H i s t o c h e m i c a l J o u r n a l 24, 461 - 634 (1992)

461

Abstracts of the 9th International Congress of Histochemistry and Cytochemistry MAASTRICHT,

THE

NETHERLANDS,

30 AUGUST

-- 5 SEPTEMBER,

1992

PLENARY LECTURES LI

In Situ Hybridization Coming of Age. M. van der Ploeg, T. Raap, R. Dirks and J. Wiegant Department of Cytoehemistty and Cytometry, Leiden University, The Netherlands. Through the combined efforts of several groups, in situ hybridization (ISH) methodology has reached a high detection sensitivity (defined as the smallest DNA target detectable); a high DNA resolution (defined as the smallest distance in kilobasepairs between two DNA targets which can be resolved microscopically) and a high multiplicity (defined as the number of different DNA targets that can be identified simultaneously). It is also possible to detect mRNA's by ISH. Single copy DNA sequences as small as 1 kb can be localised in metaphase or interphase chromosome preparations. Using chromatin of varying states of decondensation, resolutions > t 0 kb can be reached. Multiplicity - - currently about 12 targets simultaneously - - is likely to increase, particularly by use of image cytometry. Finally, the combination of ISH and immunocytochemistry allows to visualize not only the presence, absence or amplification of genes and gene transcripts, but also that of the cognate proteins or peptides. The speed, the accuracy, the relative ease of these nonradioactive procedures, and the fact that they permit to extract detailed molecular data from morphologically intact cells and chromosomes, has increased their importance in many biomedical fields.

P D G F and cytokines are currently used in these studies. Labelling with fluorescent dyes is preferred over colour staining because of its greater sensitivity. In situ hybridization for the localization, and Northern and slot blots for quantitation of the mRNAs of the different proteins complete this program. Technical aspects of these new methods will be discussed and new data from our own research will be presented. It is hoped that these methodological advances will provide a better understanding of biological processes and of the pathogenesis of human diseases.

L3

Exploring Cell Physiology with Machine-Vision Light Microscopy. D. L. Taylor Pittsburg, USA.

L4

Nerve-Immune System Interactions. D. Felten Rochester, USA.

L5

Histochemistry: From Tissues to Molecules. J. Roth Zfirich, Switzerland.

L2

Histochemistry in Cardiovascular Biology: New Frontiers. J. Schaper Max-Planck-Institute, Bad Nauheim, Germany. Histochemical methods in the conventional sense of localization of enzymes are still useful tools in histology, but in research they have been widely replaced by immunocytochemistry using monoclonal antibodies. Our own experience using these methods in cardiovascular research is concerned with: 1. studies of human myocardium afflicted by either coronary heart disease or dilated cardiomyopathy and 2. studies of angiogenesis in the chronically ischemic dog or pig heart. In addition, cells isolated and cultivated are investigated for comparison with tissue sections. Antibodies against the various proteins of the contractile machinery and o f the cytoskeleton of the cardiomyocyte, against extracellular matrix components and their receptors, antibodies for blood cell typing and against growth factors such as a F G F and

L6 Peroxisomes: Elucidation of Their Structure and Function by Cytochemistry. H. D. Fahimi Institute for Anatomy and Cell Biology (If), University of Heidelberg, D-6900 Heidelberg, FRG. The general scientific interest in peroxisomes has grown significantly in the past few years because of their involvement in several important metabolic processes and the recent discovery of more than a dozen inherited diseases characterized by the morphologic absence or biochemical dysfunction of this organelle. In my presentation the current methods for light and electron microscopic cytochemical and immunocytochemical investigation of peroxisomes are briefly reviewed and their contribution to the elucidation of the biology and pathobiology of this organelle discussed. The most common method for the detection of peroxisomes is based on the visualization of the peroxidatic activity of

462 catalase with the 3,3'-diaminobenzidine (Fahimi, J. Cell Biol. 43, 275, 1969). The application of the cerium technique recently has revealed the localization of different oxidases in distinct specific domains within the peroxisomal matrix: urate oxidase in crystalline cores, D-amino acid oxidase in central matrix and a-hydroxy acid oxidase in peripheral matrix. Moreover, the marginal plates of bovine renal peroxisomes were isolated and found to consist exclusively of a-hydroxy acid oxidase B (Zaar, V61kl, Fahimi, J. Cell Biol. 113, 113, 1991). The immunocytochemical labelling with antibodies to peroxisomal membrane proteins (Baumgart et al, J. Cell Biol. 108, 2221, 1989) and the application of computer assisted three dimensional reconstruction of proliferating peroxisomes (Yamamoto and Fahimi, J. Cell Biol. 105, 703, 1987) have elucidated the morphological basis of biogenesis of peroxisomes. Those observations together with recent biochemical studies by Lfiers and V61kl (in preparation) suggest that peroxisomes grow by forming small buds which gradually grow to regular-sized mature peroxisomes. Supported by SFB 352 of DGF, FRG. L7

Contributions of Histoehemistry in Raising Questions and Answering Some. S. S. Spicer Medical University of South Carolina, Charleston, South Carolina U.S.A. Radioautography with 3~SO4 = and cationic stains have revealed in many epithelial cells, previously unknown sulfated glycoconjugates. Labelled lectins have localized neutral glycoproteins thought to be cell specific and not priorly detected. Questions then arise about the biologic significance of these constituents. By localizing proteins of known activity, immunocytochemistry has contributed knowledge of cell function and classification. Thus, immunostaining for specific hormones settled questions about functional cell types in pituitary and pancreas. Immunolocalization of carbonic anhydrase (CA) clarified mechanisms of acidification in stomach and alkalinization in proximal duodenum and pancreatic ducts. Two types of CA-rich intercalated cells can be distinguished in distal nephron: one immunopositive also for basal band III protein and an acidifier of urine, the other

Plenary lectures devoid of band III protein and an alkalinizer. Demonstration of CA in distinctive fibrocytes under urothelium and in inner ear identified a subclass of fibrocytes active in ion transport. Immunocytochemistry of isozymes CA I, II and III revealed variable distribution of these enzyme subtypes and pointed to possible linkage between an isozyme and a compatible ion transport mechanism. A mouse line rendered genetically deficient in CA II lacked immunoreactivity for the enzyme and evidenced arteriolar calcification, establishing this line as an experimental disease model.

L8 New Trends in Immunohistochemistry. P. K. Nakane Department of Anatomy HI, Nagasaki University School of Medicine, Nagasaki 852, Japan. Unlike chemical and physical matters, living organisms are with a fixed genetic program. When the program is comprehended, one can deduce what were the PAST activities, what are the PRESENT activities and what will be the FUTURE activities of the cells and tissues. Immunohistochemical methods have contributed greatly for the comprehension of the program during the past three decades. By localizing proteinaceous antigens immunohistochemically, one recognizes what genes had been transcribed; and by localizing mRNA by in situ hybridization, one identifies what genes are being transcribed. A new trend is to expand application of immunohistochemistry to prognosticate what genes would have been transcribed in cells if the cells were not killed. Several approaches have been conceptualized to peak the FUTURE. One is to differentiate active and inactive chromatins in nuclei and identify genes in each of them. The differentiation of the chromatins may be accomplished either by selective extraction or capitalizing on the accessibility of exogenous proteins such as nuclease, polymerase, antibodies to the active chromatin but not to the inactive chromatins. The identification of genes in each chromatin is done then by utilizing methods such as in situ polymerase chain reaction, in situ hybridization, in situ nick translation, histochemistry and immunohistochemistry in combination. Those genes in the active chromatin but are not being transcribed are those genes which will be or can be transcribed in the FUTURE.

463

Invited symposia INVITED SYMPOSIA

S1. I N S I T U HYBRIDIZATION FROM BENCH TO BEDSIDE

S1.1

Genomics and molecular cytogenetics. Jr. W. Gray

Abstract not received

S1.2

PRINS (Primed In Situ Labelling) of Nucleic Acids. J. Hindkjaer, J. Koch, S. Kolvraa, H. Fischer, J. Mogensen, S. Pedersen and L. Bolund Institute of Human Genetics, Aarhus University, Denmark.

PRINS is a method for sequence specific labelling of nucleic acids in situ, which may be faster, milder and more sensitive than traditional in situ hybridization. Short unlabelled DNA probes are hybridized to the complementary nucleic acid sequences in situ. Labelled nucleotides are then incorporated by a suitable polymerase with the probe as primer and the target nucleic acid as template, thus labelling the site of hybridization. Since the probes are unlabelled, one can use very high probe concentrations giving high hybridization efficiency without background problems. The labelling is independent of probe length since the sensitivity is determined by the primer extension. This allows the detection of minor sequence variations, especially since the match at the 3' end of t h e probe is crucial for priming. Synthetic oligonucleotides can be used as primers, obviating the need for cloned probes. PRINS allows the visualization of unique DNA sequences in combination with high quality Q-banding for cytogenetic mapping. Preliminary results indicate that relaxation of torsional strain in the DNA template during chain elongation greatly improves the degree of labelling. The PRINS method can also be used to visualize mRNA variants, thus permitting functional cytogenetic analyses and, possibly, the detection of somatic mutations in expressed genes at the single cell level. Finally, PRINS is so mild, that it opens up for flow fluorimetric analysis and sorting of cells and chromosomes with respect to their contents of specific nucleic acid sequences.

$1.3

Quantitative Reverse Cytogenetics: A Powerful New Method for Molecular Cytogenetic Analysis of Solid Tumors. A. Kallioniemi, O. Kallioniemi, D. Sudar, D. Rutovitz, J.

W. Gray, F, Waldman and D. Pinkel Div. Molecular Cytometry, Dept. Lab. Med., U.C. San Francisco, CA 94143 - 0808, USA.

Cytogenetic studies in solid tumors are difficult because of the low yield of mitoses. Molecular studies of tumor DNA, in turn, can only probe one defined genomic region at a time. We have developed a powerful new method for surveying the entire genome for DNA sequence copy number variation in a single hybridization. The method is based on the competition

between biotinylated total tumor DNA and digoxigeninlabeled normal genomic reference DNA during hybridization to normal metaphase chromosomes. The relative amount of bound tumor and normal DNA at a given locus in the target chromosomes is dependent on the relative abundance of those sequences in the two DNA samples. After immunofluorescent staining with avidin-FITC (green) and anti-digoxigenin Rhodamine (red), variation of DNA sequence copy numbers in the tumor are detected as variations in the ratios of green and red fluorescence along each chromosome. Using five fibroblast cell lines with X chromosome aneusomies ( 1 - 5 copies) we verified that there was a linear correlation between the green/red ratio of the X chromosome and the degree of aneusomy. Using the method, several genetic changes such as aneusomies, deletions and amplifications, some in previously unknown loci, have been found in cancer cell lines and primary tumors.

S1.4 Visualizing the Multistep Process of Human Tumorigenesis by In Situ Hybridization and Immunohistochemistry. W. N. Hittelman, D. H. Shin, N. Voravud, J. Y. Ro, J. S. Lee and W. K. Hong Departments of Medical Oncology and Pathology, Univ. of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA.

Human aerodigestive tract tumor development has been proposed to be a multistep process driven by carcinogeninduced genetic alterations. To determine the relationship between genetic changes and their phenotypic consequences at the tissue level during tumorigenesis, paraffin-embedded head and neck squamous cell carcinoma specimens containing adjacent premalignant lesions have been examined for chromosome polysomy (by in situ hybridization), dysregulation of proliferation (PCNA immunohistochemistry), and epidermal growth factor receptor expression. Normal appearing epithelium adjacent to the tumor showed increased frequencies of cells with polysomies for chromosomes 7 and 17 compared to normal control epithelium. The fraction of cells exhibiting polysomy increased as tissue passed through hyperplasia, dysplasia, to tumor. Similarly, proliferative dysregulation was increasingly observed during histological progression. Interestingly, increased PCNA expression was first observed in the parabasal region of the epithelium, suggesting a defect in the downregulation of proliferation. Increased PCNA labelling in the basal layer occurred later in histological progression and was, in some cases, associated with an increased EGFR expression. These results suggest that a field cancerization process is associated with tumorigenesis in the aerodigestive tract, and markers of genetic change and proliferative dysregulation might serve as intermediate markers for chemopreventive trials. Moreover, this in situ approach should allow one to determine the tissue consequences of specific genetic alterations during tumor development.

