Clinical value of protein expression of kallikrein-related peptidase 7 (KLK7) in ovarian cancer.

Expression of the kallikrein-related peptidase 7 (KLK7) is dysregulated in ovarian cancer. We assessed KLK7 expression by ELISA and quantitative immunohistochemistry and analyzed its association with clinicopathological parameters and patients' outcome. KLK7 antigen concentrations were determined in tumor tissue extracts of 98 ovarian cancer patients by ELISA. For analysis of KLK7 immunoexpression in ovarian cancer tissue microarrays, a manual quantitative scoring system as well as a software tool for quantitative high-throughput automated image analysis was used. In immunohistochemical analyses, expression levels of KLK7 were not associated with patients' outcome. However, in multivariate analyses, KLK7 antigen levels in tumor tissue extracts were significantly associated with both overall and progression-free survival: ovarian cancer patients with high KLK7 levels had a significantly, 2-fold lower risk of death [hazard ratio (HR)=0.51, 95% confidence interval (CI)=0.29-0.90, p=0.019] or relapse [HR=0.47, 95% CI=0.25-0.91, p=0.024), as compared with patients who displayed low KLK7 levels. Our results indicate that - in contrast to earlier findings - high KLK7 antigen levels in tumor tissue extracts may be associated with a better prognosis of ovarian cancer patients.

Effects of simultaneous knockdown of HER2 and PTK6 on malignancy and tumor progression in human breast cancer cells.

Breast cancer is the most common malignancy in women of the Western world. One prominent feature of breast cancer is the co- and overexpression of HER2 and protein tyrosine kinase 6 (PTK6). According to the current clinical cancer therapy guidelines, HER2-overexpressing tumors are routinely treated with trastuzumab, a humanized monoclonal antibody targeting HER2. Approximately, 30% of HER2-overexpressing breast tumors at least initially respond to the anti-HER2 therapy, but a subgroup of these tumors develops resistance shortly after the administration of trastuzumab. A PTK6-targeted therapy does not yet exist. Here, we show for the first time that the simultaneous knockdown in vitro, compared with the single knockdown of HER2 and PTK6, in particular in the trastuzumab-resistant JIMT-1 cells, leads to a significantly decreased phosphorylation of crucial signaling proteins: mitogen-activated protein kinase 1/3 (MAPK 1/3, ERK 1/2) and p38 MAPK, and (phosphatase and tensin homologue deleted on chromosome ten) PTEN that are involved in tumorigenesis. In addition, dual knockdown strongly reduced the migration and invasion of the JIMT-1 cells. Moreover, the downregulation of HER2 and PTK6 led to an induction of p27, and the dual knockdown significantly diminished cell proliferation in JIMT-1 and T47D cells. In vivo experiments showed significantly reduced levels of tumor growth following HER2 or PTK6 knockdown. Our results indicate a novel strategy also for the treatment of trastuzumab resistance in tumors. Thus, the inhibition of these two signaling proteins may lead to a more effective control of breast cancer.

Clinical utility of level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1.

The prognostic and/or predictive value of the cancer biomarkers, urokinase-type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor [PAI]-1), determined by ELISA in tumor-tissue extracts, was demonstrated for several cancer types in numerous clinically relevant retrospective or prospective studies, including a multicenter breast cancer therapy trial (Chemo-N0). Consequently, for the first time ever for any cancer biomarker for breast cancer, uPA and PAI-1 have reached the highest level of evidence, level-of-evidence-1. At present, two other breast cancer therapy trials, NNBC-3 and Plan B, also incorporating uPA and PAI-1 as treatment-assignment tools are in effect. Furthermore, small synthetic molecules targeting uPA are currently in Phase II clinical trials in patients afflicted with advanced cancer of the ovary, breast or pancreas.

Characteristics of the level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1.

In cancer, the serine protease urokinase-type plasminogen activator, its inhibitor (plasminogen activator inhibitor type-1) and the receptor (CD87), among other proteolytic factors, are involved in tumor cell dissemination and turnover of the extracellular matrix. Unsurprisingly, a battery of very uniform data, amassed since the end of the 1990s, has put these members of the plasminogen activation system into the forefront of prognostic/predictive cancer biomarkers relevant to predict the clinical course of cancer patients and their response to cancer therapy. The present review focuses on the molecular characteristics of the disease forecast biomarkers urokinase-type plasminogen activator and plasminogen activator inhibitor type-1, and techniques to quantitatively assess these cancer biomarkers, in the context of potential clinical application and personalized disease management.

Emerging biomarkers in breast cancer care.

Currently, decision-making for breast cancer treatment in the clinical setting is mainly based on clinical data, histomorphological features of the tumor tissue and a few cancer biomarkers such as steroid hormone receptor status (estrogen and progesterone receptors) and oncoprotein HER2 status. Although various therapeutic options were introduced into the clinic in recent decades, with the objective of improving surgery, radiotherapy, biochemotherapy and chemotherapy, varying response of individual patients to certain types of therapy and therapy resistance is still a challenge in breast cancer care. Therefore, since breast cancer treatment should be based on individual features of the patient and her tumor, tailored therapy should be an option by integrating cancer biomarkers to define patients at risk and to reliably predict their course of the disease and/or response to cancer therapy. Recently, candidate-marker approaches and genome-wide transcriptomic and epigenetic screening of different breast cancer tissues and bodily fluids resulted in new promising biomarker panels, allowing breast cancer prognosis, prediction of therapy response and monitoring of therapy efficacy. These biomarkers are now subject of validation in prospective clinical trials.

The effect of antibacterial acting extracorporeal shockwaves on bacterial cell integrity.

BACKGROUND: Antibacterial effects of extracorporeal shockwaves (ESWs) have been demonstrated in vitro against bacteria under static and dynamic growth conditions. This study assessed the effects of ESWs on the cell wall integrity of bacteria. MATERIAL/METHODS: Standardized suspensions of Staphylococcus aureus were exposed to various shockwave impulses (2000-12,000) of different energy flux densities (EFD, 0.38-0.96 mJ/mm(2)). Bacterial suspensions of equal concentration that had been permeabilized (to >99%) with isopropanol were used as positive controls. The bacteria of all groups were stained with Sytox Green nucleic acid stain. The fluorescence of the shockwave-treated, permeabilized, and untreated suspensions was measured and compared for bacterial survival, quantified by colony-forming units after plating. RESULTS: Although ESWs showed a significant energy-dependent antibacterial effect that reduced CFUs in the treated suspensions by between 56% and 99%, only maximum energies (4000 impulses at 0.96 mJ/mm(2) and 12,000 impulses at 0.59 mJ/mm(2)) were followed by a significant increase in fluorescence compared with the untreated control (p<0.05). However, the fluorescence of these treated groups was still far less than that of the alcohol-permeabilized positive control groups (p<0.05). Lower energies and impulse rates did not show increased intracellular uptake of the fluorescent dye (p>0.05). CONCLUSIONS: This is the first study to assess bacterial cell wall permeability after ESW treatment. It was found that the permeabilization of bacterial cells after ESW treatment was far less than expected due to the corresponding antibacterial effect. Other mechanisms, such as intracellular effects, might be involved in bacterial killing after ESWs and still must be elucidated.

Y-box-binding protein YB-1 identifies high-risk patients with primary breast cancer benefiting from rapidly cycled tandem high-dose adjuvant chemotherapy.

PURPOSE: To investigate the potential of Y-box-binding protein YB-1, a multifunctional protein linked to tumor aggressiveness and multidrug resistance, to identify patients with breast cancer likely to benefit from dose-intensified chemotherapy regimens. PATIENTS AND METHODS: YB-1 was immunohistochemically determined in 211 primary tumors from the prospective, randomized West German Study Group WSG-AM-01 trial in high-risk (> or = 10 involved lymph-nodes) breast cancer (HRBC). Predictive impact of YB-1 was assessed by multivariate survival analysis, including time-varying factor-therapy interactions. RESULTS: At median follow-up of 61.7 months, patients receiving rapidly cycled tandem high-dose therapy (HD; two cycles [2x] epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2) every 14 days, followed by 2x epirubicin 90 mg/m(2), cyclophosphamide 3,000 mg/m(2), and thiotepa 400 mg/m(2) every 21 days) had better disease-free survival (DFS; hazard ratio [HR] = 0.62; 95% CI, 0.44 to 0.89) and overall survival (OS; HR = 0.59; 95% CI, 0.4 to 0.89) than those receiving conventional dose-dense chemotherapy (DD; 4x epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2), followed by 3x cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), and fluorouracil 600 mg/m(2) every 14 days). High YB-1 was associated with aggressive tumor phenotype (negative steroid hormone receptor status, positive human epidermal growth factor receptor 2 and p53 status, high MIB-1, unfavorable tumor grade) and poor OS (median 78 v 97 months; P = .01). In patients with high YB-1, HD yielded a 63-month median DFS (P = .001) and a 46-month median OS advantage (P = .002) versus DD. In multivariate models, patients with high B-1 receiving HD (v DD) had one third the hazard rate after 20 months for DFS and one sixth after 40 months for OS. CONCLUSION: In a randomized prospective cancer therapy trial, for the first time, a strong predictive impact of YB-1 on survival has been demonstrated: enhanced benefit from HD (v DD) therapy occurs in HRBC with high YB-1. Future trials could therefore address optimal chemotherapeutic strategies,taking YB-1 into account.

Immunohistochemical evidence of ubiquitous distribution of the metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines.

Immunohistochemical evidence of ubiquitous distribution of the metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, and spleen) and on a cell microarray of 31 tumor cell lines of different origin, as well as trophoblast cells and normal blood lymphocytes and granulocytes. IDE protein was expressed in all the tissues assessed and all the tumor cell lines except for Raji and HL-60. Trophoblast cells and granulocytes, but not normal lymphocytes, were also IDE-positive.

Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues.

Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and breast cancer tissue. Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed Western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Applying the four IDE-directed antibodies, we demonstrated IDE expression at the protein level, by means of immunoblotting and immunocytochemistry, in each of the tumor cell lines analyzed. Insulin-degrading enzyme protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in each of the cell lines and tissues assessed. In conclusion, we performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells thus extending our knowledge on the cellular and tissue distribution of IDE, an enzyme which to date has mainly been studied in connection with Alzheimer's disease and diabetes but not in cancer.

Intrathecal trastuzumab (Herceptin) and methotrexate for meningeal carcinomatosis in HER2-overexpressing metastatic breast cancer: a case report.

Leptomeningeal carcinomatosis represents a rare manifestation of metastatic breast cancer (MBC). We herewith report on a patient suffering from HER2 overexpressing MBC who received intrathecal methotrexate and trastuzumab for meningeal carcinomatosis. A 48-year-old woman was diagnosed with breast cancer in December 2002. Following surgery, six cycles of adjuvant FE100C plus irradiation and, subsequently for 1 year, trastuzumab were given. As a result of disseminated metastatic spread in October 2005, the patient received whole-brain radiotherapy for symptomatic central nervous system involvement, and was put on several trastuzumab-based combination regimens (capecitabine, vinorelbine, paclitaxel). In June 2006, the patient developed clinical signs of terminal cone involvement with overflow incontinence and paraparesis of the legs. Immediate radiation led to partial relief from clinical symptoms. Subsequently, the patient was put on the tyrosine kinase inhibitor lapatinib and capecitabine (August to October 2007), but on November 6th the patient suffered again from overflow incontinence and weakness of the legs. Failing to respond to lapatinib, the patient received gemcitabine/cisplatin and, additionally, was recommenced on intravenous trastuzumab. Owing to progressive leptomeningeal disease, the patient received repeated doses of intrathecal methotrexate and trastuzumab. Within 2 weeks and four intrathecal treatments, cerebrospinal fluid cytology showed the absence of tumor cells. Moreover, a striking clinical improvement with resolution of the paraparesis of the legs and overflow incontinence was observed. This case report gives details regarding the clinical course of a breast cancer patient who received intrathecal trastuzumab and methotrexate via lumbar puncture for meningeal carcinomatosis of HER2-overexpressing MBC.

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. METHODS: HIF-1alpha immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2-4 h) of hypoxic exposure (< 0.66% O2), reoxygenation (24 h, 20% O2), and radiation (0, 2, 5 and 10 Gy). RESULTS: HIF-1alpha expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. CONCLUSION: Our data suggest that both, short-term (approximately 4-8 h) and long-term (approximately 20-24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of hypoxic tumour cells during fractionated radiotherapy could counteract the increased PAI-1 levels.

Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells.

One aim during oncological radiation therapy is to induce reoxygenation in hypoxic tumours in order to enhance radiosensitivity and ultimately increase cell death. In squamous cell carcinomas of the head and neck (SCCHN), hypoxia is considered a pivotal physiological modulator for malignant progression, whereby the plasminogen activation system is involved in overlapping functions such as the shaping of the extracellular matrix, cell proliferation and signal transduction. Since little is known about reoxygenation and the plasminogen activation system in SCCHN, three human SCCHN cell lines (BHY, FaDu, and CAL27) and a non-transformed control cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated for 24 h at normoxic conditions. The mRNA expression of the urokinase-type plasminogen activator (uPA), the plasminogen activator inhibitor type-1 (PAI-1) and the urokinase-type plasminogen activator receptor (uPAR) was assessed by means of real-time semi-quantitative RT-PCR, and the protein expression was determined by immunoenzymometric quantification (ELISA). Both hypoxia and reoxygenation induced statistically significant changes in uPA, PAI-1 and uPAR mRNA and protein levels in the various cell lines investigated, showing that oxygen tension is a strong modulator of the plasminogen activation system in vitro. However, no uniform correlation pattern was found between the mRNA and protein levels analysed over all three time-points (24, 48, and 72 h) and oxygen treatment variants (N, H, R) nor according to oxygen treatment conditions over all three time-points. Changes in oxygen tension could therefore be modulating the fragile balance between the various components of the plasminogen activation system in SSCHN ultimately leading to an increased tumour matrix disruption, alterations in cell invasiveness, and the dissemination of tumour cells to distant organs.

