This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd +2 ) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd +2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd +2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd +2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd +2 transformed cells have both similarities and differences in their phenotype.

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Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14 N-or 15 N-iodoacetamide. Absolute quantitation was achieved using 15 Niodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue-and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptornegative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity. Molecular & Cellular

The nuclear lamina is primarily composed of type-V intermediate filaments, the A- and B-type lamins, which give mechanical support to the nuclear envelope, contribute to heterochromatin organization and regulate a myriad of nuclear processes. Over a dozen human diseases, collectively named laminopathies, are associated with mutations in the genes that encode the nuclear lamins. Although the etiology of the laminopathies is well understood, the molecular mechanisms by which they lead to disease remain unclear. Also poorly understood are the mechanisms by which the lamins contribute to a variety of nuclear and cellular functions. The identification of proteins associated with the lamins is likely to provide insight into these fundamental mechanisms. In recent years a unique method for identifying protein-protein and proximity-based interactions has emerged, called BioID (proximity-dependent biotin identification). BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry (MS). BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. The application of BioID is amenable to insoluble proteins such as lamins that are often refractory to study by other methods and is capable of identifying weak and/or transient interactions. In this review, we discuss the use of BioID as a powerful tool to help elucidate novel interacting partners of lamins.