Invited s y m p o s i a

464

$2. I N S T R U M E N T A T I O N

IN CYTOMETRY

AND

MORPHOMETRY

$2.1 Image Information

$2.3 Detection and Quantitation of Fluorescent Signals

G. Brugal Laboratoire TIM3, Universit~Joseph Fourier, Grenoble, France.

H. Tanke, N. P. Verwoerd, E. J. Hennink, A. K. Raap, W. C. R. Sloos and V. Vrolyk Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.

$2.2 Morphometry in a Working Laboratory Environment

$2.4 Three-dimensional Reconstruction and Quantitation

G. Slavin St. Bartholemew's Hospital Medical College, London, UK.

M. Robert-Nicoud Grenoble, France.

$3. DEVELOPMENTS

IN DIAGNOSTIC

MOLECULAR

$3.1

Histochemical Techniques in Molecular Pathology. R. DeLellis Boston, USA.

$3.2

Polymerase Chain Reaction In Situ, Technical Aspects and Potential Application. H. Wolf Boston, USA.

$3.3 Interphase Karyotyping in Tumor Pathology. A. H. N. Hopman, M. Vallinga, P. Poddighe and F. C. S. Ramaekers Dept of Molecular Cell Biology and Genetics, University of Limburg, 6200 MD Maastriet, The Netherlands.

$3.4 Mutation of the p53 Gene and Accumulation of the p53 Protein. Common Steps in Human Cancer. D. P. Lane, C. Midgley, S. Picksley and C. Stephen CRC Laboratories, University of Dundee, Dundee, DDI 4HN. The human gene for p53 is located on chromosome 17p and

$4. I N T R A V I T A L

PATHOLOGY

encodes a 393 amino acid nuclear phosphoprotein. Somatic mutation of this gene is found at high frequency in most human cancers. Germ line mutations in the p53 gene are responsible for the Li-Fraumeni cancer family syndrome and may also be involved in other high risk cancer families. The normal protein is a sequence specific DNA binding protein that has many of the properties of a transcription factor. We have expressed p53 in bacterial systems and used it to make new monoclonal and polyctonal antibodies. These new antibodies allow the detection of p53 in routine clinical samples including formalin fixed paraffin embedded sections, cytology samples and by ELISA in tumour extracts. The results of these assays for p53 protein have been compared with the sequence of the p53 gene in matched samples. Many point mutations in p53 are associated with accumulation of the protein and alteration of its conformation. The nature of this conformational change has been defined using phage epitope mapping libraries. Mutation in the p53 gene may not be the sole cause of protein accumulation and in a variety of systems we have established that other genetic events are required. A range of genotoxic agents stabilise the normal p53 protein and this may explain the growth arrest induced by these agents. The acute sensitivity of tumour ceils to these drugs may then result from the absence of functional normal p53 in tumours. Since in these cells there is no growth arrest and the agents are thus more toxic. This model may also explain the high frequency of chromosomal aberation in tumours with mutant p53.

MICROSCOPY

$4.1 Imaging Organeile and Cytoskeleton Dynamics. D. G. Weiss Inst. Zool., Techn. University Munich, D-W8046-Garching, FRG.

The developments in microelectronics and computer design that enable one to digitize and process microscope images in "real time", i.e. at video rates, led to a renaissance of light microscopy in the form of video microscopy. Since living cells could now be studied in great detail that previously was only

accessible by electron microscopy of dehydrated samples, cell motility is one field benefitting considerably from these developments. The study of the motion of identified intracellular organelles and microtubules (25 nm) which are far smaller than the limit of resolution of light microscopy has become possible by video-enhanced contrast (VEC) microscopy. Such studies have revealed that in animal cells microtubules are motile and highly dynamic and serve as tracks for organelle movement. VEC microscopy allowed for the first time to detect and assay the activity of microtubule-

Invited symposia dependent motor enzymes (cytoplasmic dynein, kinesin) so that they could be purified and characterized. Since in plant cells organelle movements occur along actin filaments we tested their possible involvement in animal cells using preparations of cytoplasm extruded from squid giant nerve fibers. We found a new type of organelle motility that did not follow microtubules but used tracks that were invisible by VEC microscopy. The presence of a network of actin filaments (typically 6 nm diameter) was demonstrated in these regions by video-intensified fluorescence microscopy (VIM) of unfixed preparations stained with rhodamine-phalloidin. The new type of motile activity along "invisible" filaments was shown to be dependent on the presence of the actin filament network but moving organelles were also able to switch from actin filaments to microtubules (Kuznetsov, Langford, Weiss: Nature 356, April 23, 1992). We can say that video microscopy allowed us to go beyond the classical limits of light microscopy: it enabled us to see smaller objects than before, to work at extremely low fluorescence intensities, and to generate contrast where none could be generated by conventional techniques. Our findings indicate that in the cytoplasm of neurons tubulin-based and actomyosin-based motilities coexist and may collaborate to generate organelle movements. (Supported by DFG)

$4.2

Quantitative Studies of Microcirculation in Transparent Invertebrates using Video Imaging and Fluorescent Probes, R. Paul Institute of Zoology, University of Diisseldorf, FRG. The physiology of systemic interactions in animals is a complex field. Mostly only one variable is measured by the applied techniques, and the frequent invasiveness of them tend to negatively influence normal function. The new video imaging techniques together with fluorescent probes are valuable tools for the physiologist to make systemic functions visible and to overcome problems related to former techniques. Essential for their application is the existence of transparent organisms which are abundant among waterliving invertebrates, but there are also transparent fish and transparent developmental stages in higher vertebrates. During the process of developing and applying basic 'optophysiological' techniques, we focused first on the relationships between blood gas transport and cell function. A former study (Paul et al., 1991: Naturwissenschaften 78, 134-135) in the tarantula Eurypelma californicum has surprisingly shown functional adaptations of haemolymph distribution in the open vascular system (revealed by immunofluorescence microscopy using a monoclonal antibody specific for the oxygen carrier haemocyanin) on the oxidative capacity of muscle cells (studied by succinic dehydrogenase activity). Microcirculation in the muscle tissues

465 of the transparent spider Pholcus phalangioides has now been studied using the strong autofluorescence of haemolymph cells and fluorescent microspheres: depending on muscle type, flow patterns and velocities vary. Haemolymph flow in an open circulatory system is probably directed by the varying adhesion of tissue sheaths as revealed by contrast enhancement techniques.

$4.3

Vital Microscopy of Chromatin for the Study of Chemically Induced Disturbances of Mitosis In Vitro. D. Schiffmann, H. Stopper and U. De Boni* Institute of Toxicology, W-8700 Wu'rzburg, Germany; ~Dept. of Physiology, University of Toronto, MSS IA8 Toronto, Canada. Some carcinogens (e.g asbestos, synthetic estrogens) have been reported as non-genotoxic. Evidence has accumulated that such compounds cause mitotic disturbances resulting in displacement, nondisjunction and subsequent loss of chromatin. Nonrandom loss of specific genomic elements has already been observed in a number of transformed cell lines and tumors. Most likely, these phenomena are associated with the loss of tumor suppressor genes and therefore responsible for the development of malignancy without involvement of gene mutations. - - We have developed a new method to monitor the arrangement and distribution of chromatin elements in living cells throughout the course of mitosis: The cells are labelled with a DNA-specific stain (e.g. Bisbenzimide) and kept at 37~ on an inverted fluorescence microscope. The cells are monitored using a 100 x oil immersion phase contrast objective. UV flux is reduced by use of neutral density filters to a level permitting analysis of spatial position of chromatin elements, but avoiding UV-damage to cells. A high resolution Silicon Intensifier Target Camera (SIT) is required for visualization. This camera is connected to a video image processor system which enhances video signals for increased contrast. This method allows a detailed analysis of the origin of mitotic disturbances and their relation to the various phases of mitosis. - - Our results indicate that different functional elements (kinetochores, tubulin, chromatin structure etc.) can be affected in different mitotic stages depending on the mode of action of the inducing agent. As an example, the synthetic estrogen diethylstilbestrol causes dislocation of chromatin elements as early as prophase which persist throughout mitosis and form micronuclei during cytokinesis. In contrast, tubulin-related metaphase disturbances are repaired, presumably at a "mitosis checkpoint" resulting in normal exit from mitosis.

$4.4

Relating IntraceUular Motility with Locomotion. G. Dunn London, UK.

466

$5. D E V E L O P I N G

Invited symposia CONCEPTS

IN CELL DIFFERENTIATION

S5,1 Epithelial Differentiation Markers W. Franke Heidelberg, Germany.

$5.2 The Proliferation Marker K i - 67: Diagnostic & Prognostic Value and Molecular Characterization. J. Gerdes, C. Schlfiter, M. Duchrow, C. Wohlenberg, M. H. G. Becker, G. Key and H.-D. Flad Div. Molecular Immunology, Forsehungsinstitut Borstel, D-2061 Borstet.

It iS now almost ten years since a unique monoclonal antibody was described which reacted selectively with the nuclei of proliferating cells a. A recent data base search revealed that 560 publications referring to this antibody and its applications have appeared since that time. These papers confirmed the value of antibody K i - 67 for the assessment of cell growth in clinical samples, and in particular for assessing the growth fraction of human neoplasms (for review sees). Despite these well-documented diagnostic and prognostic applications of K i - 6 7 , the antigen that is defined by this antibody was not known. Recently, we could demonstrate that K i - 67 detects a double band (345kD & 395kD) in Western blots of lysates obtained from proliferating cells, which was absent in lysates obtained from quiescent cells 3. Using immunocloning approaches, we have isolated and sequenced a 1095 bp eDNA fragment encoding for parts of the Ki - 67 antigen 3. Using this K i - 67 DNA as probe in plaque hybridization techniques and chromosomal walking and anchor priming with PCR, we have now cloned, sequenced, and aligned in an open reading frame the entire K i - 6 7 eDNA of 9700 bp. The structure of this eDNA is thus far unique: The centre is formed by a 6845bp exon, in which a highly conserved motif of 62 bp is repeated for 16 times. Using synthetic peptides, we could clearly demonstrate that this 62 bp element encodes for the epitope recognized by antibody K i - 67. We have now produced a number of new monoclonal antibodies by immunizing mice with a recombinant K i - 6 7 gene product (see abstract of Michael H. G. Becker et al.). One of these antibodies can be applied on paraffin sections (see abstract of Giorgio Cattoretti et al.). 'Gerdes, J., Schwab, U., Lemke, H., Stein, H., Int., J Cancer 1983; 31:13-20. 2Gerdes, J., Seminars in Cancer Biology, 1:199-206. 3Gerdes, J., Li, L., Schlfiter, C., et al. Am. J. Pathol. 1991; 138:867-873.

S5.3

Transforming Growth Factor fls in Early Muriue Development. C. M u m m e r y , H. Slager, K. Lawson and J. van den Eijnden-Van Raaij Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht.

Cell-to-cell interactions are important in the development of all complex organisms, but molecules that may mediate these regulatory interactions have only recently been identified. In the early murine embryo, peptide growth factors with their respective receptors are prime candidates as mediators of such

AND PROLIFERATION

interactions on the basis of their differential regulation in time and space. Using embryonal carcinoma (EC) and embryonic stem (ES) cells as models for early differentiation, we have described changes in RNA expression and protein secretion of the three isoforms of transforming growth factor/3 (TGF//~_3) as endodermal and mesenchymal derivatives form. TGF/~'s are multifunctional regulators of growth and differentiation that can alter the expression of a spectrum of genes depending on the nature of the target ceil. They have been implicated in mesoderm formation in explants of Xenopus embryos; we present preliminary evidence for a similar effect on the promotion of mesoderm formation in EC and ES cells, particularly for TGFfl2. Using a specific antibody, we have also shown that TGF//2 is expressed in the early embryo in a manner predicted by the results in EC and ES cells and further that microinjection of neutralizing antibodies may alter development. The implications of these findings will be discussed.

