expression was found in colchicine-induced apoptosis in retinoic acid-differentiated human neuroblastoma cells. In cryostat sections of postmortem human brain, fractin staining was observed in the processes and cell bodies of degenerating neurons and plaque-associated microglia in Alzheimer's disease. Here, double-labeling with TUNEL and fractin antibody could be used to identify cells with an apoptotic phenotype. It was speculated that fractin immunostaining might also be useful in other pathological conditions.

We now report the use of this polyclonal fractin antibody in identifying apoptotic cells and apoptotic bodies in different human cell types. Using antigen retrieval (AR) and immunohistochemistry, the antibody could be applied with formalin-fixed and paraffin-embedded tissue in addition to cryostat sections, allowing retrospective studies on archival paraffin-embedded material. Apoptotic cells and apoptotic bodies were visualized in different epithelial and mesenchymal cells, as well as in tumors of epithelial and mesenchymal origin. In these (tumor) cells, weak and moderate fractin immunostaining was seen in cells with light microscopic features of apoptosis, such as nuclear shrinkage and chromatin condensation, whereas fragmented apoptotic bodies showed strong immunostaining (Fig. 1).

We tested different AR protocols, including microwaving AR with 10 mm citrate buffer, pH 6 (Cattoretti et al, 1993); pressure cooking AR with 1 mm EDTA, pH 8 (Pileri et al, 1997); and overnight heating AR at 70šC in 10 mm Tris HCl buffer, pH 9 (Koopal et al, 1998). The pressure cooker method with EDTA, pH 8, proved to give most consistent results, even in tissue fixed in formalin for several days, although strong and diffuse fractin staining was only found in tissue fixed in formalin for up to 24 hours.

The polyclonal fractin antibody was obtained by injecting rabbits with a synthetic peptide (K-YELD) representing the last five amino acids of the C terminus of the 32-kd actin fragment produced during apoptosis, |gb-actin residues 240-245 (YELPD), coupled via the K-residue to a carrier protein (purified protein derivative of tuberculin) (Yang et al, 1998). Stock solutions of the antibody were made by resuspension of 40 ul lyophilized rabbit antiserum in 40 ul distilled water. Immunostaining was performed as follows: 3-um paraffin sections were mounted on 3-aminopropyltriethoxysilane-coated slides. After AR and blocking of endogenous peroxidase by incubation with 0.3% hydrogen peroxide for 30 minutes, the slides were incubated with the fractin antibody, diluted 1:1000 in 1% bovine serum albumen. Visualization was achieved with a one step direct peroxidase detection system. Slides were incubated with goat anti-rabbit peroxidase conjugated antibody (DAKO, Copenhagen, Denmark), diluted 1:50 in 1% human serum in 1% bovine serum albumen for 30 minutes, followed by chromogen development with 3-3`-diamminobenzidine tetrahydrochloride and counterstaining with hematoxylin. The sections were washed 3x with PBS after each step. Negative controls omitting the primary antibody were included.

In human tumors of epithelial origin, eg, basal cell carcinoma of the skin, apoptotic bodies were typically seen within the proliferative compartment of the tumor. In colonic adenocarcinoma (Fig. 1A) many apoptotic bodies were present within luminal debris in glandular tumor formations, and focal staining was also seen in the cytoplasm of carcinoma cells lining these formations. In soft tissue sarcomas, areas with apoptotic bodies also harboured some tumor cells with cytoplasmic fractin staining (Fig. 1B). In areas with replacement fibrosis in myocardium in ischemic heart disease with end-stage heart failure, apoptotic bodies were found only in interstitial mesenchymal cells, whereas cardiomyocytes, containing abundant sarcomeric and nonsarcomeric actin filaments, were always negative. In lymphoid tissue (appendix, tonsil, and lymph node), apoptotic bodies were abundantly present in hyperplastic follicles in the cytoplasm of starry sky macrophages, but also in between the follicle center cell population (Fig. 1C). In a liver allograft with chronic rejection, many apoptotic bodies were seen ingested by Kupffer cells (Fig. 1D), and in a lymph node draining a colonic adenocarcinoma, apoptotic bodies were observed in the cytoplasm of scattered macrophages.

The preferential staining of fractin in apoptotic bodies supports the general concept that caspase cleavage of actin occurs in the late, irreversible phase of apoptosis. It still has to be established whether actin cleavage by caspases is a universal mechanism in apoptosis in different pathological conditions.

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