OX3 9DS, UKhAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT -PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P ¼ 0.0002; hAG-3, P ¼ 0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P ¼ 0.003). Yeast two-hybrid cloning identified metastasis-associated GPIanchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.
B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.
Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.
The mRNA levels of hyal-1, hyal-2, LUCA3 and PH20, the 4 hyaluronidases with demonstrated endoglucosaminidase activity, were extensively profiled in normal and tumor tissues and cell lines, using dot blot analysis and quantitative PCR. In normal tissues, hyal-1, hyal-2 and LUCA3 all showed unique patterns of mRNA expression, but were generally of widespread distribution, whereas PH20 mRNA was restricted to testes. In a small set of breast tumor samples, no elevations in hyal-1, hyal-2 or LUCA3 mRNA were seen. Hyaluronidase activity measured by a novel assay or zymography was also not elevated in sera from a number of breast cancer patients, compared to sera from normal volunteers. In ex vivo xenograft tumor cell lines, however, hyal-1 or hyal-2 mRNA levels were frequently elevated, whereas LUCA3 was only infrequently elevated and PH20 not at all. The hyaluronidases are a family of degradative endoglucosaminidase enzymes 1 whose substrate, hyaluronic acid (HA), is a major constituent of the extracellular matrix (ECM). 2 Cellular interactions with the underlying ECM are crucial in development, 3 for the maintenance of normal cellular functions and in response to injury and infection. In situations where malignant cell growth and subsequent metastasis occur, the degradation of the ECM is required. HA, a large polysaccharide of approximately ten thousand disaccharide repeats, is known to inhibit cell differentiation, promote proliferation, regulate cell motility and HA synthesis occurs before mitosis. 1 Increased serum or tissue associated levels of HA have been correlated with tumor progression 2 and metastatic behavior. 4 In some types of cancer such as bladder, breast and colorectal, the serum levels of HA have been found to be of prognostic value, 5-8 although conflicting results have been obtained from patients with breast cancer. 9 The hyaluronidases that degrade HA have also been implicated in tumor progression and metastasis, as well as angiogenesis. 10 Hyaluronidase-1 (hyal-1) was originally isolated from human plasma, has a pH optimum of 3.8 and cleaves HA to small (Ͻ20 kDa) MW fragments. 11 Hyaluronidase-2 (hyal-2) is also an acidactive enzyme that digests HA polymers down to 20 kDa sizes. 12 Hyal-2 has been shown to be located in lysosomes 12 but is also reported to be a GPI-anchored cell surface protein. 13 PH20 was identified as a GPI linked sperm acrosomal enzyme with a neutral pH optimum and cleaves HA to smaller (Ͻ20 kDa) MW fragments. 1 More recently, the hyaluronidase LUCA3 (Barry et al., in preparation) and meningioma expressed antigen 5 (MGEA5, chromosome 10) have also been shown to have activity at neutral pH. 14 Hyal-4 and the pseudogene Hyal-P1, both of which co-localize to chromosome 7q31.3 with PH20, 15 have not yet been demonstrated to possess enzymatic activities.Elevated mRNA levels or increased hyaluronidase enzymatic activity have been associated with invasion and metastasis for ovarian and endometrial cancers, 16 progression of prostate cancer, 17 bladder cancer, 18 colorectal carcino...
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