Cytokeratins are particularly versatile tools in tumor biology both experimentally and clinically. Previously regarded as a rigid intracellular skeleton, the cytokeratins are now known to provide a more dynamic structural integrity within epithelial cells. This more flexible nature of cytokeratin function occurs through rapid shifts in assembly/disassembly states, geared by phosphorylations/dephosphorylations in the 'head' and 'tail' regions of the monomeric filaments. When released from proliferating or dying tumor cells, they provide a useful marker for epithelial malignancies, as evidenced by the number of available immunochemical assays for cytokeratins. Another significant area of interest that highlights their utility is the deposition of cytokeratins in the necrotic areas of tumors, providing an effective target for radioimmunotherapy and radioimmunolocalization. Moreover, the appearence of cytokeratin neodeterminants seems to be a very early event during apoptosis. Indeed, the involvement of specific cytokeratin degradation patterns resulting from the activity of caspases during apoptosis highlights yet another developing field of research devoted to these highly pleomorphic and complex structures.

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Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

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Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

Alkaline Phosphatase↗

PubMedDate not supplied

Tumor marker workshops.

Since 1996, the nine ISOBM Workshops have so far characterized more than 300 monoclonal antibodies to a variety of tumor markers that include CA125, AFP, PSA, MUC1, Cytokeratins, Sialyl Le(a), hCG, CEA, ALP, and more recently SCC, and S100. Besides the basic characterization of antibodies and their epitope configurations, several workshops have also addressed specific problems associated with multiple antigen variants. These workshops have been able to make significant advances well beyond those possible through any normal collaboration study. The data and impact of these workshops with their summary reports are reviewed.

Alkaline Phosphatase↗

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Interfering with cancer: a brief outline of advances in RNA interference in oncology.

RNA interference (RNAi) is a potent and ubiquitous gene-silencing mechanism that is generating considerable excitement in the fields of molecular biology and gene therapy. It is now in widespread use for loss-of-function analysis in many diseases including cancer. Nevertheless, RNAi is still in its infancy, with new discoveries appearing on a monthly basis. This article presents a brief outline of the history and recent advances in RNAi with a specific focus on its potential in oncology.

Genetic Therapy↗

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Cytoskeletal basis for contractility of outer hair cells in the normal adult human organ of Corti: comparisons with vestibular hair cells.

The present study is the first consecutive analysis of the adult normal human organ of Corti and vestibular hair cells with regard to the expression of F-actin, actin-associated proteins (alpha-actinin, alpha- and beta-spectrins, vinculin and tropomyosin), beta-tubulin and the calcium-binding protein synaptophysin. The expression of these cytoskeletal and their associated proteins in man is largely similar to, although not identical with, that previously described for several other mammalian species. However, a few very unusual staining patterns were found. In several long outer hair cells a rod of F-actin extended from the infracuticular area to the cell nucleus. Fluorescence for tropomyosin occurred both in the cuticular plates of the outer and inner hair cells, and in the area of close apposition between the base of the outer hair cell and the apical part of Deiter's cell. In contrast, the vestibular hair cells showed immunoreactivity for tropomyosin only in the cuticular plates.

Actinin↗

PubMedDate not supplied

The human spiral ganglion.

The adult normal human spiral ganglion (SG) was analyzed with regard to ultrastructure and immunohistochemistry. The cytoskeleton of the SG cells was found to comprise F-actin, intermediate filaments (IFs) and microtubules (MTs). The IF subgroups (cytokeratins, Cks; neurofilaments, NFs, vimentin, glial fibrillary acidic proteins, GFAP; desmin) displayed characteristic staining patterns. Ck No. 8 was found in all SG cells, whereas vimentin was lacking. GFAP stained only a small subpopulation of SG cells (type 2?). The light (68 kD) and medium-sized chains of NFs occurred in all SG cells and axons, whereas the 200-kD NF subunit was only found in the axonal hillock of (type 2?) SG cells, but in no other part of the cytoplasm, and regionally in nerve fibres. MAP-1 and MAP-2 occurred in all SG cells but only MAP-1 was found in the nerve fibres. The calcium-binding protein synaptophysin (SY) was expressed only in SG cells, in contrast to the S-100 which occurred more generally in the labyrinth. The neuropeptides VIP and substance P were identified in all SG cells, in contrast to NPY which was expressed in a small subpopulation of SG cell (type 2?). Staining for neuron-specific enolase (NSE) identified most (type 1?) but not all SG cells. The cell surface glycoprotein Thy-1 was expressed in SG cells in a way similar to that described for neurons in the CNS. The SG cells express a high degree of cytoskeletal complexity, allowing one to distinguish between type 1 and type 2 cells. The cell bodies and their adjacent nerve fibres show characteristic features of calcium-binding proteins, surface membrane glycoproteins, NSE and neuropeptides but the basic pattern is still similar to neurons in the CNS.

Actins↗

PubMedDate not supplied

A new specific method for measuring S-100B in serum.

The S-100 family of proteins are acidic calcium and zinc binding low molecular weight proteins mainly present in astrocytes and in a population of oligodendrocytes of the CNS. S100b, an acidic low weight and zinc binding protein, has attracted considerable interest due to its release into the cerebrospinal fluid and blood from brain tissue following brain damage and from malignant melanomas. A new simple two-step incubation assay has now been elaborated in which two catcher and one tracer monoclonal antibodies are used. The specificity of this assay is high because all the MAbs used bind exclusively to S-100B, as shown by real-time biospecific interaction analyses. Moreover, the working range of the assay is 0.2-60 micrograms/L with a CV of less than 10%; the resulting high sensitivity has been confirmed by clinical studies. Time dependence, shaking conditions, lower limit of detection limits, effects of dilution, hook effect, recovery, impression as intra- and interassay variations, and crossreactivities with S-100A1 were tested in order to obtain a highly reproducible assay. Sera from healthy blood donors and patients undergoing cardiopulmonary bypass operations were tested with the assay. Several of the patients undergoing open heart surgery presented measurable values in this IRMA S-100 assay, indicating cerebral effects of open heart surgery. The test may be used for postoperative monitoring of these patients.

Animals↗