$5.4 Characterisation of Human Mitotic Cyclins in the Cell Cycle. J. AT. Pines The Wellcome/CRC Institute, Tennis Court Rd., Cambridge, UK.

The mitotic cyclins are proteins implicated in the induction of mitosis, that are marked by their accumulation in interphase followed by dramatic destruction at mitosis in each cell cycle. We have been characterising A- and B- type human cyclins during the cell cycle. We have shown that the levels of cyclins A and B 1 are controlled at both the level of transcription and post-translationally. Cyclin A synthesis begins at the start of S phase, whereas cyclin B1 synthesis starts in the middle of S phase. Both cyclins are stable throughout interphase but are rapidly and specifically destroyed in mitosis. The human A and B1 type cyclins both associate with and activate protein kinases of the cdc2 family; cyclin A associates with p33 cdkaand cyclin B binds to p34cdc2 itself. In collaboration with Dr. J. Nevins (Duke University), we have demonstrated that the cyclin A-p33 Cd~zcomplex is associated with the transcription factor E2F and that this complex also contains the p107 retinoblastoma-related protein. The associated protein kinase activities of cyclin A and B1 appear to be under different forms of regulation. The cyclin B-p34r complex accumulates in an inactive form throughout G2 phase, and is only activated at the beginning of mitosis when p34 ~c2 is dephosphorylated on residues T14 and Y15. By contrast the cyclin A-p33 cd~2 complex is active as soon as it is formed in S phase. Human cyclins A and B1 differ in their sub-cellular location. Cyclin A is a nuclear protein from the time of its synthesis onwards. In mitosis cyclin A associates with chromosomes in prophase but not in metaphase, and it is degraded before the cells enter anaphase. On the other hand, cyclin B1 accumulates in the cytoplasm throughout late S and G2 phases. Most of the cyclin B1 rapidly moves at the start of mitosis, before nuclear lamina breakdown, tn the nucleus, cyclin B1 binds to condensed chromosomes and to the mitotic spindle in prophase and metaphase. Cyclin B1 is abruptly destroyed at the metaphase-anaphase transition, and thus the inactivation of the cyclin Bl-p34 c~c2 kinase activity is implicated as one of the major control steps in the exit from mitosis.

Invited symposia

467

$6. TRENDS IN A U T O R A D I O G R A P H Y

$6.1 Electron Microscopic Radioautography Using Cryo-Techniques. T. Nagata Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan.

The techniques of demonstrating diffusible compounds in electron microscopic radioautography consist of two methods, i.e. precipitation fixation and freezing fixation. We have developed cryo-techniques in combination with dry-mounting radioautography at the electron microscopic level during these 30 years. Materials used were cultured cells or tissues obtained from various organs of mouse and rat, such as the liver, pancreas, eye and others which were labelled with 3H-thymidine, 3H-uridine, 3H-leucine, 3H-glucose, 3H-glycerol, 3H-labelled drugs, and various inorganic radionuclides. The cells and tissues were quickly frozen either in iso-pentane cooled with liquid nitrogen at - 1 6 0 ~ or in contact with copper blocks cooled with liquid nitrogen at -196~ After the cryofixation, the following procedures can be divided into 3 categories, i.e., dry cryosectioning with a cryoultramicrotome followed by freeze-drying, freeze-drying and embedding, and freeze-substitution and embedding. The embedded tissues were dry-sectioned without using any water and they were drymounted with Konica NR-H2 emulsions by a wire-loop method using dioctyl sodium sulfosuccinate to prevent bursting while they are air-dried. The dry films were exposed, developed and observed in intermediate high voltage electron microscopes at accelerating voltages of 2 0 0 - 4 0 0 kV. As the results, various kinds of diffusible compounds were demonstrated to localize diffusely in the cytoplasm and nuclei or specifically located over some cell organelles.

S6.2

Cell Kinetic Studies of Various Cell Populations (Normal and Tumor Cells) using 3H-Thymidine Radioautography. B. Maurer-SchuRze and 1. D. Bassukas inst. f. Med. Strahlenkunde, University of Wffrzburg, FRG.

3H-thymidine (3H-TdR) autoradiography is still the main method applied in studies of cell proliferation and mode of growth in normal and tumor cell populations. Out of the various cell kinetic methods using 3H-TdR the determination of the labelling index (LI = number of labelled cells to all cells of a cell population) is most frequently applied. It provides a rough estimate of the proliferative activity of a cell population and is a prognostic indicator in the case of various malignancies. More detailed cell kinetic parameters can be obtained by the percent-labelled-mitoses method, the grain count halving method and the double labelling method with 3H- and ~4C-TdR. The duration of the cell cycle and its phases as well as the mode of growth under normal and perturbed conditions have been studied for a great variety of normal and tumor cell types using these methods. Combined with some mathematical modelling regulation of proliferation and differentiation in

various tissues as well as kinetic compartments throughout cell lineages can be explored. Also the extent of the growth fraction (GF) and the exchange of cells between GF and nonGF can be studied quantitatively by these methods. The mechanism of action of cytotoxic drugs, irradiation and other therapeutic regimens have been studied in a great number of ascites and solid animal tumors. Recently cell kinetic methods have been applied to study cell proliferation of human tumors after transplantation into nude mice. It has been shown that the cell kinetic parameters change after xenotransplantation, particularly the GF of human tumor cell populations increases considerably. This has implications on the conclusions which generally are drawn from the nude mouse model to the conditions in human beings.

$6.3

Receptor Autoradiography Combined with Immunocytochemistry. M. Sar Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599, USA.

A combined technique of autoradiography and immunocytochemistry has been developed in our laboratory to study interaction between steroid hormones and neuropeptides, protein hormones, neurohormones or neurotransmitters in the brain. In this procedure autoradiograms prepared after radiolabelled compound are processed for immunostaining with antibodies to neuropeptides, neurotransmitter synthesizing enzymes (TH, DBH) or receptor proteins. For colocalization of steroid hormone and antibodies to neuropeptide autoradiograms are prepared from the animals treated with colchicine prior to the administration of radiolabelled steroid horrnone. With the application of this technique, dopaminergic neurons of the hypothalamus, nor adrenergic neurons of the lower brain stem, gabanergic neurons of the preoptic area have shown to contain [3HI estradiol in their nuclei. [3HI progestin has also been localized in dopaminergic neurons of the hypothalamus and DBH containing neurons of the nucleus tractus solitarius. Colocalization of [3HI estradol with GHRH, SRIF, B-endorphin, neuropeptide-Y and ANF cells has been observed but not with LHRH neurons. Demonstration of steroid hormone association with its receptor protein in brain and uterus was possible utilizing autoradiogram of [3HI estradiol or [3H] progestin and antibodies to estrogen or progesterone receptor proteins. Furthermore, immunocharacteristic of steroid hormone target cells in rat anterior pituitary has been identified. (Supported by NIH Grant NS-17479).

$6.4 High Resolution Autoradiography in Electron Microscopy. M. A. Williams Dept of Biomedical Science, University of Sheffield, UK.

Invited symposia

468

$7. RECENT TRENDS IN EM CYTOCHEMISTRY

$7.1

Recent Developments and Applications of PostEmbedding Quantitative Immunocytochemistry. M. Bendayan and L. Ghitescu Department of Anatomy, Universit( de Montr(al, Montr(al, Quebec, Canada.

In recent years two alternative immunogold probes have been introduced. The protein G-gold and the protein A/G-gold complexes. These probes are by various aspects superior to the protein A-gold or the IgG-gold complexes because of their broader spectrum of reactivity with immunoglobulins of various mammalian species and particularly with the mouse monoclonal antibodies. They have also been found to be excellent for immunoblot analysis and therefore represent highly versatile and convenient probes providing labellings of high resolution. They were thus applied for quantitative immunocytochemistry in the study of the dynamics of cellular phenomenon such as vascular permeability and endocytotic activities. For this, hapten-tagged serum proteins were injected into the circulation and traced at different time points by post-embedding immunogold techniques. Various serum proteins including albumin, immunoglobulins and transferrin were tagged with dinitrophenol, fluorescein or digoxigenin prior to their introduction into the circulation. They were revealed by the protein A / G gold complex in conjunction with anti-hapten antibodies in various tissues and cells allowing for the study of the dynamics of the transendothelial transport through vascular walls, the glomerular filtration and the renal epithelial endocytotic activity. These studies have demonstrated that the hapten-tagged proteins combined to quantitative high resolution post-embedding immunocytochemistry represent a powerful approach for the morphological study of cellular dynamics under very favourable conditions in terms of resolution and physiological significance. Supported by the Medical Research Council of Canada.

$7.2

Application of Cryofixation, Freeze-Drying and Low Temperature Embedding for Immunocolloidal Gold (IEM) Localization of Sarcoplasmic Reticulum (SR) Proteins in Muscle. A. O. Jorgensen*, A. C.-Y. Shen*, S. A. LittlC and L. J.

McGuffeC *Dept. of Anatomy & Cell Biol., U. of Toronto, Canada and ~Dept. of Pharm., U. of New Mexico, N.M, U.S.A.

To optimize the use of monoclonal and site specific antibodies as probes for immunolocalization of intracellular antigens it is essential to also optimize the maintenance and accessibility of epitopes of the respective antigen. To this end we have previously reported on the use of cryofixation, freeze-drying and low temperature embedding in Lowicryl K4M (Jorgensen & McGuffee, J. Histochem. and Cytochem. 35, 723 (1987)) for IEM localization of SR proteins (Ca~+-ATPase and calsequestrin) and T-tubular proteins [ei-DHPR(Jorgensen et al, J. Cell Biol. 109, 135 (1989) and TS28 (Jorgensen et al., J. Cell Biol. 110, 1173 (1990)] in muscle tissue. To improve the

simultaneous visualization of colloidal gold particles and muscle membranes (SR, T-tubules and sarcolemma) muscle tissues were low temperature embedded in LR White instead of in Lowicryl K4M. Applications of this specimen preparation to the localization of SR proteins including Ca z+release channels (ryanodine receptor and IP3 receptor) will be presented. Supported by MRC of Canada (AOJ), HSFC Ontario (AOJ) and NIH HL 37015 (L.J.M.).

$7.3

Organelle Plasticity in Phagocytic Leukocytes. J. M. Robinson Ohio State University, Columbus, OH, 43210, U.S.A.

Certain organelles within phagocytic leukocytes display remarkable structural plasticity upon activation with appropriate stimuli. Human neutrophils contain a novel compartment which is characterized by the presence of the enzyme alkaline phosphatase (AlkPase). In resting cells this enzyme is present in small membrane-bound compartments within the cytoplasm and to a lesser extent on the cell surface. Upon stimulation with a chemotactic peptide or an active phorbol ester the AlkPase activity is rapidly upregulated to the cell surface. This process is unusual in that the small AlkPasepositive compartments fuse and then form tubular arrays which become continuous with the cell surface. The formation of these tubular structures is sensitive to the microtubuledepolymerizing drug nocodazole. Murine macrophages undergo a number of remarkable structural alterations upon activation with stimuli such as phorbol esters. The lysosomal compartment in these cells display elaborate alterations in structure following phorbol stimulation. These structures rapidly elongate and form elaborate tubular/reticular structures which have a close spatial association with cytoplasmic microtubules. Rapid alterations in macrophage microtubules, which may be important in the regulation of the formation of the tubular/reticular structures, also occurs during this time period.

$7.4

Cytochemistry, Autoradiography, In Situ Nucleic Acid Hybridization: Complementary Tools for Studying the Functional Organization of Cell Nucleus. F. Puvion-Dutilleul and E. Puvion Laboratoire de Biologic et Ultrastrueture du Noyau (UPR272, CNRS) BP n ~ 94801 Villejuif Cedex, France.

