Xu Dong Zhou

Keratin 6 Expression Correlates to Areas of Squamous Differentiation in Multiple Independent Isolates of As+3-Induced Bladder Cancer

This laboratory has shown that arsenite (As+3) exposure can cause the malignant transformation of the UROtsa human urothelial cell line. This single isolate formed subcutaneous tumors with a histology similar to human urothelial cell carcinoma. The tumors also displayed areas of squamous differentiation of the urothelial cells, an infrequent but known component of human bladder cancer. In the present study, five additional independent isolates of As+3‐transformed urothelial cells were isolated and each was shown to produce subcutaneous urothelial cell tumors with a characteristic histology very similar to those described in the initial report. That there were underlying phenotypic differences in the six independent isolates was demonstrated when they were assessed for their ability to form tumors within the peritoneal cavity. It was shown that two isolates could form hundreds of small peritoneal tumor nodules, one isolate a moderate number of tumor nodules, and three isolates no or only one tumor nodule. The peritoneal tumors were also characterized for their degree of squamous differentiation of the urothelial cells and, while areas of squamous differentiation could be found, such differentiation was substantially reduced compared to subcutaneous tumors. Immunostaining for keratin 6 was tested as a potential marker for malignant urothelial cells that had undergone squamous differentiation. Keratin 6 was shown to consistently stain only cells having some evidence of squamous differentiation. Keratin 16 was shown to follow the staining pattern of keratin 6. The isolates and tumor heterotransplants were all examined for keratin 6, 16 and 17 mRNA and protein expression. Copyright

Absence of Metallothionein 3 Expression in Breast Cancer is a Rare, But Favorable Marker of Outcome that is Under Epigenetic Control

Cadmium (Cd2+), a known carcinogen, mimics the effects of estrogen in the uterus and mammary gland suggesting its possible involvement in the development and progression of breast cancer. This lab showed through analysis of a small set of archival human diagnostic specimens that the third isoform of the classic Cd2+ binding protein metallothionein (MT-3) is not expressed in normal breast tissue, but is expressed in some breast cancers and that expression tends to correlate with a poor disease outcome. The goals of this study were to verify that overexpression of MT-3 in a large set of archival human diagnostic specimens tends to correlate with poor disease outcome and define the mechanism of MT-3 gene regulation in the normal breast epithelial cell. The results showed that MT-3 was expressed in approximately 90% of all breast cancers and was absent in normal breast epithelium. The lack of MT-3 staining in some cancers correlated with a favorable patient outcome. High frequency of MT-3 staining was also found for in situ breast cancer suggesting that MT-3 might be an early biomarker for breast cancer. The study also demonstrated that the MCF-10A cell line, an immortalized, non-tumorigenic model of human breast epithelial cells, displayed no basal expression of MT-3, nor was it induced by Cd2+. Treatment of the MCF-10A cells with the demethylation agent, 5-aza-2′-deoxycytidine, or the histone deacetylase inhibitor, MS-275, restored MT-3 mRNA expression. It was also shown that the MT-3 metal regulatory elements are potentially active binders of protein factors following treatment with these inhibitors suggesting that MT-3 expression may be subject to epigenetic regulation.

SPARC Gene Expression is Repressed in Human Urothelial Cells (UROtsa) Exposed to or Malignantly Transformed by Cadmium or Arsenite

SPARC belongs to a class of extracellular matrix-associated proteins that have counteradhesive properties. The ability of SPARC to modulate cell-cell and cell-matrix interactions provides a strong rationale for studies designed to determine its expression in cancer. The objective of this study was to determine if SPARC expression was altered in cadmium (Cd(2+)) and arsenite (As(3+)) induced bladder cancer and if these alterations were present in archival specimens of human bladder cancer. The expression of SPARC was determined in human parental UROtsa cells, their Cd(2+) and As(3+) transformed counterparts and derived tumors, and in archival specimens of human bladder cancer using a combination of real time reverse transcriptase polymerase chain reaction, Western blotting, immunofluorescence localization and immunohistochemical staining. It was demonstrated that SPARC expression was down-regulated in Cd(2+) and As(3+) transformed UROtsa cells. In addition, the malignant epithelial component of tumors derived from these cell lines were also down-regulated for SPARC expression, but the stromal cells recruited to these tumors was highly reactive for SPARC. This finding was shown to translate to specimens of human bladder cancer where tumor cells were SPARC negative, but stromal cells were positive. Acute exposure of UROtsa cells to both cadmium and arsenite reduced the expression of SPARC through a mechanism that did not involve changes in DNA methylation or histone acetylation. These studies suggest that environmental exposure to As(3+) or Cd(2+) can alter cell-cell and cell-matrix interactions in normal urothelial cells through a reduction in the expression of SPARC. The SPARC associated loss of cell-cell and cell-matrix contacts may participate in the multi-step process of bladder carcinogenesis.

Kindlin-2 Expression in Arsenite- and Cadmium-transformed Bladder Cancer Cell Lines and in Archival Specimens of Human Bladder Cancer

OBJECTIVE To confirm a microarray study that suggested that Kindlin-2 might play a role in the development and progression of bladder cancer. There has been no previous examination of Kindlin-2 expression in human bladder cancer. METHODS A combination of real-time polymerase chain reaction, Western analysis, and immunohistochemistry was used to characterize Kindlin-2 expression in arsenite (As(+3))- and cadmium (Cd(+2))-transformed human cell lines, their tumor transplants in immunocompromised mice, and in archival specimens of human bladder and bladder cancer. RESULTS The results show that the Kindlin-2 expression patterns in the cell lines were not duplicated in the tumor tissues. However, it was shown that Kindlin-2 was expressed in the stromal element of all the transplanted tumors and archival specimens of human bladder cancer. It was also shown that a small number of high-grade invasive urothelial cancers have focal expression of Kindlin-2 in the tumor cells. CONCLUSION Kindlin-2 is expressed in the stromal component of most, if not all, human bladder cancers. Kindlin-2 is not expressed in normal urothelium. Kindlin-2 is expressed in a small subset of high-grade invasive bladder cancers and may have potential as a prognostic marker for tumor progression.

Cadmium, Vectorial Active Transport, and MT-3–Dependent Regulation of Cadherin Expression in Human Proximal Tubular Cells

Previous studies from this laboratory have implicated the expression of the third isoform of metallothionein (MT-3) in the maintenance of proximal tubular vectorial active ion transport. It was shown that HK-2 cells have no expression of MT-3 and do not form domes in culture; whereas, the human proximal tubular (HPT) cells and HK-2 cells stably transfected with MT-3 [HK-2(MT-3)] form these structures. In the present study, this association was further explored by determining the effect of MT-3 expression on the expression of the E -, P -, N -, K -, and Ksp-cadherins. It was demonstrated that the HPT cells and HK-2(MT-3) cells had significant elevations in the expression of messenger RNA and protein for the E -, P -, and Ksp-cadherins compared with that of the HK-2 cells transfected with the blank vector [HK-2(blank vector)]. In contrast, the HK-2(blank vector) cells had significantly elevated expression of N- and K-cadherin compared with both the HPT and HK-2(MT-3) cell lines. These patterns of cadherin expression provide strong evidence that MT-3 might be involved in epithelial to mesenchymal transition that is postulated to occur during several disease states and in the mesenchymal to epithelial transition that occurs during normal kidney morphogenesis. A final goal of the study was to determine if Cd(+2) exposure influenced vectorial active transport of the proximal tubular cells and if this might occur through alterations in the expression of MT-3. It was shown that exposure to Cd(+2) eliminated vectorial active transport by the proximal tubular cell lines, but that Cd(+2) exposure did not reduce the expression of the MT-3 protein. The study shows that the level of MT-3 expression in HPT cells influences transepithelial resistance and cadherin expression but does not influence the Cd(+2)-induced loss of vectorial active transport.

Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

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 of 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 those of 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.

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.

Beclin-1 expression in normal bladder and in Cd2+ and As3+ exposed and transformed human urothelial cells (UROtsa)

The expression of beclin-1 in normal human bladder and in Cd(2+) and As(3+) exposed and transformed urothelial cells (UROtsa) was examined in this study. It was shown using a combination of real-time PCR, Western analysis and immunohistochemistry that beclin-1 was expressed in the urothelial cells of the normal bladder. It was also demonstrated that the parental UROtsa cell line expressed beclin-1 mRNA and protein at levels similar to that of the in situ urothelium. The level of beclin-1 expression underwent only modest alterations when the UROtsa cells were malignantly transformed by Cd(2+) or As(3+) or when the parental cells were exposed acutely to Cd(2+) or As(3+). While there were instances of significant alterations at individual time points and within cell line-to-cell line comparisons there was no evidence of a dose-response relationship or correlations to the phenotypic properties of the cell lines. Similar results were obtained for the expression of the Atg-5, Atg-7, Atg-12 and LC3B autophagy-related proteins. The findings provide initial evidence for beclin-1 expression in normal bladder and that large alterations in the expression of beclin-1 and associated proteins do not occur when human urothelial cells are malignantly transformed with, or exposed to, either Cd(2+) or As(3+.).

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.