Abundance of IgG4+ plasma cells in isolated reactive lymphadenopathy is no indication of IgG4-related disease.

OBJECTIVES: IgG4-related disease is a recently recognized condition that can be associated with lymphadenopathy, with several histologic patterns and increased absolute number and ratio of immunoglobulin G4 (IgG4)-positive plasma cells. However, these findings are considered to be not exclusively specific for IgG4-related disease. METHODS: The occurrence of the histologic patterns reported in patients with isolated lymphadenopathy was studied and correlated with the clinical presentation to determine their predictive value for IgG4-related lymphadenopathy. RESULTS: We found cases meeting all histologic criteria for IgG4-related lymphadenopathy, without clinical signs of IgG4-related disease. The only pattern that was not seen in this series was an inflammatory pseudotumor-like picture. CONCLUSION: Without a clinical suspicion of IgG4-related disease, these morphologic patterns and high numbers of IgG4-positive plasma cells should be interpreted with care to avoid an erroneous diagnosis of IgG4-related disease.

Molecular interactions of ErbB1 (EGFR) and integrin-ß1 in astrocytoma frozen sections predict clinical outcome and correlate with Akt-mediated in vitro radioresistance.

INTRODUCTION: Treatment of astrocytoma is frequently hampered by radioresistance of the tumor. In addition to overexpression of ErbB1/EGFR, functional crosstalk between receptor tyrosine kinases and cell adhesion molecules may also contribute to therapy resistance. METHODS: Acceptor photobleaching FRET was implemented on frozen sections of clinical astrocytoma to check the role of ErbB1-integrin-ß1 interaction. U251 glioma subclones were obtained by introducing extra CHR7 material or the ErbB1 gene to test the relevance and mechanism of this interaction in vitro. RESULTS: Grade IV tumors showed higher ErbB1 and integrin-ß1 expression and greater ErbB1-integrin-ß1 heteroassociation than did grade II tumors. Of these, the extent of molecular association was a single determinant of tumor grade and prognosis in stepwise logistic regression. In vitro, integrin-ß1 was upregulated, and radiosensitivity was diminished by ectopic ErbB1 expression. Great excess of ErbB1 provided colony forming advantage over medium excess but did not yield better radiation resistance or faster proliferation and decreased to medium level over time, whereas integrin-ß1 levels remained elevated and defined the extent of radioresistance. Increased expression of ErbB1 and integrin-ß1 was paralleled by decreasing ErbB1 homoassociation and increasing ErbB1-integrin-ß1 heteroassociation. Microscopic two-sided FRET revealed that pixels with higher ErbB1-integrin-ß1 heteroassociation exhibited lowed ErbB1 homoassociation, indicating competition for association partners among these molecules. Boosted Akt phosphorylation response to EGF accompanied this shift toward heteroassociation, and the consequentially increased radioresistance could be reverted by inhibiting PI3K. CONCLUSION: The clinically relevant ErbB1-integrin-ß1 heteroassociation may be used as a target of both predictive diagnostics and molecular therapy.

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230.

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence (916)NFNHIHKRIRRVADKYLSG(934) comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin alpha/beta mechanism employing importins alpha1 and alpha3 but not alpha5. This transport was inhibited by wheat germ agglutinin and GTPgammaS. The sequence (1414)SKKTNRGSQLHKYYMKRRTL(1433), a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin alpha1/beta or alpha3/beta complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

ErbB-directed immunotherapy: antibodies in current practice and promising new agents.

The ErbB family is well known for its significance in oncogenesis. As bad prognostic markers, overexpressed or mutated ErbB1 and ErbB2 have an important role in the molecular diagnosis of various cancers, but as membrane proteins, expressed often selectively in tumor tissues, they can be targeted with kinase inhibitors or therapeutic antibodies. In addition to trastuzumab, the first humanized antibody that was approved for the therapy of solid tumors by the FDA, now many humanized antibodies are in late clinical trials, or already approved. Conjugation of antibodies with radioactive isotopes, cytotoxic agents or liposomes loaded with chemotherapeutic drugs led to improved therapeutic efficiency over their parent "unarmed" antibodies. Novel, engineered antibody derivates with better pharmacodynamic properties are also available and allow the targeting of ErbB1 or ErbB2 positive cancers in a wider patient population. In this review, we discuss the biological and clinical background of ErbB targeting, and describe the most successful antibodies against ErbB1 (cetuximab, panitumumab, matuzumab, nimotuzumab, ICR62, mAb 528, ch806 and MDX-447) and ErbB2 (trastuzumab, pertuzumab, MDX-H210, 2B1, C6.5xscFv(NM3E2), ertumaxomab and FRP-5 derivates) that are in clinical trials or already approved, along with the various relevant conjugation and engineering strategies. Recent data pertinent to the prevalent problem of clinical resistance to treatment with trastuzumab are also discussed.

Na+/Ca2+ exchanger inhibitors modify the accumulation of tumor-diagnostic PET tracers in cancer cells.

