A novel role for BRCA1 in regulating breast cancer cell spreading and motility.

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain-interacting proteins. Ezrin-radixin-moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA1(1634-1863) acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA1(1634-1863) in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

Monoclonal antibody 14C5 targets integrin alphavbeta5.

This study identifies and characterizes the antigen recognized by monoclonal antibody (mAb) 14C5. We compared the expression of antigen 14C5 with the expression of eight integrin subunits (alpha1, alpha2, alpha3, alphav, beta1, beta2, beta3, and beta4) and three integrin heterodimers (alphavbeta3, alphavbeta5, and alpha5beta1) by flow cytometry. Antigen 14C5 showed a similar expression to alphavbeta5 in eight different epithelial cancer cell lines (A549, A2058, C32, Capan-2, Colo16, HT-1080, HT-29, and SKBR-3). Specific binding of P1F6, an anti-alphavbeta5 specific antibody, was blocked by mAb 14C5. After transient expression of alphavbeta5 in 14C5-negative Colo16 cells, mAb 14C5 was able to bind a subpopulation of alphavbeta5-positive cells. We evaluated the tissue distribution of the 14C5 antigen in colon (n = 20) and lung (n = 16) cancer tissues. The colon carcinoma cells stained positive for 14C5 in 50% of tumors analyzed, whereas bronchoalveolar lung carcinoma and typical carcinoid were not positive for the antigen. More common types of non-small cell lung cancer, i.e., squamous (n = 5) and adenocarcinoma (n = 3), stained positive in 2 of 5 squamous carcinomas and in 1 of 3 investigated adenocarcinoma. Colon (95%) and lung (50%) carcinoma tissues showed extensive expression of antigen 14C5 in the stroma surrounding the tumor cells and on the membrane of the adjacent fibroblasts. We show for the first time that mAb 14C5 binds the vascular integrin alphavbeta5, suggesting that mAb 14C5 can be used as a screening agent to select colon and lung cancer patients that are eligible for anti-alphavbeta5-based therapies.

Anti-biotin antibodies offer superior organelle-specific labelling of mitochondria over avidin or streptavidin.

A number of endogenously biotinylated proteins are found in both cytosol and mitochondria of mammalian cells from many tissues, including liver, spleen, pancreas, kidney, and intestine. Therefore, caution should be taken when using the biotin detection system. Endogenous biotin can interfere with staining systems that employ the use of biotin-avidin- or biotin-streptavidin-based detection systems and may therefore result in high, non-specific background staining. Here, we show that this endogenous biotin reactivity can be deliberately exploited and used as a specific mitochondrial marker in both light and electron microscopy as well as for identifying mitochondrial fractions on Western blot.

Biodistribution and planar gamma camera imaging of (123)I- and (131)I-labeled F(ab')(2) and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor.

UNLABELLED: Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab')(2) and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. METHODS: F(ab')(2) and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of (125)I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of (131)I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these (123)I-labeled 14C5 fragments for radioimmunodetection (RID). RESULTS: Saturation binding experiments showed highest affinity for (125)I-labeled F(ab')(2) fragments (K(d)=0.37+/-0.10 nmol/L) and lowest affinity for (125)I-labeled Fab fragments (K(d)=2.25+/-0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t(1/2)alpha) value for Fab, F(ab')(2) and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t(1/2)beta value for Fab, F(ab')(2) and mAb 14C5 was 439, 627 and 4067 min, respectively. (131)I-Fab fragments showed highest tumor uptake 3 h after injection (2.4+/-0.8 %ID/g), (131)I-labeled F(ab')(2) showed highest tumor uptake 6 h after injection (4.7+/-0.7 %ID/g) and for (131)I-labeled mAb highest tumor uptake was observed at 24 h (10.7+/-2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than (123)I-mAb 14C5. CONCLUSION: Fab and F(ab')(2) fragments derived from intact mAb 14C5 have significant potential for diagnostic and therapeutic applications and may provide new tools in mAb-based radiopharmaceuticals for targeting non-small cell lung cancer.

In vitro and in vivo targeting properties of iodine-123- or iodine-131-labeled monoclonal antibody 14C5 in a non-small cell lung cancer and colon carcinoma model.

PURPOSE: The monoclonal antibody (mAb) 14C5 is a murine IgG1 directed against a yet undefined molecule involved in cell substrate adhesion found on the surface of malignant breast cancer tissue. mAb 14C5 is able to inhibit cell substrate adhesion and invasion of breast cancer cells in vitro. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast, no expression of the antigen 14C5 occurs. The aim of this study was to investigate the in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 as a novel agent for radioimmunodetection and radioimmunotherapy. EXPERIMENTAL DESIGN: Internalization of mAb 14C5 was investigated with 125I-labeled mAb 14C5 and by confocal laser scanning microscopy. Biodistribution studies of 131I-labeled mAb 14C5 and planar gamma imaging were done in nude mice bearing an A549 (non-small cell lung carcinoma) or a LoVo (colon carcinoma) tumor. RESULTS: Internalization studies with both A549 and LoVo cells showed that 125I-labeled mAb 14C5 is slowly internalized with approximately 30% of the initially bound mAb 14C5 internalized after 2 hours at 37 degrees C. Internalization of mAb 14C5 could be visualized with confocal laser scanning microscopy. In vivo, radioisotope uptake peaked at 24 hours for both tumor models (n = 5) with no significant difference in percentage of injected dose/g tissue (A549 10.4 +/- 0.8 and LoVo 9.3 +/- 0.8). Via planar gamma camera imaging, A549 lung tumors as well as LoVo colon tumors could be clearly visualized. CONCLUSIONS: The in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 are promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing antigen 14C5-expressing tumors.

