Donald A. Sens

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

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 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-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-receptor-negative 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.

Immunolocalization of band 3 protein in normal and cystic fibrosis skin.

Current evidence indicates that the defect in cystic fibrosis (CF) involves chloride transport in various epithelial cells. The sweat gland, one site of altered chloride transport in CF, was examined immunocytochemically for localization of a chloride-channel membrane protein, designated band 3 protein. Immunoreactivity was observed in sweat duct cell membranes of both normal and CF samples, whereas secretory coil regions were entirely unreactive. No difference was observed in the pattern or intensity of immunoreactivity between the two groups at the light microscopic (LM) level of resolution.

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

BackgroundNeuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As+3 and Cd+2 sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.ResultsIt was shown that both As+3 and Cd+2 exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As+3 or Cd+2. Localization studies showed that ENO2 in the MCF-10A cells transformed by As+3 or Cd+2 had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.ConclusionThe results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As+3 or Cd+2. The findings also suggest a possible link between As+3 and Cd+2 exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As+3 and Cd+2.

Short and long term gene expression variation and networking in human proximal tubule cells when exposed to cadmium

Cadmium (Cd2+) is a known nephrotoxin causing tubular necrosis during acute exposure and potentially contributing to renal failure in chronic long-term exposure. To investigate changes in global gene expression elicited by cadmium, an in-vitro exposure system was developed from cultures of human renal epithelial cells derived from cortical tissue obtained from nephrectomies. These cultures exhibit many of the qualities of proximal tubule cells. Using these cells, a study was performed to determine the cadmium-induced global gene expression changes after short-term (1 day, 9, 27, and 45 μM) and long-term cadmium exposure (13 days, 4.5, 9, and 27 μM). These studies revealed fundamental differences in the types of genes expressed during each of these time points. The obtained data was further analyzed using regression to identify cadmium toxicity responsive genes. Regression analysis showed 403 genes were induced and 522 genes were repressed by Cd2+ within 1 day, and 366 and 517 genes were induced and repressed, respectively, after 13 days. We developed a gene set enrichment analysis method to identify the cadmium induced pathways that are unique in comparison to traditional approaches. The perturbation of global gene expression by various Cd2+ concentrations and multiple time points enabled us to study the transcriptional dynamics and gene interaction using a mutual information-based network model. The most prominent network module consisted of INHBA, KIF20A, DNAJA4, AKAP12, ZFAND2A, AKR1B10, SCL7A11, and AKR1C1.

Long-term α1B-adrenergic receptor activation shortens lifespan, while α1A-adrenergic receptor stimulation prolongs lifespan in association with decreased cancer incidence

The α1-adrenergic receptor (α1AR) subtypes, α1AAR and α1BAR, have differential effects in the heart and central nervous system. Long-term stimulation of the α1AAR subtype prolongs lifespan and provides cardio- and neuro-protective effects. We examined the lifespan of constitutively active mutant (CAM)-α1BAR mice and the incidence of cancer in mice expressing the CAM form of either the α1AAR (CAM-α1AAR mice) or α1BAR. CAM-α1BAR mice have a significantly shortened lifespan when compared with wild-type (WT) animals; however, the effect was sex dependent. Female CAM-α1BAR mice lived significantly shorter lives, while the median lifespan of male CAM-α1BAR mice was not different when compared with that of WT animals. There was no difference in the incidence of cancer in either sex of CAM-α1BAR mice. The incidence of cancer was significantly decreased in CAM-α1AAR mice when compared with that in WT, and no sex-dependent effects were observed. Further study is warranted on cancer incidence after activation of each α1AR subtype and the effect of sex on lifespan following activation of the α1BAR. The implications of a decrease in cancer incidence following long-term α1AAR stimulation could lead to improved treatments for cancer.

Increased neuron specific enolase expression by urothelial cells exposed to or malignantly transformed by exposure to Cd2+ or As3+

Neuron specific enolase (ENO2, γ-enolase) is a biomarker used to help identify neuroendocrine differentiation in tumors. This laboratory has shown that ENO2 might be a biomarker for exposure to cadmium and arsenite. In this study these observations are extended to the urothelial cell, where environmental exposures are strongly linked to urothelial cancer. The UROtsa urothelial cell line and its Cd²⁺- and As³⁺-transformed counterparts were used as the model. Acute exposure of the UROtsa cells to both As³⁺- and Cd²⁺-caused significant increases in ENO2 expression. Treatment with the histone deacetlyase inhibitor was also shown to significantly increase the expression of ENO2 mRNA. The expression of ENO2 was significantly elevated in the Cd²⁺- and As³⁺-transformed UROtsa cells and tumor transplants. In contrast, ENO1, was unaffected by exposure to As³⁺ or Cd²⁺. Immunofluorescence showed ENO2 associated with both the nucleus and cytoplasm and cytoplasmic ENO2 co-localized with ENO1. The findings extend the evidence suggesting a link between As³⁺ and Cd²⁺ exposure and neuroendocrine differentiation in tumors. The results suggest that ENO2 might be a biomarker of human exposure to Cd²⁺ and As³⁺ that operates through histone modification.