A number of new preparative methods for electron microscopy have been developed during the last twenty years. Used in parallel, they provide complementary information and make possible the study of the composition of defined cellular structures at in situ level. Cells infected with adenovirus type 5 (Ad5) are very suitable to study the function-structure relationships since the development of Ad5 induces reproducible nuclear alterations. In order to elucidate the precise location of the progeny AdS genomes in HeLa

Invited symposia nuclei and to determine their sites of transcription and replication, we present the techniques which have already brought conclusive data in the study of adenovirus infection or which could provide new informations in the future, and we examine their advantages and limits. Several methods concomitantly detect both cellular and viral nucleic acids in a section. The osmium ammine complex of Cogliati and Gautier clearly reveals different degrees of compaction of DNA in the nuclear compartments, but does not permit the identification of the various types of Ad5 DNA. The method of high resolution autoradiography provides functional data. A short pulse labelling reveals the

469 sites of nucleic acid synthesis whereas additional chase periods reveal the dynamics of the incorporated radioactive molecules. In situ hybridization which reveals defined nucleic acid sequences is a powerful tool for localizing Ad5 and their specific transcripts in biological material. We use biotinlabelled viral DNA probes and ultrathin sections of Lowicrylembedded infected cells. With appropriate pro-treatments of sections, in situ hybridization provides specific identification of the different types of viral DNA: total viral DNA, or only its single-stranded (ss) portions, or only its double-stranded (ds) portions. Viral RNA molecules can also be specifically detected.

$8. CYTOCHEMISTRY OF N E U R O N A L MESSENGERS

$8.t

Cytochemistry of Neuronal Messengers. T. Hokfelt Department of Histology and Neurobiology, Karolinska Institute, S-104 O1 Stockholm, Sweden.

The complexity of the nervous system including the diversity of neurons and glial cells with an array of processes extending over shorter or longer distances requires detailed morphological analysis at the light and electron microscopic levels. During the last decades a number of powerful histochemical methods have been developed suitable for analysis of the nervous system providing complementary data to earlier light and electron microscopic structural analysis. These methodologies include histochemical methods of various types, for example formaldehyde induced fluorescence, immunohistochemistry and in situ hybridization. This can be combined with retrograde and antrograde tracing methods which allow definition of transmitter specific projections. In the present symposium examples of some of these techniques will be presented encompassing both the most recent developments in tracing techniques and in in situ hybridization allowing the use of both radioactive and none radioactive probes and double labelling experiments. Moreover, it has been recognized that so called immediate early genes can be visualized both with immunohistochemistry and in situ hybridization providing a new tool to map functional circuits in the nervous system.

$8.2 New Cytochemical Approaches to Tracing of Neuronal Pathways. F. G. Woutertood Department of Anatomy, Vrije Universiteit, A msterdam, The Netherlands. Traditionally, neuroanatomy has been dealing with the cytoarchitectonic delineation of nuclear areas in the brain and with the tracing of fiber pathways that connect these areas. The Golgi silver impregnation method was used to study the shape and dendritic geometry of individual neurons. Knowledge of function was based on electrophysiological, and mare recently, on histochemical data. The application of immunohistochemistry has fundamentally changed our approach to brain research as well

as our view of the brain. Areas in the brain are nowadays studied using primarily immunohistochemical markers, whereas the traditional cytoarchitectonic stains are applied only for the comparison of the newly described areas with brain maps determined in the classical way. New tracing methods based on immunocytochemistry have been developed, as well as combinations of tracing methods and immunocytochemistry to determine the neurochemical substances involved in the pathways and the target neurons. Golgi impregnation has been supplanted by intracellular injection in slices of fixed brain. Most powerful are combinations of these methods. Knowledge of the neuron populations, the neuroactive substances that the cells contain, and their connectivity has provided new openings towards the functional interpretation of brain areas and the interconnecting pathways,

$8.3 Functional Neurochemical Mapping Using c-Fos, c-Jun and Other Immediate-Early Gene Markers. S. P. Hunt Molecular Neurobiology Unit, MRC Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QH, UK. Immediate-early genes (IEGs) were originally identified as a class of genes which were rapidly and transiently expressed in cells following stimulation by growth factors but which were not themselves dependant upon new protein synthesis for their induction. Many of these IEG products have subsequently been shown to be transcription factors, binding to DNA. The subsequent demonstration of IEG expression within the central nervous system (CNS) led to the suggestion that genes such as c-fos and c-jun could be involved in triggering the molecular changes underlying long term change. At the same time, evidence was accumulating to suggest that, at least in neuronal cell lines, IEG expression could be increased by depolarization of the cell membrane and that the involvement of growth factors or second messengers was by no means essential. This led to the hypothesis that the appearance of, for example, Fos-like proteins could be used as activity markers at the single cell level within the nervous system. However it is now clear that IEG expression within the CNS is not simply a result of neuronal activation but is related to the long term consequences of stimulation. The appearance of IEG mRNA

470

Invited symposia

or protein is dependant upon numerous criteria which vary according to the brain area under study. In the spinal cord, for example, activation of high threshold nociceptive sensory afferents, but not low threshold 'touch' afferents is necessary to activate c-fos and other IEGs in neurons of the dorsal horn. Moreover, this pattern can change and evolve with time and may be causally linked to changes in peptide gene expression which can follow injury. In contrast to these rapid changes in gene expression, recent evidence now suggests a role for c-jun in the regenerative response of neurons, appearing only at around 24 h following axon damage. The analysis of IEG

$9. C Y T O C H E M I S T R Y

expression is therefore emerging as a powerful new tool in our understanding of events following stimulation of the CNS.

$8.4 Molecular Anatomy of Neuronal Interactions by Combined In Situ Hybridization and Immunocytochemistry. B. Bloch Bordeaux, France.

OF PLANTS

$9.1

Cytoskeletal Dynamics in Living Plant Cells. P. Hepler, A. Cleary, U. Meindl, P. Wadsworth, G. Wasteneys and D. Zang Dept. of Biology University of Massachusetts, Amherst, MA, USA.

$9.2

Imaging Calcium Dynamics in Living Plant Cells. N. D. Read Molecular Signalling Group, Institute of Cell and Molecular Biology, Edinburgh University, Edinburgh EH9 3JH, UK.

Living systems have fashioned a complex network of signalling capabilities around Ca 2+. The direct demonstration of signal response coupling via Ca 2+ requires the measurement and localization of changes in cytosolic free Ca 2+ ([Ca2+]~) in living cells. In the last few years we have had considerable experience in imaging [Ca2% dynamics in a wide range of plant and fungal cells. We are presently employing three different technologies towards this end: ratio imaging of cells loaded with fluorescent dyes (Indo-1 and Fura-2); confocal microscopy of cells loaded with fluorescent dyes (Fluo-3 and Calcium Green); and imaging, using a photon counting camera, of luminescence from plants genetically transformed with the aequorin gene. This paper will cover three aspects of our recent work: (1)problems with loading fungal hyphae with fluorescent dyes; (2) confocal imaging of [Ca2% during red light-mediated photomorphogenesis, and the use of caged Ca -'+ and caged InsP3 to dissect apart and define components of the signal transduction pathway; and, (3) imaging [Ca2+]~in whole seedlings subjected to cold shock, touch and wounding. $9.3

Cell Wall Subunit Affinodetection and Topochemistry of the Assembled Composite. B. Vian Laboratoire des Biomembranes et Surfaces Cellulaires Vdgdtales, ENS, 46 rue d'Ulm, 75231 PARIS Cedex 05, France.

Higher plant cell walls are mutifunctional constructions built up from cellulose and an heteromolecular matrix. In the recent years we have focused on the fact that many cell walls have an ordered architecture revealing a helicoidal pattern (1). The paper presents recent data concerning: a) the interactions of wall polymers in heteromolecular fractions from controlled dissociated walls; b) the emerging sites of the polymers in the cell and the spatial distribution of cellulose and of some

matrix polymers in helicoidal walls from both elongating and non elongating cells. Wall subunits were targeted mainly by means of affinity probes bound to fluorescent or gold markers (2). The probes were the enzyme cellobiohydrolase, CBH1 for cellulose, monoclonal antibodies JIM 5 or JIM 7 for homogalacturonan sequences according to their degree of esteriflcation (3), and a polyclonal antibody for targeting hydroxyproline-rich structural glycoproteins (HRGPs). Affinity labelling was complemented by subtractive cytochemistry and by the labelling of the available anionic groups by means of cationic gold particles. On the heteromolecular fractions the available residual charges appeared present along the microfibrils organizing an anionic surfacting coat.Data confirm that co-distribution and a strong association exist between cellulose and some major matrix components, thus strengthening the morphogenetic role of the latter. (1) Roland, J. C., Reis, D., Vian, B. & Roy (1989). Biol. Cell 67, 209 - 220 (2) Vian, B. & Roland, J. C. (1991) . Biol. Cell 71, 4 3 - 5 5 (3) Knox, J. P., Lindstead, P. J., King, J., Cooper, C. & Roberts, K. (t990). Planta 181, 512-521.

$9.4 Morphological Dynamics of the Endoplasmic Reticulum Imaged in Living Plant Cells. H. Quader Zellenlehre, Fakultdt ffir Biologie, University of Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg 1, Germany.

Isolated vesicles of the endoplasmic reticulum (ER) as obtained by cell fractionation procedures, hardly represent the morphological situation in vivo of this continuous, highly anastomosing membrane meshwork. Three morphologically distinct modifications of the ER can be distinguished in so different plant cells as epidermal cells, root hairs, protonema cells of mosses, by fluorescence microscopy employing the vital fluorochrome DiOC6 or video-enhanced differentialinterference-contrast microscopy: two tubular forms, a peripheral network closely associated with the plasma membrane and long tubular strands deeper in the cytoplasm, and flat sheet-like lamellae fitted into or closely associated with the peripheral network. These modifications reversibly change into one another very rapidly depending on the environmental conditions. At the extreme, the ER may occur entirely in tubular form, e.g. at low temperature (35~ interfering with the cellular calcium distribution, or elevating the cytosolic pH.

Invited s y m p o s i a

471

The tubular ER moves along actin filaments with myosin most likely functioning as linking and motor protein. Protons and calcium seem to play a major regulatory role in ER shaping. Both cations affect the ER morphology either directly by causing the fusion of parts of the tubular ER to flat sheets or

S10, ENZYME

HISTOCHEMISTRY:

IN SITU

the decay of the latter to tubular elements, or indirectly by destabilizing the link between the actin filaments and the ER. The movement of the ER and its 3D-arrangement has been studied in different cell types by eonfocal laser scanning microscopy.

STUDY

$10, I

Enzyme Histochemistry: I n Situ Study of Cell Metabolism. C. J, F. Van Noorden Laboratory of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, The Netherlands. The efforts made in the past two decades to validate quantitative enzyme histochemical methods enable the analysis of kinetic parameters of enzymes in their cellular environment. These parameters can be essentially different from biochemically determined parameters in dilute solutions with protein concentrations of 0.1% or less. Protein concentrations in mammalian cells are usually 15-25~ A particular enzyme of interest may be diluted in the cytoplasm but a large variety of other macromolecules take up a substantial fraction of the total cytoplasmic volume. Enzymes perform their biological task in the crowded cytoplasm in a different way than in a dilute solution. Moreover, many enzymes can be found either in a soluble form or associated with membranes or the cytoskeleton as part of the regulation of their activity. Quantitative histochemical methods for the demonstration of catalytic activities of enzymes in unfixed tissue sections or cells allow the study of these posttranslational regulation mechanisms. For glucose-6phosphatase it was found that both KM and Vma~values were significantly different in periportal and perieentral zones of rat liver. In fact, KM values were found to increase linearly with V .... values, irrespective of sex and feeding condition, showing that the enzyme exists in a high affinity configuration at low enzyme concentrations. At high enzyme concentrations the enzyme transforms to a low affinity configuration by a hysteretic mechanism (Jonges et al., J. Biol. Chem. 267, 1991: 4878-4881). A similar kinetic behaviour was also described for Ca2+-ATPase in rat muscle (Blanco and Sieck, Histochem. J. 24, 1992: in press). These findings clearly indicate that the in situ analysis of cellular metabolism reveals a different kinetic behaviour of enzymes than in dilute solutions and may be a more reliable method to study the metabolism as it occurs in vivo.

