Trophoblast apoptosis in human placenta at term as detected by expression of a cytokeratin 18 degradation product of caspase

CONTEXT: Apoptosis occurs in the normal placenta. The monoclonal antibody M30 is directed against a novel epitope of cytokeratin 18 (CK18) that is formed by caspase cleavage early in the apoptotic cascade, and this antibody may therefore be useful for evaluating trophoblast apoptosis. OBJECTIVE: We undertook the present study to evaluate the use of monoclonal antibody M30 to assess trophoblast apoptosis in placenta at term. METHODS: We stained paraffin-embedded placental tissues from 15 deliveries at term with M30. We compared positive M30 staining and CK18 staining (as detected by a monoclonal antibody directed against CK18) of trophoblasts in serial slides. We also compared apoptotic rates as detected by M30 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) in 7 of the placentas. RESULTS: In fields of villous tissue, most M30-positive cells were CK18-positive syncytiotrophoblasts. Approximately half of M30-positive cells occurred as focal positive staining in the syncytial layer, and half occurred as abundant staining of syncytiotrophoblasts in areas with increased intervillous or perivillous fibrinoid. We found very few M30-positive cells in villous stroma. In decidual/basal plate tissues, most (two thirds) of the M30-positive cells were CK18-positive extravillous trophoblasts, whereas one third were syncytiotrophoblasts of anchoring villi. Since TUNEL detects apoptosis in both epithelial and nonepithelial cells, more cells were positively stained with TUNEL than with M30 in some tissue fields. However, our observations suggest that M30 was more sensitive than TUNEL in recognizing apoptotic trophoblasts and had less nonspecific staining than TUNEL. CONCLUSION: We recommend the use of monoclonal antibody M30 for apoptosis studies in placental tissues. This antibody is easy to handle, the staining obtained seems specific, and the nonspecific staining seems negligible.     

Apoptosis and expression of bcl-2 protein in invasive ductal adenocarcinoma of the pancreas

Apoptosis plays a central role in the development and/or progression of cancer. There are several methods for detection of apoptotic cells in tissue sections including light and electron microscopy, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNEL) and immunohistochemical detection of proteins associated with apoptosis. Apoptosis was assessed by the monoclonal antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18 that becomes available at an early caspase cleavage during apoptosis. Expression of bcl-2 protein was evaluated, because bcl-2 protein plays an important role in the regulation of apoptosis. Twenty-six invasive ductal adenocarcinomas of the pancreas were studied immunohistochemically with antibodies M30 and bcl-2. The mean apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells number) was 2.75%. High AI (> 10%) was observed in 4 cases of the 26 pancreatic carcinomas (15%). Protein bcl-2 was expressed in 3 cases (11.5%). The AI did not correlate with the expression of protein bcl-2. In conclusion, the detection of neo-epitope in cytokeratin 18 by monoclonal antibody M30 can be used for quantification of apoptotic cells with immunohistochemical techniques in tissue sections. It is a new approach to evaluate apoptosis in pancreatic carcinomas. The low positivity of bcl-2 expression in pancreatic adenocarcinomas suggests that bcl-2 protein does not play a central role in pancreatic tumorigenesis and cancer progression.     

General suitability of techniques for in situ detection of apoptosis in small intestinal epithelium

The present study was designed to evaluate different techniques for the in situ detection of apoptosis in human and rat small intestinal epithelium. The techniques included light microscopy (LM) and transmission electron microscopy (TEM) observation of epoxy resin-embedded tissue, scanning electron microscopy (SEM), TUNEL assay, and antibodies directed against caspase cleavage products of caspase 3, cytokeratin 18 (CK 18), and apoptotic single-strand DNA (ssDNA). All techniques, if the labeling was positive, showed apoptotic cells exclusively at the villus tip. LM and TEM were the most reliable and revealed morphological signs typical of cells that have died via apoptosis. SEM indicated the extension of the process. The antibody recognizing cleaved caspase 3 could be considered an appropriate marker for apoptotic epithelial cells in human and rat small intestine. However, the majority of epithelial cells lining the proximal small intestinal villus contained only low levels of intact CK 18. Therefore, sufficient amounts of cleaved CK 18 for immunohistochemical detection were not generated during apoptosis, rendering the application of the antibody inappropriate. The antibody detecting formamide-denatured ssDNA in apoptotic cells was both suitable and reliable; however, the particular staining procedure used compromised the tissue preservation. In comparison to this, the TUNEL assay was less reliable. Although it was performed with a commercially available ready-to-use kit, its application conditions had to be adjusted for each specimen on the basis of the findings produced by other techniques.     

Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope

Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.     

Lytic infection with vaccinia virus activates caspases in a Bcl-2-inhibitable manner

Vaccinia virus (VV) is considered to cause lytic infection of most cells, with lysis being regarded equivalent to necrosis. Activation of caspases has not been associated with necrosis. However, we observed the activation and activity of caspases in epithelial cells HeLa G and BSC-40 lytically infected with VV. Using three different flow-cytometric approaches, we characterized the distinct stages of caspase cascade in VV-infected cells: a cleaved, activated form of caspases detected using a fluorescent pan-caspase inhibitor; caspase activity assayed by cleavage of a non-fluorescent substrate into a fluorescent product; caspase-specific cleavage of death substrates characterized by a fluorescent antibody detecting a neo-epitope in cytokeratin-18. All of these approaches yielded an increased fluorescent signal in VV-infected cells compared to mock-infected controls. Additionally, the signal was decreased by the expression of Bcl-2. The cleavage of cytokeratin-18 was confirmed by western blotting, but another key protein involved in apoptosis, PARP, was not cleaved in VV-infected lytic cells. The necrotic phenotype of the cells was confirmed by increased cell membrane permeability and/or decreased mitochondrial membrane potential. In conclusion, our data suggest that VV infection of the epithelial cells HeLa G and BSC-40 initiates the apoptotic program, however, apoptosis is not completed and switches into necrosis.     

Detection of the M30 neoepitope as a new tool to quantify liver apoptosis: timing and patterns of positivity on frozen and paraffin-embedded sections

One of the first stages of apoptosis is cytokeratin cleavage mediated by caspases, which is associated with the expression of a neoepitope, the cleavage site of cytokeratin 18, identifiable by the M30 monoclonal antibody. The aim of this study was to evaluate the timing of neoantigen expression and its modifications in the various morphologic stages of apoptosis on frozen and paraffin-embedded sections from liver biopsies of patients with chronic hepatitis or transplanted liver. The appearance of this neoepitope coincides with the gradual disappearance of cytokeratins, with the appearance of nuclear DNA fragmentation, and with the presence of Councilman bodies. The staining patterns on paraffin-embedded sections of liver specimens were similar to those found in frozen sections, with a reduced sensitivity. The M30 antibody is correlated with apoptosis, and its specificity for epithelial cells makes this method the first choice for routine evaluation of apoptosis in liver epithelial cells.     

Melanosis of the appendix: common in the paediatric age group

AIMS: The objective of this study was to assess apoptotic activity in gestational trophoblastic disease (GTD) and its prognostic value in hydatidiform mole (HM). METHODS AND RESULTS: Expression of the specific caspase cleavage site within cytokeratin 18 was assessed immunohistochemically using the monoclonal antibody M30 CytoDeath in 12 spontaneous abortions, 22 partial and 57 complete HM, eight choriocarcinoma (CCA) and 28 normal placentas. The M30 immunoreactivity occurred predominantly in the syncytiotrophoblasts. A significantly higher M30 index in HM and CCA was found when compared with normal placentas and spontaneous abortions (P < 0.001). The M30 index of those HM which spontaneously regressed was significantly higher than those HM which developed persistent disease requiring chemotherapy (P < 0.001). The M30 index correlated with another apoptotic index previously detected by TdT-mediated dUTP nick-end labelling (TUNEL) (P = 0.007) and the proliferation index assessed by the Ki67 antigen (P = 0.034). CONCLUSIONS: We conclude that apoptosis is important in the pathogenesis of GTD. Assessment of apoptotic activity in HM by the M30 index may be considered as an alternative prognostic indicator for predicting the clinical behaviour.     