AIM: To establish the effects of Na(+)/Ca(2+) exchanger (NCX) blockers on 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) and (11)C-choline accumulation in different cancer cells. METHODS: The tumor cells were incubated with NCX inhibitors, and the uptakes of (18)FDG and (11)C-choline were measured. Flow cytometric measurements of intracellular Ca(2+) and Na(+) concentrations were carried out. The presence of the NCX antigen in the cancer cells was proved by Western blotting, flow cytometry and confocal laser scanning microscopy. RESULTS: The NCX is expressed at a noteworthy level in the cytosol and on the cytoplasmic membrane of the examined cells. Incubation of the cells with three chemically unrelated NCX blockers (bepridil, KB-R7943 or 3',4'-dichlorobenzamil hydrochloride) resulted in an increase in the intracellular Ca(2+) concentration, with a simultaneous decrease in the intracellular Na(+) concentration. The treatment with the NCX inhibitors increased the energy consumption of the tumor cells by 50-100%. Thapsigargin abolished the NCX-induced (18)FDG accumulation in the cells. The NCX blockers applied decreased the (11)C-choline accumulation of all the investigated cancer cells by 60-80% relative to the control. CONCLUSION: A possible masking effect of NCX medication must be taken into consideration during the diagnostic interpretation of PET scans.

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

BACKGROUND: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals. METHODS: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230. RESULTS: PI4K230 gives a prominent signal in the nucleolus of ethanol fixed rat brain cryosections and of several cell types in addition to its presence in the nucleus and cytoplasm. The PI4K230 immunoreactivity of the nucleolus is masked in PFA fixed cells, but it can be restored by treatment of PFA fixed cells with hot wet citrate buffer or by washing the cryosections with PBS prior to PFA fixation. Nucleolar PI4K230 occurs in a Triton X-100 resistant complex. Treatment of COS-7 cells with siRNA targeting PI4K230 and permeabilized B50 cells with DNase or RNase results in the loss of PI4K230 signal from the nucleolus. CONCLUSION: These experiments suggest the participation of PI4K230 in a DNase and RNase sensitive complex with a unique localization and function in the nucleolus.

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: local stimulation using magnetic microspheres as assessed by quantitative digital microscopy.

BACKGROUND: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. METHODS: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. RESULTS: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher K(d) for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. CONCLUSION: ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution.

Quantitative and functional assay of MDR1/P170-mediated MDR in ascites cells of patients with ovarian cancer.

BACKGROUND: MDR1-associated P-glycoprotein-dependent multidrug resistance is a common cause of chemotherapy failure in patients with advanced ovarian cancer. Here, we describe a clinical method for simultaneously assessing the expression and function of the MDR1/Pgp in tumour cells from ascites of patients with malignant ovarian carcinoma. MATERIALS AND METHODS: Cells from ascites from 35 patients were collected. The expression and function of Pgp were detected by flow cytometry. For functional study, rhodamine 123 was used. RESULTS: Using the Pgp-specific UIC2 and MM6.15 antibodies, we demonstrated the presence of Pgp in 10-79% (38.9+/-20, 7; n=35) of the CA 125-positive cell subpopulations. The results of the functional assay showed strong correlation with the level of Pgp expression (r=0.976; p=3.2x10(-5)). CONCLUSION: Direct detection of the expression level and function of MDR1/Pgp in the ascites provide useful information for the more efficient treatment of malignant diseases by proper adjustment of the chemotherapeutic protocol.

Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line.

Overexpression of erbB2 in breast tumors is associated with poor prognosis and is a target of receptor-oriented cancer therapy. Trastuzumab (Herceptin), a monoclonal antibody against a membrane-proximal epitope in the extracellular region of erbB2, shows a therapeutic effect against a fraction of erbB2-amplified breast tumors. Unfortunately, resistance to Herceptin is common, and its cause is as yet unclear. Here we investigated the properties of erbB2 in a Herceptin-resistant cell line, JIMT-1, established from a breast cancer patient showing erbB2 gene amplification and primary resistance to Herceptin. The expression profile of erbB proteins, Herceptin-induced erbB2 internalization, and down-regulation in JIMT-1 were similar to those in Herceptin-sensitive lines. However, the mean number of Herceptin Mab binding sites in JIMT-1 was 1/5 that of the expressed erbB2 molecules, although 5% to 10% of the cells showed a approximately 10-fold higher Herceptin binding than the main population. Herceptin Fab and Mab 2C4, an antibody binding to an epitope in the ectodomain further removed from the membrane, bound more efficiently to JIMT-1 cells than Herceptin Mab, implying that erbB2 was partly masked. The expression of MUC4, a membrane-associated mucin that according to reports contributes to the masking of membrane proteins, was higher in JIMT-1 than in Herceptin-sensitive lines, and its level was inversely correlated with the Herceptin binding capacity of single cells. Knockdown of MUC4 expression by RNA interference increased the binding of Herceptin. Western blotting showed a low level of proteolytic processing, shedding, and tyrosine phosphorylation of erbB2 in JIMT-1. The latter finding may explain its Herceptin-resistant phenotype characterizing both the low and high Herceptin binding subpopulations. We conclude that masking of erbB2 in JIMT-1 leads to diminished Herceptin binding and isolation of erbB2 from its normal interaction and activation partners.