St0.2 In Situ Measurements of Enzyme Activities in the Brain. P. Kug/er Dept. of Anatomy, University of Wr~rzburg, Wurzburg, FRG. Catalytic enzyme histochemistry applied to brain sections allows both localization and quantification of enzyme activities (Kugler, P., Int. J. Biochem. 23: 6 5 7 - 6 6 1 , 1991). This study refers to the histochemistry of dehydrogenases in

OF CELL

METABOLISM

the rat hippocampus which is a well defined brain area. We have studied three mitochondrial enzymes involved in glutamate and GABA metabolism, i.e. NAD-linked isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH) and GABA transaminase (GABAT). It was shown that 1) the use of appropriate tetrazolium salts in combination with tissue stabilizers, exogenous electron carriers and inhibitors of the respiratory chain offered satisfactory conditions for the qualitative and quantitative evaluation of dehydrogenase activities; 2 ) G D H activity was restricted to astrocytes, whereas ICDH and GABAT seemed to be localized in both neurons and astrocytes; 3 ) i n spite of different levels of activities and cellular localizations ICDH, GDH and GABAT were significantly correlated in their hippocampal distribution. It is concluded that the enzymes demonstrated might be involved in a layer-specific cooperation to produce or catabolize glutamate and GABA, and thus to control aminoacidergic transmission. - - This study was supported by the Deutsche Forschungsgemeinschaft. S10.3

Measurements of Local Substrate Concentrations in Tissue Sections using a Bioluminescence Technique. S. Wa[enta and W. Mueller-Klieser Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, D-6500 Mainz, FRG. The metabolic status of malignant tumors are characterized by a pronounced spatial variation in tissue oxygenation, in pH and in the fraction of proliferating cells (Vaupel et al., Cancer Res. 49: 6449, 1989). The resulting heterogeneous metabolic micromilieu represents a crucial problem in non-surgical cancer therapy. To further describe the metabolic status of such tissues, a bioluminescence technique was developed allowing for the detection of local ATP, glucose, and lactate concentrations in consecutive cryosections. Cryosections were made from rapidly frozen tumors, picked up on cover glasses and were heat inactivated for t0 minutes at 100~ For measurement, a solution containing all necessary enzymes and cofaetors for substrata specific luminescence reaction is frozen in a mould and covered with the tissue section. After controlled thawing the enzyme reaction starts, and the luminescence is registered with high spatial resolution by an imaging photon counting system (Argus 100, Hamamatsu Europe, Herrsching, FRG) connected to a microscope (Axiophot, Zeiss, Oberkochen, FRG). The resulting intensity distributions are transferred into concentration values using appropriate tissue standards with known substrata contents. The evaluation of local concentrations in viable and necrotic tissue areas is performed by comparision with parallel sections stained with H.E. using a specially designed computer software. (Supported by the BMFT 01 ZO 8801)

472

Invited symposia

s10.4 Immunocytochemical Detection of Cathepsins and Cystatins at LM and EM Level to Elucidate their Physiological Functions. N. Katunuma Tokushima, Japan.

S l l . H I S T O C H E M I S T R Y OF R E C E P T O R S Sll.1

Histochemistry of Receptors: An Up-Date on Ligand Binding Autoradiography. J. M. Palacios Research & Development Department, Laboratorios Almirall, Cardener, 68-74, 08024 Barcelona, Spain.

Receptor autoradiography (RA) visualizes, at the regional or light microscopic level, radiolabelled receptor binding sites. Like its parent technique, high affinity binding analysis, receptor autoradiography is well established as an essential widely used research methodology. When used in combination with image analysis, RA provides spatial mapping of receptor localization at moderately high resolutions, in combination with full quantitation of ligand affinity and/or receptor densities. RA suffers from a number of limitations. A major problem is discrimination of sites of origin for receptors. Demonstration that a given receptor originates in a specific cell population could only be made indirectly. Another limitation of RA is not inherent to the technique itself, but arises from the lack of selectivity of many ligands. Ligands previously considered as selective for a receptor population do, in fact, bind to several subtypes of the same receptor class. Advances in the understanding of the molecular biology of receptors, such as the cloning of the genes coding for a large and ever-increasing number of receptor molecules have produced developments in receptor analysis. Knowledge of the sequence of the mRNA coding for receptor proteins has made possible the development of nucleic acid probes. These can be used in combination with the technique of in situ hybridization to visualize perikarya that express the mRNA for a particular receptor with unprecedented selectivity, allowing direct demonstration of sites of active synthesis of receptor proteins. Combination of in situ hybridization and receptor autoradiography with highly selective liagands provides precision in characterizing receptor populations. Sll.2

Molecular Neuroanatomy of Receptors and Enzymes - - Targets for Psychoactive Drugs. G. Richards Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland.

In order to provide a more rational basis for developing psychoactive drugs, an in-depth knowledge of the structure, cellular expression and regulation of their molecular targets is essential, particularly considering the anatomical complexity of the CNS. In neuroscience research the application of quantitative receptor/enzyme radioautography with image analysis and in situ hybridization histochemistry - - high

resolution protein and transcript mapping techniques, respectively - - has been instrumental in this regard. To exemplify this research, the characteristics of two drug targets in the CNS - - GABAA receptors and monoamine oxidases - will be described. They are the recognition sites of future generation drugs for the treatment of anxiety, epilepsies and sleep disturbances on the one band, and depression, Parkinson's disease and Alzheimer's disease on the other. GABAA receptors in the CNS - - the targets for the therapeutic effects of benzodiazepines - - are transmembrane heterooligomeric (pentameric?) glycoproteins which probably exist in structurally and functionally diverse forms. The 5 polypeptide subunits (a,/3,y,6,p) and a total of 15 variants, which have been cloned to date, have a distinct pattern of mRNA expression in the CNS, creating new opportunities for drug development - - namely their targeting to specific subpopulations of GABAA receptors. Monoamine oxidases (MAO-A and MAO-B) - - isoenzymes identified by substrate selectivity, inhibitor sensitivity and primary structure - - oxidatively deaminate neurotransmitterand xenobiotic amines in the CNS and periphery. In human brain, serotonin and noradrenaline are preferentially metabolized by MAO-A, the trace amines phenethylamine and methylhistamine by MAO-B but dopamine, tyramine and octopamine by both enzymes. Paradoxically, MAO-A is expressed in noradrenergic neurons but MAO-B in serotoninergic and histaminergic neurons as well as in glia.The physiological role of MAOs - - in transmitter inactivation - - as well as their pathophysiological roles - - in neurodegenerative diseases - - provide drug targets for selective, reversible inhibitors with unique and even unexpected therapeutic profiles.

Sll.3 Mapping of Receptors for Catecholamines and Peptides. R. Elde, A. P. Nicholas, V. A. Pieribone and T. H6kfelt Department of Histology & Neurobiology, Karolinska Institutet, S-104 O1 Stockholm, Sweden. Several receptors have been recently cloned, including adrenergic and peptide receptors, the latter providing further evidence for a functional role of peptides in the nervous system. We have synthesized oligonucleotide probes against a l b , a2b (brain and kidney) /31 and /32 adrenoreceptor mRNAs, as well as to the neurotensin and substance P receptors. Both central nervous system tissue and peripheral organs have been analysed. In the brain, differential distribution patterns were observed. For example, a l b mRNA was seen in cerebral cortex, thalamus, dorsal and medullary raphe, as well as in liver, heart and kidney. Brain a2b mRNA was found in the hippocampus, spinal grey matter, striatum, cerebral and cerebellar cortices, as well as in

Invited s y m p o s i a

473

s3nnpathetic and dorsal root ganglia and in the adrenal medulla. fll mRNA was seen in cerebral cortex and heart, while/32 mRNA was in hippocampus, trachea and lung. The kidney a2b probe labelled kidney, but not brain. Neurotensin mRNA was observed in several brain regions with particularly high levels in the doparnine cells of the substantia nigra and in the cholinergic forebrain cells. Substance P receptor mRNA had a wide distribution in brain and spinal cord, for example in the caudate nucleus and in the medulla oblongata. These results provide

S12. NEUROHISTOCHEMISTRY

complementary information to earlier binding studies and open up possibilities to study regulation of receptor synthesis.

Sll.4 Sorting of Glucose Transferase in Mammalian Cells. Jo W. Slot Dept of Cell Biology, ]~4edical School, University of Utrect, The Netherlands.

OF DEVELOPMENT

5t2.1

Spatio-Tempora| Patterns of Hormone and Hormone Transcription Factor Gene Expression During the Development and Mature Function of the Neuroendocdne System. L. W. Swanson Department of Biological Sciences, Hedco Neuroscience Building, me 2520, University of Southern California, Los Angeles, California, 90089-2520, USA.

The core of the neuroendocrine system consists of the hypothalamus and pituitary, and the mechanisms that interrelate these two organs. We have used this system as a model for studying the spatio-temporal patterns of gone expression that may be involved in establishing a functional system during the development of the rat embryo, as well as in the modulation of gone expression during different functional states of the adult organism. In the embryo, we have examined the spatio-temporal pattern of expression for the various genes that are involved in the synthesis of the classical anterior pituitary hormones and it was found that there is an interesting compartmental pattern of expression, which is also characterized by distinct temporal patterns of expression. In addition, we have correlated these patterns with the pattern of expression of two putative transcription factors that are thought to be involved in regulating the expression of growth hormone, thyrotropin releasing hormone, and prolactin. In the adult rat, it is now becoming clear that individual hypothalamic neurosecretory neurons can not only synthesize multiple neuropeptides, but that the genes regulating the synthesis of these neuropeptides may be differentially regulated by different physiological and behavioral factors that fall under the broad category of stress. 512.2

Molecular Neuroanatomical Indications for Gene Repair in Post-Mitotic Vasopressin Neurons of Brattleboro Rats. F. W. Van Leeuwen Netherlands Institute for Brain Research, Amsterdam, The Netherlands. The homozygous Brattleboro rat (di/di) is a mutant strain of the Long Evans rat which exhibits a recessively inherited hypothalamic diabetes insipidus. The frame-shift mutation is due to a single base deletion in exon B of the vasopressin (VP) gone and results in a different C-terminus of the VPprecursor. This altered VP precursor cannot be translocated at the endoplasmic reticulum since it remains anchored in the

AND AGING

membranes of this organetle (Schmale etal., Eur. J. Biochem. 182, 625, 1989). This precursor therefore never reaches the Golgi apparatus and does not undergo axonaI transport from the magnocetlular hypothalamic nuclei to the terminals in the neural lobe. An intriguing finding was that a small but linearly increasing number of solitary hypothalamic neurons (up to 3%) of the di/di rat undergoes a switch to a heterozygous phenotype. In addition to the mutant VP precursor they express the wild-type VP precursor, which is axonally transported (Van Leeuwen et al., PNAS 86, 6417, 1989). The molecular event generating this somatic reversion occurring in neurons after termination of their proliferation (foetal day 15) is presently being worked out. Preliminary data (Evans et al., Soc. Neurosci. 17, 380, 1991) indicate that the revertant phenotype is due to single base insertions in the region of the deletion in exon B. It has been reported in the ostrich that at the very place of the G deletion even an amino acid insertion occurs indicating that this area can represent a potential "hotspot recombinational" area similar to the rosy locus of drosophila rnelanogaster and the gene responsible for Duchenne muscular dystrophy. In conclusion, the di/di rat model has shown that alterations at the level of DNA of postmitotic neurons are possible.

512.3

Axonai RNA in the Hypothalamo-neurohypophyseal System: Structures and Functional Implications. G. 1=:.Jirikowski Dept. of Neuropharmacology, Scripps Res. Inst., La Jolla, USA. mRNAs coding for oxytocin (OT) or vasopressin (VP) have been detected in axonat secretory vesicles in the rat hypothalamoneurohypophysial system. Magnocellular hypothalamic neurons of Brattleboro rats are capable of accumulating, transporting and translating exogenous VP RNA, thus leading to a temporary correction of diabetes insipidus. Poly A ( - ) RNA proved to be much more effective than poly A( + ) RNA. In further studies we used primary cultures of dissociated fetal rat hypothalamus !n order to investigate the fate of radiolabelled VP RNA added to the culture medium. Autoradiograms revealed that a portion of neurons in vitro accumulates RNA in processes and perikarya. This group of cells includes the VP immunoreactive perikarya but is not limited to this ceil type. We could further demonstrate in such experiments that depolarization of cells with 50mM KC1 results in increased amounts of radioactivity in the culture medium, indicating a stimulus dependent release of RNA. This effect was in part Ca + + dependent. Synthetic Arg-VP

474

Invited symposia

also stimulated release of incorporated exogenous VP RNA. Our data indicate that hypothalamic neurons can accumulate, transport and translate exogenous RNA and that RNA can also be secreted in a stimulus-dependent manner. It is possible that exchange of RNA occurs in the hypothalamus under physiological conditions thus representing a novel way of interneuronal signalling.