Assessment of apoptosis by M30 immunoreactivity and the correlation with morphological criteria in normal colorectal mucosa, adenomas and carcinomas

Aims : To investigate the monoclonal antibody M30 for the assessment of apoptosis in colorectal tissues. Although Terminal deoxyribonucleotidyl transferase mediated nick end labelling (TUNEL) and in‐situ end labelling (ISEL) are the methods most often used to demonstrate and quantify apoptosis in histological tissue sections, the interpretation and specificity of these techniques have been controversial. Immunohistochemistry using the monoclonal antibody M30 that recognizes caspase‐cleaved cytokeratin 18 is considered to be a promising alternative but has yet to be validated against a generally accepted standard. Methods and results : Paraffin sections of normal colonic mucosa (n = 30), normal mucosa obtained from resection margins from carcinomas (n = 30), colorectal adenomas (n = 84) and carcinomas (n = 40) were studied. Apoptosis of epithelial cells was assessed by M30 immunoreactivity and morphological criteria and expressed as a proportion of the total number of cells counted (apoptotic index). Mean apoptotic indices using M30 were 0.18 ± 0.04% in normal mucosa, 0.42 ± 0.04% in adenomas and 1.97 ± 0.24% in carcinomas. Using morphological criteria, these indices were 0.23 ± 0.03%, 0.62 ± 0.06% and 1.78 ± 0.19%, respectively. Apoptotic counts were higher in normal mucosa obtained from resection margins than in genuinely normal mucosa using the M30 antibody. Apoptotic indices obtained by M30 immunoreactivity and morphological criteria were positively correlated (r = 0.71, P < 0.01). Conclusion : Assessment of apoptotic cells by M30 immunoreactivity correlates well with morphological criteria. Apoptotic indices increase in the course of the adenoma–carcinoma sequence. Apoptosis in normal mucosa obtained from resection margins differs from genuinely normal mucosa necessitating caution when interpreting studies of apoptosis in normal colonic mucosa. Our findings support the use of the M30 method in the study of apoptosis in colorectal tissues.     

Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: a comparative study with cytokeratin 8

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N -acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.     

The clinical use of a P63/cytokeratin7/18/cytokeratin5/14 antibody cocktail in diagnostic breast pathology

An antibody cocktail directed against p63, cytokeratin (CK)5/14, and CK7/18 is reported to be useful in distinguishing noninvasive from invasive breast lesions and for the characterization of intraductal epithelial proliferations. However, limited studies evaluate its use in clinical practice. A retrospective review of breast material at a university medical center identified cases that were immunostained with the above antibody cocktail. Additional p63 immunostaining alone was performed to further determine the utility of the antibody cocktail in the evaluation of invasion. Of 50 breast cases identified, the antibody cocktail was used to confirm or exclude invasion in 44 (88%). Twenty-two (50%) of these had easily identifiable p63/CK5/14-positive myoepithelial cells, whereas the remainder lacked such staining, confirming the diagnosis of invasive carcinoma. In 27 cases with available diagnostic material for additional p63 immunostaining, the cocktail better highlighted myoepithelial cells by staining nuclei and cytoplasm. Easier identification of invasion was also facilitated by CK7/18 expression in invasive foci, especially those composed of single cells. Ten cases were immunostained to help determine the nature of an intraductal proliferation. The cocktail demonstrated a mosaic staining pattern of both CK7/18- and CK5/14-positive epithelial cells in 3 (30%) cases consistent with usual hyperplasia; homogenous CK7/18 expression in the remaining cases supported the diagnosis of atypical ductal hyperplasia or carcinoma in situ. In summary, the p63/CK7/18/CK5/14 cocktail stain appears to be a useful tool in diagnostic breast pathology, in the evaluation of possible invasion, particularly in the setting of minute foci of invasion as well as in epithelial proliferations.     