Metallothionein isoform 3 expression in human skin, related cancers and human skin derived cell cultures

Human skin is a well known target site of inorganic arsenic with effects ranging from hyperkeratosis to dermal malignancies. The current study characterizes the expression of a protein known to bind inorganic, As(3+), metallothionein 3 (MT-3). Expression of this protein was assessed immunohistochemically with a specific MT-3 antibody on human formalin-fixed, paraffin-embedded biopsy specimens in normal skin, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Assessment in normal skin using nine normal specimens showed moderate to intense MT-3 staining in epidermal karatinocytes with staining extending into the basal cells and moderate to intense staining in melanocytes of nevi. MT-3 immunoexpression was shown to be moderate to intense in 12 of 13 of SCC, low to moderate in 8 of 10 BCC, and moderate to intense in 12 melanoma samples. MT-3 expression in cell culture models (normal human epidermal keratinocytes, normal human melanocytes, and HaCaT cells) showed only trace expression of MT-3, while exposures to the histone deacytalase inhibitor, MS-275, partially restored expression levels. These results indicate that the epidermis of human skin and resulting malignancies express high level of MT-3 and potentially impact on the known association of arsenic exposure and the development of skin disorders and related cancers.

Comparison of expression patterns of keratin 6, 7, 16, 17, and 19 within multiple independent isolates of As+3- and Cd+2-induced bladder cancer

This laboratory has generated a series of seven cadmium (Cd+2)- and six arsenite (As+3)-transformed urothelial cancer cell lines by exposure of parental UROtsa cells to each agent under similar conditions of exposure. In this study, the seven Cd+2-transformed cell lines were characterized for the expression of keratin 6, 16, and 17 while the six As+3 cell lines were assessed for the expression of keratin 7 and 19. The results showed that the series of Cd+2-transformed cell lines and their respective transplants all had expression of keratin 6, 16, and 17 mRNA and protein. The expression of keratin 6, 16, and 17 was also correlated with areas of the urothelial tumor cells that had undergone squamous differentiation. The results also showed that four of the six As+3-transformed cell lines had expression of keratin 7 and 19 mRNA and protein and produced subcutaneous tumors with intense focal staining for keratin 7 and 19. The other two As+3-transformed cell lines had very low expression of keratin 7 mRNA and protein and produced subcutaneous tumors having no immunoreactivity for keratin 7; although keratin 19 expression was still present. The peritoneal tumors produced by one of these two cell lines regained expression of keratin 7 protein. The present results, coupled with previous studies, indicate that malignant transformation of UROtsa cells by Cd+2 or As+3 produce similar patterns of keratin 6, 7, 16, 17, and 19 in the resulting series of cell lines and their respective tumors.

Aminoglycoside Antibiotics Alter the Electrogenic Transport Properties of Cultured Human Proximal Tubule Cells

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD) and short-circuit current (Isc). These electrical parameters were used in the present study to determine if aminoglycoside exposure altered electrogenic sodium transport by HPT cells. The results of this determination demonstrated that exposure to gentamicin, at levels below that producing cell necrosis, caused a marked reduction in Isc and that this reduction followed the known in vivo nephrotoxicities of the aminoglycosides streptomycin, gentamicin, and neomycin. It was concluded through a similar analysis on a total of 14 isolates of HPT cells that the aminoglycosides repeatably reduced the electrogenic sodium transport of HPT cells. It was further determined that this alteration in electrogenic transport by gentamicin was mediated through exposure of the drug to the basolateral cell surface and that apical exposure had little effect. Evidence was obtained against the involvement of Na+, K+-ATPase, adenosine 3,5-cyclic monophosphate, and sodium-coupled substrate transport in this alteration in electrogenic transport by the aminoglycosides. The basolaterally located Na+: CO3 -2:HCO 3 -1 symporter is a possible site for aminoglycoside-induced nephrotoxicity.

Immunocytochemistry of band 3 protein in kidney and other tissues of control and cystic fibrosis patients

ABSTRACT: Current evidence indicates that the underlying genetic defect in cystic fibrosis (CF) results in defective chloride transport, and more specifically, chloride impermeability. However, recent consideration has been given to a possible defect in band 3 protein at CF-affected sites. In an effort to determine a possible role for band 3, an anion exchange channel protein, a series of immunocytochemical localizations was performed. Immunocytochemical staining for the anion channel band 3, 43K, in normal and CF human kidney was confined to erythrocytes, glomerular podocytes, the basal region of half of the cells of the initial connecting segment of cortical collecting ducts, and a minority of cells in medullary ray-collecting ducts. Erythrocytes alone evidenced immunoreactivity for band 3 protein in human pancreas, submandibular gland, trachea, and lung. In all cases, specimens from patients with CF stained like those from control subjects for band 3. Abnormal CI- transport in CF tissues is not reflected in altered band 3, 43K, localizations.