S12.4 Histochemistry of the Human Hypothalamus in Development and Aging. D. F. Swaab Netherlands Institute for Brain Research, Amsterdam, The Netherlands.

S13. I M M U N O H I S T O C H E M I S T R Y : N E W S O L U T I O N S FOR O L D P R O B L E M S S13.1

Modern Microscopical Imaging Methods. J. M. Polak and A. E. Bishop Department of Histochemistry, Royal Postgraduate Medical School, DuCane Road, London W12 ONN, UK.

The huge advances made in the understanding of neuroendocrine and endothelial cell biology by the advent of immunocytochemistry are being built upon with the development of new investigative morphological methods. Immunocytochemical techniques have been refined and are now applied, at the light and electron microscopical levels, in the study of not only bioactive products but also their precursor or abnormal molecular forms. Gene expression can be examined in histological preparations by in situ hybridization. The results of this technique, viewed together with data from immunocytochemistry of stored products, can reveal not only sites of expression of specific nucleic acid sequences but also the rates of product synthesis. In vitro autoradiography can be used to localize, characterize and quantify binding sites on the surface of neuroendocrine cells, and if the nucleotide sequence has been disclosed the mRNA can also be localised at the cellular level. In combination, these methods provide the investigator with the means not only to identify neuroendocrine and endothelial cells but also to derive morphofunctional information on their biology.

S13.2 Immunocytochemistry in Combination with other Techniques. G. V. Childs Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX 77555-0843, USA.

For several decades immunolabelling has been a valuable tool for identifying sites of synthesis, storage or binding of antigens. As multiple labelling techniques were improved, one could determine sites of multiple antigens in the same tissue sections or cell population. However, with immunolabelling alone, one could not distinguish functionally different sites,

S14. C Y T O C H E M I C A L

MARKERS

such as sites of binding from sites of storage. Thus, target cells might be mislabelled as source cells. During the past decade, the sensitive technology developed by immunocytochemists has been adapted to allow the detection of probes other than antibodies. These probes include biologically active ligands that detect target cells, or complimentary DNA, RNA, or oligonucleotide probes to detect mRNA and sites of synthesis. With basic dual detection systems the researcher can now detect expression of multiple functions in a given cell or tissue section. The first part of the presentation illustrates how affinity cytochemistry combined with immunocytochemistry is used to study physiological changes in pituitary target cells for releasing hormones. The second part of the presentation will show how in situ hybridization can be combined with immunolabelling to identify the source of a regulatory factor in the pituitary that inhibits the release of follicle stimulating hormone (follistatin). Dual in situ hybridization and immunolabelling labelling studies can also be used to detect and characterize multipotential pituitary cells. Studies of plasticity in the pituitary cell population during the estrous cycle show that some of these augment the gonadotropes just before the midcycle surge. Supported by NIH R01 HD 15472, NIH R01 DK 39553; NSF IBN-9117897. S13.3

Quantitative Immunocytochemistry. T. Cowen and T. Andrews Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London UK.

s13.4 Recombinant Monoclonal Antibodies in an In Vitro Immune System. H. Hoogenboom Cambridge Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK.

AS DIAGNOSTIC

S14.1 Problems and Possible Solutions using Cytochemieal Markers in Toxicology. G. R. Bullock K125.10.16, Ciba-Geigy A G, CH-4OO2-Basel, Switzerland.

The question often arising in toxicology is how to establish

TOOLS

IN TOXICOLOGY

early predictors for potential toxicological changes. Previously much reliance was placed on gross a)morphological changes such as alterations in structure, deposition of abnormal material e.g. amyloid and b) histochemical changes such as up/down-regulation of enzymes. In addition, cell proliferation leading to benign/malign tumours was and is of paramount importance or alternatively cell death as in

Invited symposia

475

irreversible neuronal toxicity. All of this, however, is classical toxicology as we know it. Now there exists many opportunities to incorporate the newer methodology deriving from molecular biology and allied techniques. Whilst more knowledge has been gained re abnormal cell constituents such as the appearance of Glial Fibrillary Acidic Protein (GFAP) in the brain, greater emphasis should be placed on more subtle changes such as induction of c-fos protein following treatment with neurotoxic compounds or cardiotoxicity following DNA intercalation by adriamycin. Predictors for cell proliferation (cyclins), cell differentiation, breaks in nucleic acids and mitotic aberrations (by 'in situ' hybridization) and sensitive cellular changes such as upregulation of growth factors are all now within the remit of the toxicologist. These developments will be discussed in particular with regard to cardiovascular and brain toxicology and the potential for reducing the drug test period.

S15. A D V A N C E D

MICROSCOPICAL

Imaging of Cells and Tissues by Confocal Microscopy. B. A m o s .MRC Laboratory o f Molecular Biology, Hills Rd, Cambridge, CB2 2QH, UK.

S15.2

Time Resolved Microscopy. H. Tanke, N. P. Verwoerd, J. Bonnet, C. R. G. van de Geest and E. J. Hennink Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.

2-Photon Imaging in Biological Microscopy. W. Webb Ythaea, USA.

P. 1t. Bach Faculty of Science Polytechnic of East London, Romford Rd, London E15 LZ4, UK.

S14.3 The Use of Isolated Cells in Toxicity Testing. A. J. Guillouzo Hdpital de Pontchaillou, 35033 Rennes, France.

S14.4 The Use of Bone Proteins as Cytochemical Markers of Bone Metabolism. P. Clezardin Lyon, France.

TECHNIQUES

$15.1

$I5o3

s14.2 Application of Fluorescent Probes and Confocal Scanning Laser Microscopy Data for Toxicology.

S15.4 Atomic Force Microscopy for High Resolution Imaging of Biological Surfaces. J. H. Hob and P. K. Hansma Department of Physics, University of California Santa Barbara, Santa Barbara, California 93106, USA. The Scanning Tunneling Microscope started a revolution in microscopy that has led to the emergence of a family of Scanning Probe Microscopes. These instruments have in common the feature of passing a probe in close proximity to a surface, and collecting spatially resolved information about properties such as tunneling current, physical topography, ion conductance or temperature. One of the most promising members of this family of instruments for biological applications is the Atomic Force Microscope (AFM, also known as the Scanning Force Microscope, SFM). The AFM has now been used to image samples ranging from whole cells to single ion channels. On hard flat samples this microscope can often resolve atoms, though on soft biological materials the lateral resolution is currently in the range 1-50 nm. Its ability to image in fluids, including physiological buffers, and to gather dynamic data point to a bright future in biology. In addition to imaging, the AFM also can be used for experimental manipulation of membranes and molecules, and can measure a variety of local physical and biophysical surface properties. Future technological developments, particularly in the area of tip structure and composition, will improve resolution and extend the types of local properties that can be examined.

476

Minisymposia

MINISYMPOSIA

M16. APOPTOSIS M16.1 Ultrastructural Localisation of BCL-2 and the Epstein-Barr Virus Protein BHRF-1 to the Outer Mitochondrial Membrane. P. Monaghan, D. Robertson and T. F. Hickish Institute of Cancer Research, Haddow Laboratories, 15, Cotswold Road, Sutton, Surrey SM2 5NG, England. The BCL-2 gene was first recognised in follicular B-cell lymphomas with the t(14; 18) translocation. This translocation brings the BCL-2 gene to the immunoglobulin heavy chain locus, leading to de-regulation and overexpression of the mRNA and protein. The gene codes for a 26KDa protein of unknown function. Light microscope immunolocalisation of BCL-2 has demonstrated the protein in a variety of normal tissues cells which are characterised by apoptic cell death suggesting that the BCL-2 protein protects cells from apoptosis. Because of the uncertain function of the BCL-2 protein it is important to determine its subcellular location. Previous cell fractionation studies have proposed an inner mitochondrial membrane location for BCL-2. We have determined the locatisation of the BCL-2 protein in lymphoma and breast tumour cell lines and biopsies by cryo-sectioning, low temperature embedding and freeze substitution. In all cases BCL-2 was predominantly associated with the outer mitochondrial membrane (approx. 50% of label). A closely related protein BHRF1 with a 40% homology to the BCL-2 gene, but unknown function also localises to the outer mitochondrial membrane.

M16.2 A New Method to Detect Apoptosis in Paraffin Sections: In $itu End-Labelling of Fragmented DNA. J. Wijsman, R. Jonker, C. J. Cornelisse and J. H. van. Dierendonck Departments of Surgery and Pathology, University of Leiden and TNO Radiobiological Institute (RJ), Rijswijk, The Netherlands. A new staining technique to facilitate the detection of apoptosis in formalin-fixed paraffin embedded tissue sections is described. As extensive DNA fragmentation is one of the characteristics of apoptosis, these breaks can be visualized by an in situ endlabelling technique (ISEL). After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated in DNA breaks by polymerase and subsequently visualized by DAB staining via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in prostate and uterus after castration, tumors, lymph node follicles and embryos. Apoptotic cells could be discriminated from areas of labelled necrotic cells, in which DNA degradation occurs as well. We used H&E stained sections of involuting prostates of castrated rats to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL labelled cells and the fractions of apoptotic cells morphologically determined in adjacent H&E stained sections.

M16.3 Endonuclease Activation in Apoptotic Cells. V. G. Evans and I. D. Bowen School of Pure and Applied Biology, University of Wales College of Cardiff, UK. The classic diagnostic feature of cells undergoing apoptosis or programmed cell death is the appearance of DNA fragments, cut to multiples of 200bp by a Ca z+ and Mg 2+ dependent endonuclease. A cytochemical method was used to check results which showed that this endonuclease had been activated independently of apoptosis-inducing treatment in murine thymocytes. Samples, air-dried on microscope slides, were fixed in Bouin's fixative and then put through the Feulgen reaction. Apoptotic cells show dark purple stained condensed chromatin and are easily distinguishable from normal or necrotic cells. Results of the cell counts showed the expected levels of apoptosis, depending on treatment. Endonuclease activation, visualized as DNA laddering on agarose gels, occurred in the identical samples regardless of treatment. Rapid endonuclease activation has been reported in the literature, as has the induction of apoptosis by various stress factors. Here it is proposed that endonuclease activation can occur independently from the genetically governed programmed cell death as a response of cells which are susceptible to the induction of apoptosis and which are undergoing fast-acting, irreversible damage. Therefore, endonuclease activation should be regarded as indicative of apoptosis only in conjunction with other morphological or biochemical features of apoptotic cells.

M16.4 Cell Death and Growth Arrest: Integral Components of Tissue Homeostasis. P. A. Hall, P. J. Coates, B. Ansari, J. Kearsey, J. Whitaker and N. Sawhney Dept of Histopathology, UMDS, St Thomas's Campus, London, UK. That the regulation of populations of cells in normal tissues involves control of cell proliferation and differentiation has long been realised. In addition, it has been well documented that pathological processes may be associated with abnormalities of these processes. There is now considerable information concerning the mechanisms of cell proliferation and differentiation, but these are only one facet of the mechanisms that regulate tissue homeostasis. We argue that considerable attention should be placed on two other primary controls of cell number and function in tissues: namely programmed cell death and growth arrest. Recently molecular biological methods have led to the identification of genes involved in these processes and the generation of antibodies that recognise the products of these genes. For example, recent data points to a role for genes such as bcl-2, myc and p53 in programmed cell death. In addition two novel cDNAs (RP-2 & RP-8) have been described that specifically expressed by thymocytes undergoing programmed cell death and a number

Minisymposia

477

of genes are expressed in growth arrest (eg. TI-I, prohibitin, gas & gadd). We have been defining the distribution of expression of such genes in normal and pathological tissues, as well as in vitro model systems, using molecular methods including in situ hybridisation. In addition, we have been generating novel antibodies raised against genetically engineered recombinant proteins. Of considerable interest is the observation that the in vivo or in vitro treatment of cells with chemotherapy, radiation or heat shock can induce programmed cell death and the expression of growth arrest genes.

radiation-induced apoptosis. Radiation induced apoptosis could almost completely be blocked in IL-3 dependent cell lines after addition of IL-3. Double labelling of a cell surface marker and the DNA strand breaks allowed for further analysis of subpopulations of cells. This method proved valuable in the fields of AIDS and of hyperthermia research.

