The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.The familial breast and ovarian cancer susceptibility gene product BRCA1 functions in multiple cellular processes that include DNA repair, transcriptional regulation, cell cycle control, and apoptosis (1-4). One possible biochemical function that could contribute to the cellular functions of BRCA1 is the ubiquitin (Ub) 1 ligase activity that arises when BRCA1 forms a RING heterodimer with BARD1 (5-10). Ub-protein isopeptide ligases (E3) catalyze the formation of poly-Ub chains on substrate proteins via isopeptide bonds that link the C-terminal Gly residue of one Ub molecule (activated in an ATP-dependent manner by the enzyme E1) to the ⑀NH 2 group of a Lys side chain in another Ub molecule (11). The most common poly-Ub chain is linked through Lys-48 of Ub and serves as a signal for rapid degradation of substrates by the proteasome-dependent proteolysis pathway (12). However, recent studies have revealed roles other than proteolysis for polyubiquitination (13). While Lys-48-and Lys-29-linked chains mediate proteasomedependent degradation (12, 14), Lys-63-linked chains are a signal for endocytosis, IB kinase activation, ribosome modification, and DNA repair (15-21). Therefore characterization of the poly-Ub chain linkage is important to predict biological function of Ub ligases.Several groups have characterized the type of poly-Ub linkages formed by the BRCA1-BARD1 heterodimer. It was reported that the poly-Ub chain built by the BRCA1-BARD1 ligase is linked through Ub Lys residues other than Lys-48, suggesting they may not serve as a degradation signal (6). Another group reported that BARD1 stimulates the formation of both Lys-48-and Lys-63-linked poly-Ub chains and that the BRCA1 autoubiquitylation by BARD1 mostly results in poly-Ub chains linked through Lys-63 (7)...

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Urinary catecholamines (CAs) and their metabolites are usually measured during the process of diagnosing pheochromocytoma (pheo), but a 24-hour urine collection is not convenient for outpatients. Since 1987 we have utilized "spot" urine metanephrine (MN) and normetanephrine (NMN) assays for management of patients with pheo or adrenal incidentaloma. MN and NMN were measured by radioimmunoassay in 82 patients with surgically proved pheo and 15 patients with incidentaloma. In 10 patients with pheo, MN and NMN were measured with fractional every-3-hour urine samples, which were accumulated and then measured as a 24-hour urinary specimen. Fractions of 3-hour MN and NMN excretion were constant (MN 98.5 +/- 9.6%, NMN 97.6 +/- 10.8%; 24-hour MN and NMN 100%). The average levels of MN and NMN in patients with pheo were 6801 ng/mg creatinine (Cr) (range 93-88,248, median 1426) and 5627 ng/mg Cr (range 219-31,528, mean 3190), whereas the MN and NMN levels in patients with incidentaloma were 123 ng/mg Cr (range 36-246, mean 133) and 251 ng/mg Cr (range 84-472, mean 220), respectively. When we selected a cutoff value for MN + NMN of 1000 ng/mg Cr, the sensitivity was 97.6% and the specificity 100% for diagnosing pheo. When the standard was set as > 500 ng/mg Cr for either MN or NMN, both the sensitivity and specificity were 100%. The assay for MN and NMN is simple and effective, not only for screening but for diagnosing pheo and managing incidentaloma.

Herein we report a 62-year-old woman with an excisable breast tumor in whom needle tract seeding was suspected during preoperative ultrasound and magnetic resonance imaging (MRI). A tumor of the right breast was observed during initial examination, and she was referred to our hospital after fine-needle aspiration cytology led to diagnosis of breast cancer, even though core needle biopsy results were negative. Mammography showed a high-density mass with a portion of the margin exhibiting very fine serrations. Ultrasonography revealed a circular mass with a border that was indistinct in some regions, and a hypoechoic band that extended from the tumor toward the skin. A mass was observed on MRI, with a linear enhancement extending on the skin side, and needle tract seeding was suspected. Fine-needle aspiration cytology revealed malignancy, and the histological appearance was consistent with mucinous carcinoma. T1cN0M0 stage I breast cancer was diagnosed, and wide excision and sentinel lymph node biopsy were performed. The skin directly above the tumor was concurrently excised to remove the biopsy puncture site. Histopathological diagnosis confirmed mucinous carcinoma, with the tumor observed to extend linearly into the subcutaneous adipose tissue in a pattern corresponding to the biopsy puncture site. The stump of the excised breast was negative for cancer cells. The possibility of tumor seeding must be considered during fine-needle aspiration cytology and biopsy. As demonstrated in this case, diagnosis of such seeding through preoperative imaging may enable extraction of the entire lesion, including the needle tract.