No abstract

Although mucins have been studied at the biochemical and biophysical level for some time, attempts to define their structures in detail were only partially successful because of their size and complexity. The advent of monoclonal antibodies reactive with these molecules introduced a new approach to structural studies by defining antigenic epitopes, by allowing purification of the mucin molecules by affinity chromatography, and by providing a means to clone genes coding for the core proteins. By their profile of reactivity with the normal and cancer-associated mucin in a particular tissue, the antibodies also defined a difference in the mucin derived from the two sources. It is now clear that this difference lies in the carbohydrate side chains, as the core proteins are identical. Because the mucins are tumor-associated antigens and the cancer mucins can express epitopes that are relatively tumor specific, this family of molecules is now being intensively studied. There is also considerable interest in elucidating the normal function of the mucin and in determining whether, through an altered structure, this function is subverted in malignancy. In the next few years we should expect that the structure of other mucins will be defined in the same detail as the product of the MUC1 gene. We should also expect to see the continued application of mucin-reactive antibodies in the clinic and the investigation of mucins as agents for immunotherapy of some cancers. As to the function(s) of these molecules, perhaps we will learn enough in the future to make a critical reappraisal of the name.

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the

Objective To investigate and catalogue systematically the phenotypic and genotypic characteristics of the commonly used prostatic cell lines using immunocytochemistry and polymerase chain reaction (PCR) of hypervariable sequences within the genome to provide a ‘fingerprint’ characteristic of each cell line. Materials and methods Malignant (LNCaP, LNCaP‐r, PC‐3, DU‐145) and benign immortalized prostatic cell lines (PNT‐1A, PNT‐1B, BPH‐1) were grown on four‐well slides, fixed and subjected to indirect streptavidin‐biotin immunocytochemistry. Twenty‐three antibodies were used in the following groups: cytoskeletal elements: cytokeratins (CK)‐5, ‐7, ‐8, ‐14 (two), ‐16, ‐18, ‐19 (three), ‐20, vimentin and desmin; MUC1 (three); cell adhesion molecules (E‐cadherin, α‐β‐and γ‐catenin); and prostatic associated proteins: prostate specific antigen (PSA), prostatic acid phosphatase (PAP) and androgen receptor (AR). For the PCR, genomic DNA was extracted from the cell lines and from SKOV3 and MCF7 (positive controls). PCR was performed on three variable regions which were then sequenced: AR exon 1 (CAG repeat polymorphism), and two areas of microsatellite instability (MSI): AR exon 8 and hypoxanthine‐guanine phosphoribosyl transferase (HPRT) exon 3. Results All cell lines were CK‐8/18 positive and most also expressed CK‐7 and ‐19. Heterogeneous CK‐20 expression was detected for the first time in prostatic cell lines. All lines were positive for vimentin and negative for desmin. MUC1 was expressed in one malignant (DU‐145) and all immortalized cell lines. E‐cadherin expression was low or absent in three lines: PNT1A, 1B and PC‐3. Only PC‐3 failed to express α‐catenin; β‐ and γ‐catenin were expressed by all lines. PSA, PAP and AR were only expressed by LNCaP and LNCaP‐r. On PCR, the CAG repeat lengths in exon 1 of the AR ranged from 19 to 27. Three pairs of cell lines had the same exon 1 CAG repeat length: LNCaP/PC‐3 (26 repeats), BPH‐1/DU‐145 (19 repeats) and PNT1 A/1B (20 repeats). Exon 8 sequences were identical except for LNCaP, which showed a single base mutation, and HPRT exon 3 sequences were all identical. There was no evidence of generalized MSI in any of the cell lines examined. Conclusions The cell lines studied fell into three broad groups according to their phenotypic characteristics: (i) prostatic marker positive (LNCaP and LNCaP‐r); (ii) high expression of most antigens (DU‐145, PC‐3 and BPH‐1); and (iii) low or absent expression of most antigens (PNT1 A and 1B). Each of the cell lines derived from PC could be identified on the basis of exon 1 and 8 AR sequence variability. DU145 and BPH‐1 had identical profiles of the three areas studied, but these cell lines are easily distinguished by their different phenotypic characteristics. PNT1A and 1B had identical genetic and similar phenotypic profiles, which is unsurprising given that they are subclones derived from the same parental line. Even so, these were separable on the basis of CK‐19 immunostaining. Using a combination of geno‐ and phenotypic markers it was...