Ml6.5 Detection of Apoptosis with In Situ Nick Translation.

M. G. Ormerod, X.-M. Sun, R. T. Snowden and G. M. Cohen MRC Toxicology Unit, Woodmansterne Road, Carshalton, Surrey SM5 4EF, England.

R. R. Jonker, J. G. J, Bauman, J. H. Wijsman* and J. W. M. Visser Institute of Applied Radiobiology and Immunology, TNO Rijswijk, and *Dept. of Surgery, Leiden University, The Netherlands. A technique was developed for the detection and quantification of apoptosis in individual cells using In Situ Nick Translation (ISNT). The technique is based on the detection of DNA degradation, caused by activation of endogenous endonucleases. This degradation is an early and specific characteristic of apoptosis. DNA breaks could readily be detected by enzymatic incorporation of labelled nucleotides, starting at DNA strand breaks. Biotin- or digoxigenin labelled nucleotides were stained and flow cytometry was used for quantification. The fluorescence of cells in apoptosis was up to 100 fold stronger than in control cells. ISNT also allowed for easy and objective detection of apoptosis in paraffin-embedded material by microscopy. The method was used to detect apoptosis in haemopoietic cells and cell lines after irradiation. The effect of radiation on the induction of apoptosis could be detected down to 0.25 Gy ),-irradiation in subpopulations of bone marrow cells and thymocytes. We showed that growth factors can inhibit

M16.6 Characterisation and Quantification of Apoptosis by F l o w Cytometry,

Conventional methods for detecting apoptotic cells (light and electron microscopy, DNA gel electrophoresis) do not tend themselves to quantification. Flow cytometric methods have the advantage that measurements are made on large numbers of individual ceils yielding good quantitative data. Cells can also be sorted for further morphological or biochemical analysis. Several authors have reported that, in a DNA histogram, apoptotic cells give a ' s u b - G l ' peak. This method is limited in its use because it uses fixed cells. In an alternative method, unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342; the apoptotic cells take up this dye more rapidly. Non-viable ceils can be enumerated at the same time by addition of propidium iodide. We have used this method to examine the effects of various inhibitors on the induction of apoptosis in an IL-3 dependent murine cell line after withdrawal of IL-3 and in rat thymocytes treated with glucocorticoids or DNA topoisomerase poisons. The basis of the assay lies in a change in the properties of the membranes of apoptotic cells. These changes have been further characterised in rat thymocytes using a series of derivatives of fluorescein diacetate.

M17. E N Z Y M E H I S T O C H E M I S T R Y M17.t

Microfluorometric Kinetic Analysis of Cathepsin B Reaction in Single Human Thyrocytes Using an Image Analysis System. L. Kayser and P. E. Hoyer Institute of Medical Anatomy, Dept A, The Panum Institute, University of Copenhagen, Denmark. Dept of Medicine E, Frederiksberg Hospital, Frederiksberg, Denmark. The activity of the cysteine proteinase Cathepsin B has been assessed in unfixed single human thyroid epithelial cells using an image analysis system (Image-l), The reaction was continuously monitored at room temperature by measuring the increase in fiuorescense intensity due to formation of a Schiff-base product formed by using N-CBZ-ala-arg-arg-4methoxy-2-naphthylamide as substrate and 5-nitrosalicylaldehyde as coupling agent (Van Noorden et al., Histochem J 1987; 19: 483). Contribution from non-specific fluorescent signals was eliminated by adjusting the video signal to zero using leupeptin - - a specific inhibitor of cathepsins B, H and L. A plot of fluorescense intensity versus time showed a lag

period of 5 ( 4 - 8 ) rain, followed by a linear increase for 5 ( 3 - 8) rain. Then at the same time as crystallization began to occur in the cytoplasm, a marked decrease in reaction rate was seen for 3 (1 - 4 ) rain followed by a new linear increase for 11 ( 8 - 14) rain. The second linear part of the curve was not as steep as the first one. In conclusion, microfluorometry can be used for kinetic analysis of Cathepsin B activity in single cells. The reaction demonstrates a biphasic pattern which may be due to a change in activity after crystallization of the final fluorescent reaction product began. M17.2

Analysis of Cathepsin B Activity in Human Pancreatic Cancer Cells with Confocal Scanning Laser Microscopy. C. J. F. van Noorden ~, I. M. C. Vogels ~, G. N. Jonges ~ and J. van Marle 2 ~Laboratory of Cell Biology and Histology, and 2Department of Electron Microscopy, Academic Medical Centre, University of Amsterdam, The Netherlands.

478 Many different proteolytic enzymes may play a role in various aspects of tumour growth and metastasis. Cathepsin B, a proteinase of the cysteine class, has been implicated in the ability of tumour cells to degrade the laminin component of the basement membrane and extracellular matrix compounds, which facilitates tumour cell invasion. Cathepsin B activity has been detected in the lysosomes of normal cells. In cancer cells, the enzyme is probably processed in a different way and its presence has not only been shown in the lysosomes, but also on the plasma membrane and extracellularly. In the present study, cathepsin B activity has been investigated in xenografts of a poorly differentiated human pancreatic tumour cell line grown in nude mice. The catalytic activity of the enzyme was analyzed with a histochemical fluorescence method instead of determining the mere presence of enzyme molecules by immunohistochemical means because large quantities of the proteinase may occur as inactive proenzymes or as enzymeinhibitor complexes. The fluorescence enzyme assay has been performed on unfixed cryostat sections using 5-nitrosalicylaldehyde as coupling agent. The formation of fluorescent final reaction product was monitored continuously during incubation using confocal scanning laser microscopy. Cathepsin B activity was found to be localized in lysosomes and at the plasma membrane of the pancreatic tumour cells. A high activity was not only determined in the border area between viable tumour cells and necrotic areas within the tumour but also in the connective tissue surrounding the tumour tissue. The activity could be monitored during incubation in each of the subcellular compartments of the tumour cells. It appeared that confocal scanning laser microscopy enables kinetic analysis of enzyme reactions in situ with a high 3D resolution, which is impossible to obtain in actually cut thin sections (0.5 - 1/am) due to the limited amounts of enzyme present in such thin sections.

M17.3

Probes for Detecting Enzymatic Activity in Single Cells. R. P. Haugland, R. Haugland, Z. Huang, K. Larison, M. N. Malekzadeh, P. Millard, J. Naleway, N. Olsen, V. Paragas, D. Robinhold, V. Singer, S. Wells, W. You and Y. Z. Zhang Molecular Probes Inc., Eugene, OR 97405, USA. We have developed several approaches for the fluorescent histochemical detection of enzymatic activity in single cells using fluorogenic substrates. In some cases we have accomplished this in living cells. The challenge has been to improve retention of the fluorescent product in the cell as well as to facilitate loading of the substrate into the cell. At least four approaches have been used: l)precipitation of an insoluble fluorescent product, 2)formation of a lipophilic product that accumulates in the cell membranes, 3) use of a fluorogenic substrate that is also glutathi0ne-reactive to yield a product with good retention in the cytoplasm of living cells and 4) formation of a fluorescent product that accumulates in intracellular organelles or binds to other cellular components. Among the enzymes that have been successfully detected by at least one of these approaches are /~-galactosidase and /3-glucuronidase (both intrinsic and as the result of gene fusions), a-fucsoidase, fl-glucosidase, hexoasaminidase, neuramidase, aryl sulfatase, alkaline and acid phosphatase,

Minisymposia various peptidases, and peroxidases. We have also demonstrated the potential of the substrates that yield fluorescent precipitates for significantly improving the detection levels in assays such as in situ hybridization. This research has been supported in part by N.I.H grant GM 38987 to R.P.H.

M17.4 Advances with Secretory Proteinases in Rat Submandibular Glands. J. R. Garrett, D. K. Shori, G. B. Proctor and K. M. Chan Oral Pathology, K.C.M.D.S., The Rayne Institute, London SE5 9NU, England.

Numerous kaUikrein-like proteinases occur in the granular tubules of rat submandibular glands. Differences in histochemical staining of the tubules using D-Val-Leu-Arg MNA (extensive and uniform) and Z-Arg MNA (irregular and patchy) suggested that the proteinases may be identifiable with different peptide substrates. The constituent proteinases in glandular homogenates or saliva can now be identified after IEF by the development of fluorescent bands in overlay membranes impregnated with fluorogenic substrates. The underlying proteinases can then be excised and eluted for further biochemical testing. We have developed methods whereby tissue kallikrein can be identified catalytically by the use of D-Val-LeuArg-AFC in the presence of SBTI and tonin by means of Z-ValLys-Lys-Arg AFC in the presence of aprotinin. Ratios of the different proteinases in sympathetic saliva have been found to be the same as in the glands but to differ from those in parasympathetic saliva, which contains a greater percentage of more glyeosylated tissue kallikrein, suggesting that it is synthesised and secreted constitutively during parasympathetic stimulation. Assessment after degranulation indicates that tissue kallikrein is resynthesised more rapidly than the other proteinases. Sexual dimorphism between secretory proteinases has been identified histochemically and biochemically, which supports the belief that hormones influence the individual synthetic pathways differently. Streptozotocin-induced diabetes reduced synthesis of all proteinases; insulin, however, evoked a more rapid recovery of tissue kallikrein than of the other proteinases, indicating that insulin exerts a more direct influence on kallikrein synthesis. Supported by a Wellcome Project Grant and K.M.R.T.

M17.5

A Novel Fluorescent Method for Detection of Subpicogram Quantities of Nucleic Acids. S. H. Hori ~, N. Kagiyama 1"2, S. Fujita z, M. Momiyama 2 and Y. Kondoh 2 ~Department of Zoology, Faculty of Science, Hokkaido University, Sapporo 060, Japan and 2AISIN Seiki Co. Ltd., Asahirnaehi, Aichi 448, Japan. Nucleic acid hybridization widely used in molecular biology usually necessitates the use of radioisotope-labelled probes. However, the usefulness of radiolabelled probes is limited by safety and disposal problems, short shelf life, radiodecay and duration of autoradiographic exposure. To circumvent these problems, we have synthesized a number of fluorochromes which can be used as substrates for alkaline phosphatase coupled to DNA probes, and tested for their usefulness. As a

Minisymposia

479

result, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) was found to be most suitable for detection of membrane-bound DNA. The target DNA was transferred to a membrane filter, hybridized with a biotin-labelled probe, treated with phosphatase-labelled antibody and then incubated with H N P P as a substrate. The fluorescent reaction product of H N P P was insoluble in buffer and precipitated on membrane filter. Under UV light (302 rim), a human single copy gene was detected on Southern blot hybridization. Sensitivity is compared with a chemiluminescent method using adamantyl1,2-dioxetane phosphate.

M17.6

Monoclonal Antibodies to Ornithine Decarboxylase: New Clues with Respect to the Active Site of the Enzyme. R. G. Schipper ~, R. G. J. Rutten ~, M. Sauerbeck 2, H. P. J. H. M. Adams 3, J. Kopitz 2, P. Bohley 2, A. A. J. Verhofstad t and G. Tesser 3 1Department of Pathology, University of Nijmegen, The Netherlands, 2Department of Physiol.-Chem., University of

M18.

