Micronucleus assay with urine derived cells (UDC): a review of its application in human studies investigating genotoxin exposure and bladder cancer risk.

The first micronucleus (MN) study with urine derived cells (UDC) appeared 30 years ago. So far, 56 investigations have been published with this method and it was shown that it can be used for the detection of chromosomal damage caused by environmental and lifestyle factors as well as by occupational exposures and certain diseases This approach may be also useful as a diagnostic tool for the detection and prognosis of bladder cancer. The test system has been improved in the last years, i.e., it was shown that, apart from MN also other nuclear anomalies can be evaluated in UDC which are found in other types of epithelial cells as well (e.g., in oral and nasal cells) and are indicative for acute toxicity (pyknosis, karyorrhexis, karyolysis, condensed chromatin) and genomic instability (nuclear buds, binucleates). Furthermore, an improved protocol with Carnoy I fixation and Papanicolaou stain was developed which enables the discrimination between cells which originate from the cervix and those from the urothelium. The evaluation of the currently available results indicates that exposures and health conditions which are associated with increased cancer rates in the bladder (and possibly also in other organs) lead to positive results in MN-UDC assays and a limited number of studies indicate that this method may be equally sensitive as other more frequently used human biomonitoring assays. The major shortcoming of the UDC-MN method is the lack of standardization; the evaluation of the current data shows that a variety of staining and fixation methods are used and that the numbers of evaluated cells vary over a broad range. These inconsistencies may account for the large inter-laboratory variations of the background frequencies. In order to improve the reliability of the method, further standardization and validation is required. Therefore an international program should be initiated in which a similar strategy could be used as in previous validation/standardization projects concerning MN-cytome assays with lymphocytes and buccal cells.

Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Insulin and the IGF system in the human placenta of normal and diabetic pregnancies.

The insulin/insulin-like growth factor (IGF) system regulates fetal and placental growth and development. In maternal diabetes, components of this system including insulin, IGF1, IGF2 and various IGF-binding proteins are deregulated in the maternal or fetal circulation, or in the placenta. The placenta expresses considerable amounts of insulin and IGF1 receptors at distinct locations on both placental surfaces. This makes the insulin and the IGF1 receptor accessible to fetal and/or maternal insulin, IGF1 and IGF2. Unlike the receptor for IGF1, the insulin receptor undergoes a gestational change in expression site from the trophoblast at the beginning of pregnancy to the endothelium at term. Insulin and IGFs are implicated in the receptor-mediated regulation of placental growth and transport, trophoblast invasion and placental angiogenesis. The dysregulation of the growth factors and their receptors may be involved in placental and fetal changes observed in diabetes, i.e. enhanced placental and fetal growth, placental hypervascularization and higher levels of fetal plasma amino acids.

Expression of functional Fas ligand in choriocarcinoma.

PROBLEM: In the course of pregnancy, fetal trophoblast cells and in that of choriocarcinoma-etiology, trophoblast derived tumor cells, invade the uterine mucosa without causing rejection by decidual leukocytes. Fas ligand (FasL, CD95L, APO-IL), a central regulator of the immune system, has been implicated in the maintenance of immune privileged sites, such as the eye, the testis and the pregnant uterus by inducing apoptosis in activated infiltrating leukocytes. In normal pregnancy FasL, which is expressed by trophoblast cells, appears to contribute to the immune privilege of the pregnant uterus. As choriocarcinoma derives from trophoblast we wanted to assess the expression of FasL in this tissue. METHOD OF STUDY: Immunohistochemistry, immunofluorescence, TUNEL-assay, Western blotting, coculture experiments and flourescence-associated cell sorter-analysis were the techniques used. RESULTS: Expression of FasL was found on cells of choriocarcinoma in paraffin sections in situ and on three choriocarcinoma cell lines such as JEG-3, JAR and BeWo. These results were confirmed by Western blotting. In coculture experiments choriocarcinoma cells induced apoptosis in a Jurkat cell line - sensitive to FasL mediated killing. However, when the Jurkat cells were pre-incubated with a Fas-blocking monoclonal antibody, apoptosis was abolished to a great extent. CONCLUSION: Our findings show that choriocarcinoma cells express FasL and this aforementioned molecule is biologically active. We assume that FasL expression on choriocarcinoma cells may contribute to control of anti-tumor responses by inducing apoptosis in activated Fas bearing leukocytes.