European Organisation for Research and Treatment of Cancer (EORTC) Pathobiology Group standard operating procedure for the preparation of human tumour tissue extracts suited for the quantitative analysis of tissue-associated biomarkers.

With the new concept of 'individualized treatment and targeted therapies', tumour tissue-associated biomarkers have been given a new role in selection of cancer patients for treatment and in cancer patient management. Tumour biomarkers can give support to cancer patient stratification and risk assessment, treatment response identification, or to identifying those patients who are expected to respond to certain anticancer drugs. As the field of tumour-associated biomarkers has expanded rapidly over the last years, it has become increasingly apparent that a strong need exists to establish guidelines on how to easily disintegrate the tumour tissue for assessment of the presence of tumour tissue-associated biomarkers. Several mechanical tissue (cell) disruption techniques exist, ranging from bead mill homogenisation and freeze-fracturing through to blade or pestle-type homogenisation, to grinding and ultrasonics. Still, only a few directives have been given on how fresh-frozen tumour tissues should be processed for the extraction and determination of tumour biomarkers. The PathoBiology Group of the European Organisation for Research and Treatment of Cancer therefore has devised a standard operating procedure for the standardised preparation of human tumour tissue extracts which is designed for the quantitative analysis of tumour tissue-associated biomarkers. The easy to follow technical steps involved require 50-300 mg of deep-frozen cancer tissue placed into small size (1.2 ml) cryogenic tubes. These are placed into the shaking flask of a Mikro-Dismembrator S machine (bead mill) to pulverise the tumour tissue in the capped tubes in the deep-frozen state by use of a stainless steel ball, all within 30 s of exposure. RNA is isolated from the pulverised tissue following standard procedures. Proteins are extracted from the still frozen pulverised tissue by addition of Tris-buffered saline to obtain the cytosol fraction of the tumour or by the Tris buffer supplemented with the non-ionic detergent Triton X-100, and, after high-speed centrifugation, are found in the tissue supernatant. The resulting tissue cell debris sediment is a rich source of genomic DNA.

The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions.

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.

Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells.

In squamous cell carcinoma of the head and neck (SCCHN), hypoxia is considered a crucial physiological modulator for malignant progression, wherebythe plasminogen activation system is involved in overlapping functions such as moulding of the extracellular matrix, cell proliferation and signal transduction. Little is known about the effects of reoxygenation on the plasminogen activation system in SCCHN cells. Three human SCCHN cell lines (BHY, CAL27, FaDu) and a non-transformed human fibroblast cell line (VH7) were exposed to hypoxic (<0.5% O(2)) conditions for up to 72 h and subsequently reoxygenated at normoxic conditions for 24 h. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein concentration and former protein activity were determined by ELISA and complex ELISA, respectively. Reoxygenation induced significant changes in cell-associated and secreted PAI-1 protein compared to the normoxic control. Significant increase in cell-associated and secreted uPA protein after reoxygenation was only observed for some of the cell lines. Determination of uPA-PAI-1 complex formation revealed the release of active protein into the cell supernatant. The beneficial role of reoxygenation during radiation therapy is widely accepted. However, reoxygenation does not seem to counteract the effects induced by hypoxia on the plasminogen activation system. Fatally irradiated reoxygenat- ed tumour cells might still produce sufficient amounts of 'harmful' protein and thus initiate a path for invasion and metastasis for surviving tumour cells.

High hydrostatic pressure-induced cell death in human chondrocytes and chondrosarcoma cells.

BACKGROUND: In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in different ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used, although both methods have substantial disadvantages, e.g. loss of biomechanical and/or biological integrity of the bone and destabilization of the articular surface. In this regard, high hydrostatic pressure (HHP) treatment of bone is a new, advancing technology, now being used in preclinical testing to inactivate tumor cells. To find out if this technique is also suited for extracorporal inactivation of chondrocytes and chondral tumor cells, the effect of HHP on cell viability and morphology of human chondrocytes / chondrosarcoma cells was investigated in the present study. MATERIALS AND METHODS: SW1353 chondrosarcoma cells and chondrocytes were subjected to HHP in the range of 50 to 350 MPa (10 min, 37 degrees C) and, subsequently, cell viability and cell morphology assessed. RESULTS: After exposure at 350 MPa, all HHP-treated chondral cells showed explicit morphological changes, evident by membrane ruffling and bleb formation; chondrosarcoma cells treated this way were irreversibly damaged and not alive. CONCLUSION: We anticipate that, in orthopedic surgery, HHP eventually can serve as a novel, promising technical approach for cell inactivation (including tumor cells) and allow subsequent reimplantation of the osteoarticular autograft.

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.