HISTOCHEMISTRY

OF INTEGRINS

Tfibingen, FRG; 3Department of Organic Chemistry, University of Nijmegen, The Netherlands. The primary structure of rat ornithine decarboxylase, EC 4.1.1.17 (Ratus norvegicus) was used for the selection of an epitope by peptide structure calculations. The epitope, a hexadecapeptide representing ODC (34~Lys-36~ was synthesized by solid phase peptide synthesis (SPPS) using the Fmoc strategy and the p-alkoxybenzyl alcohol (Wang) resin as the support. A peptide-conjugate containing bovine serum albumin was used for the immunization of mice. Two hybridoma cettines, MPI6-2 and MP16-3, were selected by ELISA using a conjugate containing thyroglobulin. Further studies with ELISA, ODC-activity measurements and immunoblotting experiments revealed that MP16-2 and MP16-3 recognize the native enzyme but not the inhibited enzyme (i.e. ODC labelled with DFMO). These results indicate that the selected epitope represents (at least partly) the active site of the enzyme. Furthermore, MP16-2 shows affinity to purified native ODC as well as cytosotic ODC. On the contrary, MP16-3 recognized only cytosolic ODC indicating that (a) MP16-2 and MP16-3 react with different parts of the epitope and (b) the sequence recognized by MP16-3 is not available for antibody binding after purification of the enzyme.

AND EXTRACELLULAR MATRIX

M18.1 Immunoehemieal Localization of Integrin Molecules in Cutaneous Tissue during Tumor Promotion. 7". M. Oberyszyn and F. M. Robertson The Ohio State University Department of Surgery, Columbus OH 432t0, USA. Immunochemical localization techniques were used to examine the cutaneous compartments for expression of Intercellular adhesion molecule-1 (ICAM-1) following treatment of the dorsal epidermis of SENCAR mice with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate. At 24 h following exposure to 10~g TPA, the epidermal keratinocytes within the epidermis bound immunoreactive anti-ICAM-1 antibodies, however, there was no binding within the dermal compartment. Leukocytes within the dermis bound antibodies directed against Lymphocyte Function Associated antigen-I (LFA-1) but not ICAM-1 antibodies. Intravenous injection of antibodies directed against ICAM-1, LAF-I or the beta chain subunit of LFA-1 (CD-18) 2 h prior to exposure of the dorsal epidermis to TPA inhibited the leukocyte infiltration and the skin swelling normally observed at 24 h after TPA treatment, with the rank order of potency of inhibition of the antibodies anti-CD-18>anti-LFA>antiICAM-1. Taken together, these observations suggest that integrin and adhesion molecule interactions may play a pivotal role in the early stages of cutaneous inflammation induced by the potent tumor promoter TPA.

M18.2 Ultrastructural Localization of a6P4 and Laminin in Cell-Substrate and in Cell-Cell Contact Sites of a Transformed Mouse Keratinocyte Cell Line. J. Calafat, H. Janssen and A. Sonnenberg The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. The ~6P4 complex is a member of the integrin family of adhesion receptors. In a previous study we have shown that in human and in mouse epidermis the ad34 complex was localized in hemidesmosomes, and in the mouse it was also detected in small clusters on the surface of perineural myofibroblasts and Schwann cells. In all these tissues a6(/4 was always in contact with the basal membrane. This distribution is consistent with a matrix-adhesion function of this integrin. A possible ligand is laminin. However, a MoAb against the /34 subunit did not block the adhesion of ceils to proteolytic fragments of laminin. In this study we describe a transformed mouse keratinocyte ceil line RAC-11P that can be used as an in vitro model for the study of the function of a6/34. RAC-11P cells form colonies of stratified cells that resemble normal epidermis: a basal layer of epithelial cells, flattened suprabasal cells and cornified superficial cells. Using immuno-EM, a6/34 was localized on hemidesmosomes, also characterized by the labelling of the intermediate filaments with MoAb E59 against keratin. Moreover, some clusters of a6/34 were found in contact areas between two neighbouring cells and were also abundant on large filopodia. RAC-11P cells synthesize and secrete laminin. By immunoEM labelling, laminin was found in endoptasmic reticutum and, extracetlularly, on the basal side and in small clusters on the apical surface, in a pattern similar to that of ~6/34. Double

480 labelling for /34-subunit and laminin on RAC-11P (whole mounts), showed that both proteins were present in the same areas and often in the same clusters. This ultrastructural study suggests that laminin is the ligand responsible for aggregation of ad34 in clusters in cell-cell contact sites and on filopodia.

M18.3 Immunolocalization of Basement Membrane Components in Human Skeletal Muscle Cells In Vivo and In Vitro; Topographical Relation with Acetylcholine Receptors. T. van Kuppevelt, A. Benders and J. Veerkamp University of Nijmegen, Dept. Biochemistry, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Human skeletal muscle cells are completely encompassed by a basement membrane (BM), which is specialized at the site of neuromuscular junction (the synaptic BM). Immunofluorescence shows that muscle cells in vivo are surrounded by a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, type IV collagen and fibronectin. HSPGs are distinctively concentrated at the synaptic BM and so are molecules reacting with isolectin B4 from Vicia villosa. Myotubes cultured on the serum substitute Ultroser G and brain extract show a continuous layer of HSPGs, laminin and type IV collagen. At sites of acetylcholine receptor clusters, HSPGs and lectin-positive molecules are distinctively concentrated, resembling the in vivo situation. In contrast, myotubes cultured on serum-containing media do not display these characteristics. Electron microscopy reveals that myotubes cultured on Ultroser/brain extract are surrounded by a continuous BM. Proteoglycans are present on the external site of the lamina densa, and associated in a regular fashion with collagen fibrils.

M18.4 Demonstration of Adhesion Molecules on Macrophages and Glial Cells during Acute and Chronic Experimental Allergic Encephalomyelitis. J. Bauer, C. D. Dijkstra, I. Huitinga and T. Sminia Dept. Cell Biology, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

Several reports have described the expression of adhesion molecules on endothelial cells in the CNS and the role of these molecules in lymphocyte adherence and migration during experimental allergic encephalomyelitis (EAE). Hardly anything is known about the expression of adhesion molecules on glial cells during EAE. Therefore we investigated the presence of adhesion molecules in the brain parenchyma by immunocytochemistry at the light and ultrastructural level. The antibodies WTI (anti LFA-1/CD1 la), OX-42 (anti CR3/ CD1 lb) and an antibody against ICAM-1 were used. During the first attack, intensive staining for ICAM-1 and CD1 la was found on macrophages in the perivascular lesions and on activated microglial cells surrounding these lesions. During the relapse and remission phase of chronic and acute EAE respectively, staining for these adhesion molecules appeared less intense. Here, however, microglial cells all over the brain parenchyma were positively stained for ICAM-1 and LFA-1. OX-42 reactivity, which in the normal brain is present on

Minisymposia resting microglial, was enhanced during the first attack. During none of the phases of EAE were glial cells other than microglia found to express ICAM-1, CD1 la or CDI lb. From these results it can be concluded that microglial cells through their expression of ICAM-1, C D l l a and C D l l b are able to interact with each other and with cells of the immune system.

M18.5 Immuno-EM Localization of Beta-1 lntegrin in WetCleaved Fibroblasts. A. M. L. Meijne, D. M. Casey, C. A. Feltkamp and E. Roos Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

We have used immuno-EM to study the localization of/31integrin in chicken embryo fihroblasts that had spread for 3 h on fibronectin in serum-free medium. Cells were "wetcleaved" which yields a ventral substrate-associated membrane with associated cytoskeleton, that was sometimes removed with actin depolymerization buffer and high salt buffers to improve accessibility. Antibodies against the cytoplasmic domain of//1-integrin revealed/31 to be located in the cell periphery often close to talin. Furthermore, pl was abundant above fibronectin fibrils. Adhesion plaques contained /31. However, it was not distributed all over the plaque like vinculin and talin, but located in patches, in close proximity to the plaques. Results obtained after application of actin depolymerization and high salt buffers indicate that this observation is not caused by limited accessibility of integrins located under the adhesion plaque. Our observations indicate that /31-integrins are located at the periphery of adhesion plaques. If so, other membrane proteins should be responsible for matrix attachment of the center of the plaque.

M18.6 Internalization of the a6/34-Integrin from the Basal Membrane of Dispase-Detached Keratinocytes. Y. Poumay, M. Leclercq-Smekens, S. Grailly, I. Roland, A. Degen and R. Leloup Histologie-Embryologie, Faeultds Universitaires Notre-Dame de la Paix, Namur, Belgium.

The epidermal keratinocytes form a polarized tissue and can be cultured for the preparation of skin grafts which mimic features of the original tissue. Undissociated cultures sheets can be detached by incubation with dispase. In consequence of this detachment of basal cells, the culture area retracts rapidly and basal cells turn from a flattened to a columnar morphology. When these floating cultures are stored for several hours, the polarized spatial organization into superposed layers is progressively lost. Detachment in dispase at 4~ only prevents the culture retraction. Integrins are restricted to basal ceils of the epidermis and believed to determine the spatial localization of cells in this tissue. Using specific antibodies and immunofluorescent labelling, we show on cryostat sections that the integrin 06/34 is rapidly internalized from their basal membrane when basal cells retract within the first minutes following detachment, whereas

Minisymposia integrins of the VLA-subtype remain on the cell surface. Using EM and immunogold labelling of the a6 chain at 4~ on nonretracted detached cultures, we observe the label on hemidesmosome-like structures typical of keratinocyte

481 cultures. Incubation at 37~ then induces internalization of the label, concomitantly with retraction of the culture area. Studies using skin biopsies suggest a similar event in noncultured basal keratinocytes after dispase-detachment.

M19. O N C O G E N E S A N D T U M O U R S U P P R E S S O R GENES M19.1

P53 Protein Overexpression in Normal, Premalignant and Malignant Tissue of the Cervix Uteri. R. Holm ~, H. Skomedal 1, A. Helland 2, G. Kristensen 3, A.L. Borresen 2 and J. M. Nesland ~ Departments of Pathologj, Genetics2 and Gynecologj, The Norwegian Radium Hospital, Montebello, Oslo, Norway. Mutations in the p53 tumour suppressor gene play an important role in the development of many human malignancies. Because of the short half-life of normal p53 protein, only the mutated form seems to be detectable immunohistochemicaUy. The aim of the present study was to examine the overexpression of p53 protein in 238 benign and malignant cervical lesions in an attempt to define when elevated p53 protein occured in the development of cervical cancer pathogenesis. Immunohistochemicatly, no staining was seen in normal tissue, condyloma, dysplastic tissue and adenocarcinoma in situ, whereas p53 protein was identified in 7070 of squamous cell carcinomas in situ, 11070 of infiltrating adenocarcinomas and 6207o of infiltrating squamous cell carcinomas. In 9% of the cases 5070 to 50~ of tumour cells were immunoreactive for p53 protein, whereas the other positive specimens were characterized by only rare p53positive cells. In conclusion, our results demonstrated that overexpression of p53 protein is common in infiltrating cervix carcinomas, rare in carcinoma in situ and not found in dysplastic, condyloma and normal tissue, and therefore appears to be related to turnout progression.

M19.2

P53, EGFR and ERBB2 Expression in Prostatic Carcinoma. T. Visakorpi, O.-P. Kallioniemi, T. A. Koivula and J. J. Isola Tampere University Hospital, P.O. Box 2000, SF-33521 Tampere, Finland. We analyzed the expression of p53, EGFR and ERBB2 proto_n in paraffin-embedded primary prostatic carcinomas by immunohistochemistry using CM-1, MAb31G7 and MAbl antibodies for p53 (n = 137), EGFR (n = 147) and ERBB2 (n = 147), respectively. Accumulation of p53 protein was found in 17~ carcinomas, whereas normal and hyperplastic prostatic tissues were always negative. Six percent of carcinomas showed intense immunostaining in more than 20~ of carcinoma cells whereas 11% had lower levels of immunostaining. In 47o70 of carcinomas almost uniform EGFR immunoreactivity was detected, whereas in 39~ only some carcinoma cells were positive. Fourteen percent of carcinomas were EGFR-negative. All prostate hyperplasias (n = 17) tested, were strongly positive for EGFR. All prostatic carcinomas were ERBB2-negative. Both EGFR-

positivity and high level p53-immunostaining were associated with high cell proliferation activity (determined by flow cytometric S-phase analysis) and predicted poor 10-year progression-free (p