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta.

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria.

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

Radiolabeling, biodistribution, and dosimetry of (123)I-mAb 14C5: a new mAb for radioimmunodetection of tumor growth and metastasis in vivo.

UNLABELLED: This study reports on the in vitro evaluation, biodistribution, and dosimetry of (123)I-labeled monoclonal antibody (mAb) 14C5, a new antibody-based agent proposed for radioimmunodetection of tumor growth and metastasis in vivo. METHODS: (123)I-mAb 14C5 was prepared by direct iodination and tested for stability in vitro. Binding assays were performed on human SK-BR-3 and HeLa carcinoma cells to investigate the antigen expression, antibody affinity, and kinetics of tracer binding. For the biodistribution and dosimetry study, 3- to 4-wk-old NMRI mice were injected intravenously with (123)I-mAb 14C5 (148.0 +/- 7.4 kBq per mouse) and killed at preset time intervals. Organs, blood, urine, and feces were counted for radioactivity uptake, and the data were expressed as the percentage injected dose per gram tissue (%ID/g tissue) or %ID. The MIRDOSE3.0 program was applied to extrapolate the estimated absorbed radiation doses for various organs to the human reference adult. RESULTS: (123)I-mAb 14C5 was obtained in radiochemical yields of 85.0% +/- 2.5% and radiochemical purities were >97%. The iodinated antibody demonstrated good in vitro stability with 93.6% +/- 0.1% of (123)I-mAb 14C5 remaining intact at 24 h after radiolabeling. (123)I-mAb 14C5 bound to SK-BR-3 cells (dissociation constant [K(d)] approximately 0.85 +/- 0.17 nmol/L) and HeLa cells (K(d) approximately 1.71 +/- 0.17 nmol/L) with nanomolar affinity and high specificity, whereas both cell types exhibited a high CA14C5 antigen expression (maximum number of binding sites [B(max)] = 40.6 +/- 5.2 and 57.1 +/- 9.6 pmol/L, respectively). In mice, (123)I-mAb 14C5 accumulated primarily in lungs (20.4 %ID/g), liver (15.1 %ID/g), and kidneys (11.1 %ID/g) within 5 min after injection. A delayed uptake was observed in stomach (12.8 %ID/g) and urinary bladder (8.7 %ID/g) at 3 and 6 h, respectively, after injection. Radioactivity clearance was predominantly urinary, with 44.9 +/- 4.5 %ID excreted during the initial 48 h after administration (cumulative amount). The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.1200-0.1210 mGy/MBq), liver (0.0137-0.0274 mGy/MBq), uterus (0.0196-0.0207 mGy/MBq), and lower large intestine wall (0.0139-0.0258 mGy/MBq). The average effective dose resulting from a single (123)I-mAb 14C5 injection was estimated to be 0.017-0.022 mSv/MBq. CONCLUSION: (123)I-mAb 14C5 shows good in vitro biologic activity and favorable biodistribution properties for imaging carcinomas of different origin and provides an acceptable radiation dose to the patient.

Neurofibromin is actively transported to the nucleus.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors 'shuttle' between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28-49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.

The role of heregulin-alpha as a motility factor and amphiregulin as a growth factor in wound healing.

Wound healing is a complex process of which growth and motility are essential features. The aim of this study was to search for keratinocyte-derived secreted factors that may play a role in these mechanisms, and their corresponding receptors. Growth and motility factors were purified from conditioned medium from cultured primary keratinocytes. Receptor and growth factor expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured keratinocytes and tissue sections derived from chronic wounds. The most potent autocrine growth factor for keratinocytes, which it was possible to purify and sequence from keratinocyte-conditioned medium, is amphiregulin. Its receptor HER-1 is up-regulated on the membranes of keratinocytes lining the edge of the wound. From the same keratinocyte-conditioned medium, heregulin-alpha was purified as a potent motility factor for keratinocytes. Its receptor is HER-3, which is up-regulated on the membranes of keratinocytes lining the edge of the wound and on keratinocytes that had migrated towards the centre of the wound. HER-4 - another receptor for heregulin-alpha - is weakly present in occasional cells near the edge of the wound. The co-receptor for HER-3 and HER-4 is HER-2/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin-alpha is a potent motility factor for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin-alpha has already been shown to be the motility factor leading to migration of HER-2/neu-overexpressing breast cancer cells. The role of amphiregulin as a growth factor and of heregulin-alpha as a motility factor for keratinocytes in epidermal and mucosal wound healing parallels their motility and growth induction in carcinogenesis.