Circulating Breast Cancer Cells Are Frequently Apoptotic

Automatic search for cytokeratin/mucin-1 double immunofluorescence was performed to detect and characterize circulating epithelial tumor cells in patients with advanced breast cancer. The peripheral blood samples in 8 of 19 patients (42.1%) presented with cytokeratin-positive and epithelial-type mucin-positive (CK(+)/MUC1(+)) tumor cells. Detailed microscopic analysis, however, suggested that the majority of the double immunopositive cells was apoptotic according to an "inclusion type" cytokeratin staining pattern and nuclear condensation. Furthermore, apoptosis-related DNA strand breaks could be demonstrated by applying the TdT-uridine nick end labeling assay in these cells. In 3 of 8 positive samples all of the CK(+)/MUC1(+) cells displayed apoptotic features. We conclude that apoptotic cells significantly contribute to the circulating tumor cell fraction in breast cancer patients. As the predictive value of such cells for the outcome of the disease is unclear, they should be considered separately when analyzing tumor cell dissemination.     

Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation

Keratins 18 and 19 (K18/K19) are epithelial-specific intermediate filament proteins. Apoptosis induces caspase cleavage at the highly conserved K18 or K19 Asp237, which in K18 is preceded by cleavage at Asp396. We characterized the keratin N-terminal fragments that are generated upon caspase digestion of K18/K19 at Asp237 in order to study keratin dynamics during apoptosis. This was carried out by generating and characterizing antibodies selective to K18/K19 Asp237. K18 or K19 peptides that expose Asp237 in 234VEVD were used for rabbit immunization. The generated antibodies recognized cleaved but not intact K18/K19, exclusively, as determined by blotting or immunofluorescence staining of apoptotic human HT29 cells or livers isolated from Fas-Ab-injected mice. Antibodies to K18/K19 Asp237 recognized the common VEVD-motif as determined by immunoblotting of cells transfected with K18, K19 or K20. The K18/K19 VEVD-directed antibodies demonstrated sequential Asp396 then Asp237 K18 cleavage during apoptosis. Specific-keratin selectivity of the anti-Asp237 antibodies was confirmed by their inability to recognize K14 after UV-induced apoptosis in transfected cells. The Asp237-containing apoptotic keratin fragments are secreted into the medium of cultured HT29 cells and are stable up to 96 h after inducing apoptosis. Furthermore, the generated antibodies recognize keratin apoptotic fragments in sera of mice undergoing hepatocyte apoptosis and sera of patients with cirrhosis, and also recognize apoptotic cells in various epithelial human tumours. Therefore, the N-terminal caspase-generated K18 fragment is stable in tissues and biological fluids. The Asp237-directed antibodies provide a powerful tool to study apoptosis in human and mouse tissues, cells and serum, using a broad range of detection modalities.     

Cytokeratin expression in normal human thymus at different ages

Subsets of thymic epithellal cells were examined Immuno-histochemically to determine whether or not their pheno-types change during thymic growth and at early involution in terms of cytokeratin (CK) expression. Five monoclonal antibodies specific for CK4, CK8, CK13, CK18 and CK19 were used and applied for 16 neonatal, three Infantile and one adult thymus speeimen, which had been obtained at autopsy, that were normal macroscopically and microscopicaily. CK4, CK8, CK13, CK18 and CK19 were expressed simultaneously in the cortex, medulla and subcapsular area with the exception of CK4, which showed expression on the adult thymus. Light and electron microscopy showed that CK8 and CK19 expression was overlapped. Thus, It was thought that CK8 and CK19 formed complexes in the cytoplasm of thymic epithelial cells. The Immunoreactivity to CK4, CK13 and CK18 were attenuated or disappeared In the subcapsular area during the early involution stage. Interestingly, two patterns of CK18 expression were observed in the neonatal and Infantile thymus tissues, which Indicated that the thymic microenvironment was changeable even under normal conditions.     