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151 (+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787-98)

When hepatocyte regeneration after a two-thirds partial hepatectomy (PH) in rats is blocked by oral gavage of acetylaminofluorene, a proliferation of ductular cells ensues that results in a profusion of neoductules radiating from each portal tract. To examine the possibility that this population of newly emerging cells harbors cells capable of differentiating into hepatocytes, we have looked in these cells for expression of functional markers of hepatocyte commitment at both the RNA and protein levels. Expression of albumin and a-fetoprotein (a-FPI messenger RNA (mRNA) transcripts were sought in situ using antisense riboprobes, and the expression of a number of cytochrome P450 enzymes was examined immunohistochemically. Before any signs of differentiation the ductular cells strongly expressed cytokeratins 7, 8, 18, and 19 in the same manner as authentic bile ducts, but unlike the latter also expressed vimentin. In situ hybridization studies showed that small bile ducts close to the limiting plate, as well as the newly formed ducts, expressed albumin and a-fetoprotein messenger RNAs, and immunocytochemistry showed that the distribution of the respective proteins was similar. Beginning at 1 week after partial hepatectomy, areas of differentiation could be found in the new ducts, with cells resembling either columnar intestinal-type epithelia or hepatocytes. Intestinal-like cells expressed neither albumin, a-FP, nor cytochrome P450 enzymes, whereas ductular cells appearing like hepatocytes with the typical membranous distribution of cytokeratin 8 strongly expressed a variety of cytochrome P450 enzymes normally associated with functional hepatocytes. These observations further support the belief that reactive ductules, sprouted from small ducts, can represent an adaptive response of the liver to replenish lost hepatocytes, al-

Objective To assess the level and morphological distribution of cyclooxygenase (COX)-1 and -2 in human prostates and to determine any association with the Gleason grade of prostate cancer. Materials and methods The study comprised 30 samples from patients with benign prostatic hyperplasia (BPH) and 82 with prostate cancer. Immunohistochemistry was used to assess the expression of COX-1 and -2, and 13 samples were also assessed using immunoblotting (six BPH and seven cancers). Results For both BPH and prostate cancer, COX-1 expression was primarily in the ®bromuscular stroma, with variable weak cytoplasmic expression in glandular/neoplastic epithelial cells. In contrast, COX-2 expression differed markedly between BPH and cancer. In BPH there was membranous expression of COX-2 in luminal glandular cells and no stromal expression. In cancer the stromal expression of COX-2 was unaltered, but expression by tumour cells was signi®cantly greater (P=0.008), with a change in the staining pattern from membranous to cytoplasmic (P<0.001). COX-2 expression was signi®cantly higher in poorly differentiated than in well differentiated tumours (P<0.001). These results were supported by immunoblotting, which showed similar levels of COX-1 in both BPH and cancer, but four times greater expression of COX-2 in cancer than in BPH. Conclusion This is the ®rst study to assess the coexpression of COX-1 and COX-2 proteins in benign and malignant human prostates, and showed the induction and signi®cantly greater expression of COX-2 in cancer, which was also associated with tumour grade. The regular use of nonsteroidal anti-in¯am-matory drugs is associated with a reduced incidence of cancers. The present results provide the basis for a potential role for COX-2 inhibitors in the prevention and treatment of prostate cancer.

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the 'lifetime' factor tau. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger tau (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean tau of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer tau than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution.