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Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions. The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.

We have previously shown that neu oncogene-initiated rat mammary carcinomas uniquely over-express neu-related lipocalin (NRL), a member of the calycin protein superfamily. Here, we characterize the putative human homolog of NRL, neutrophil gelatinase-associated lipocalin (NGAL). ngal gene expression was found at moderate levels in only 2 of 17 human tissues examined, breast and lung. When breast cancers were examined for NGAL mRNA and protein levels, they were found to exhibit heterogeneous expression. NGAL levels varied in these tumors from undetectable to exceeding those in normal breast parenchyma. Immuno-histochemical analysis confirmed the presence of NGAL within breast carcinoma cells but detected only low levels of this protein in normal ductal epithelium. In contrast, large amounts of the protein were localized to the lumen of normal breast ducts in the vicinity of NGAL-expressing tumors. Interestingly, unlike NRL in rat mammary carcinomas, no significant association between NGAL expression and HER-2/neu activation was found in human breast tumors. In contrast, a significant correlation between NGAL expression in breast cancer was found with several other markers of poor prognosis, including estrogen and progesterone receptor-negative status and high proliferation (S-phase fraction). NGAL levels were stratified as high or low in breast cancers from a cohort of nodepositive patients with known outcome. No significant association between NGAL expression and disease-free or overall survival was observed. Int. J. Cancer (Pred. Oncol.) 79:565-572, 1998. Wiley-Liss, Inc.The isolation of genes uniquely expressed in a subset of breast cancers has the potential to identify diagnostic markers, prognostic indicators and therapeutic targets. To isolate such over-expressed genes, various model systems have been developed and characterized. For example, 2 in vivo rodent systems have been established to examine mammary carcinomas induced by neu (c-erbB-2/HER-2), the oncogene most commonly associated with the clinical progression of human breast cancer. In the first model, transgenic mice have been generated in which the expression of activated neu is targeted to the mammary gland using mammary-specific promoters (Muller et al., 1988;Bouchard et al., 1989). In the 2nd model, the activated neu oncogene has been directly and stably introduced into in situ rat mammary epithelial cells, using a replicationdefective retroviral vector (Wang et al., 1991a). Using these models, Stoesz and Gould (1995) and others (Morrison and Leder, 1994) have sought to isolate and characterize rodent genes uniquely over-expressed in neu-initiated mammary tumors. To date, using both models, only 1 gene has been cloned which is uniquely over-expressed in mammary tumors induced by neu (Stoesz and Gould, 1995). This gene, termed neu-related lipocalin (nrl), is 12-fold over-expressed in rat neu-initiated carcinomas but is not over-expressed in ras-initiated mammary carcinomas or in mammary tumors induced with chemical carcinogens. In addition, this ...

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

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