Cable-piliated Burkholderia cepacia binds to cytokeratin 13 of epithelial cells

Although the Burkholderia cepacia complex consists of several genomovars, one highly transmissible strain of B. cepacia has been isolated from the sputa of cystic fibrosis (CF) patients throughout the United Kingdom and Canada. This strain expresses surface cable (Cbl) pili and is thought to be the major strain associated with the fatal "cepacia syndrome." In the present report we characterize the specific 55-kDa buccal epithelial cell (BEC) protein that binds cable pilus-positive B. cepacia. N-terminal sequences of CNBr-generated internal peptides identified the protein as cytokeratin 13 (CK13). Western blots of BEC extracts probed with a specific monoclonal antibody to CK13 confirmed the identification. Mixed epidermal cytokeratins (which contain CK13), cytokeratin extract from BEC (which consists essentially of CK13 and CK4), and a polyclonal antibody to mixed cytokeratins inhibited B. cepacia binding to CK13 blots and to normal human bronchial epithelial (NHBE) cells. Preabsorption of the antikeratin antibody with the BEC cytokeratin fraction reversed the inhibitory effect of the antibody. A cytokeratin mixture lacking CK13 was ineffective as an inhibitor of binding. Colocalization of CK13 and B. cepacia by confocal microscopy demonstrated that intact nonpermeabilized NHBE cells express small amounts of surface CK13 and bind Cbl-positive B. cepacia in the same location. Binding to intact NHBE cells was dependent on bacterial concentration and was saturable, whereas a Cbl-negative isolate exhibited negligible binding. These findings raise the possibility that surface-accessible CK13 in respiratory epithelia may be a biologically relevant target for the binding of cable piliated B. cepacia.     

Tau truncation during neurofibrillary tangle evolution in Alzheimer's disease

The microtubule-associated protein, tau, is a highly soluble molecule that is nonetheless capable of self-association into filamentous deposits characteristic of a number of neurodegenerative diseases. This state change is thought to be driven by phosphorylation and/or C-terminal truncation events resulting in intracellular inclusions, such as the neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). Previously, we reported the existence of a novel truncation event, cleavage at aspartic acid(421), presumably by a caspase, and also described a monoclonal antibody (Tau-C3) specific for tau cleaved at this site. Here, we report the timing of this cleavage event relative to other antibody-targeted alterations in the tau molecule during the course of NFT evolution in AD. Immunohistochemical studies indicate that cleavage at aspartic acid(421) occurs after formation of the Alz50 epitope but prior to formation of the Tau-66 epitope and truncation at glutamic acid(391) (formation of the MN423 epitope). Thus, creation of the Tau-C3 epitope appears to occur relatively early in the disease state, contemporaneous with the initial Alz50 folding event that heralds the appearance of filamentous tau in NFTs, neuropil threads, and the dystrophic neurites surrounding amyloid plaques.     

Regulation of the new coexpressed CD55 (decay-accelerating factor) receptor on stomach carcinoma cells involved in antibody SC-1-induced apoptosis.

The human monoclonal antibody SC-1 was isolated from a patient with a diffuse-type adenocarcinoma of the stomach using somatic cell hybridization. The immunoglobulin (Ig)M antibody reacts specifically with diffuse- (70%) and intestinal-type (25%) gastric adenocarcinoma and induces apoptosis in vitro and in vivo. When used in clinical trials with stomach carcinoma patients, significant apoptotic and regressive effects in primary tumors have been observed with the antibody SC-1. The SC-1 receptor is a new 82 kd membrane-bound isoform of glycosylphosphatidylinositol (GPI)-linked CD55 (decay-accelerating factor, DAF). CD55 is known to protect cells from lysis through autologous complement and is coexpressed with the ubiquitously distributed 70 kd isoform. The SC-1-specific CD55 isoform is up-regulated shortly after antibody binding, followed by an internalization of the antibody/receptor-complex, whereas the membranous expression of wild-type CD55 remains unchanged. The apoptotic process is marked by cleavage of cytokeratin 18, indicating the involvement of caspase-6 in the apoptotic process. In contrast to other apoptotic pathways, a cleavage of poly(ADP-ribose)polymerase (PARP) is not observed. The expression of the cell-cycle regulator c-myc becomes up-regulated, whereas expression of topoisomerase IIalpha is down-regulated. Induction of apoptosis leads to an increase in the internal Ca(2+) concentration, which is not necessary for the apoptotic process but for the transport of newly synthesized SC-1-specific CD55 isoform to the membrane.     

Immunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies.

Carcinomas of different origin have been tested in immunofluorescence microscopy with the monoclonal murine antibodies CK1-CK4, which recognize a single cytokeratin polypeptide (human cytokeratin No. 18) present in simple but not in stratified squamous epithelia, and with the monoclonal antibody KG8.13 and guinea pig kerA antibodies, both of which recognize a variety of cytokeratins common to almost all epithelial cell types. Tumors derived from simple epithelia, including adenocarcinomas and some other tumors such as ductal breast carcinomas, were strongly stained by all three antibodies. So was a transitional carcinoma of the bladder. In contrast, basal cell epithelioma, cloacogenic carcinoma, and squamous cell carcinoma of skin, tongue, and esophagus appeared negative with CK1-CK4 but positive with the other two antibodies. Other squamous cell carcinomas derived from epiglottis and cervix uteri showed a mixture of positive and negative cells when tested with CK1-CK4, although all tumor cells were positive when tested with KG8.13 and with kerA. Thus, use of an appropriate collection of cytokeratin antibodies with different specificities not only allows tumors of epithelial origin to be distinguished from other tumor types but, in addition, allows a further subdivision of carcinomas in relation to their histologic origin.     

Anatomie der Brustdrüse

The human breast consists of lobes with a luminal glandular and a basal myoepithelial layer. Immunofluorescence studies have shown that the breast epithelium contains cytokeratin (CK)5/14-positive precursor cells which give rise to CK8/18-positive glandular or sm-actin-positive myoepithelial cells. Only some of the glandular cells contain estrogen receptors. The luminal epithelium of the lobules shows a much higher glandular differentiation than the ductal system. Diagnostically important cytokeratins of normal breast epithelium and its proliferative epithelial processes include luminal cytokeratins (CK7, CK8 and CK18) as markers of glandular differentiation and basal cytokeratins (CK5, CK14 and CK17) as markers of progenitor cells and early cells of the glandular and myoepithelial differentiation pathway. The most important myoepithelial markers are currently CD10, SMA, SMM-HC and Calponin.     

Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action

BACKGROUND: Upon Fas stimulation, procaspase-8 is recruited to the death-inducing signalling complex where autoactivation of caspase-8 occurs. Active caspase-8 can directly activate downstream caspases (e.g. caspase-3, 6, and 7) for the execution of apoptosis (mitochondria-independent pathway), while caspase-8 can also lead to executioner caspase activation through mitochondrial damage (mitochondria-dependent pathway). Caspase activation results in the dismantling of intracellular structure through specific proteolysis. RESULTS: We have found that an intermediate filament protein, vimentin, is cleaved at multiple sites by caspases at an early stage of apoptosis in Jurkat cells. The sequences of the two major cleavage sites in vimentin (IDVD/V and DSVD/F) suggested that these sites are cleaved by caspase-8 and caspase-3, respectively, or by close homologues of these proteases. The IDVD/V site can be cleaved by caspase-8 in vitro, and its cleavage is less sensitive to DEVD-CHO and Bcl-2 over-expression than that of the DSVD/F site in Jurkat cells. Over-expression of a mutant vimentin which was insensitive to caspase cleavage at these sites delayed the appearance of apoptotic nuclei in Jurkat cells. CONCLUSION: The specific cleavage of vimentin can be used as an apoptotic marker of both apical- and mitochondria-dependent caspase activation. Apoptotic cleavage of vimentin most likely results in disruption of its filamentous structure, which may facilitate nuclear condensation and subsequent fragmentation through disruption of the cytoskeletal network.