Mary Ann Sens

Metallothionein Isoform 3 Overexpression Is Associated with Breast Cancers Having a Poor Prognosis

The third isoform (MT-3) of the metallothionein gene family is unique in that it has a limited tissue distribution, is not induced by metals, has a neuronal growth inhibitory activity, and sequesters zinc more effectively under zinc-depleted conditions. The goal of the present study was to determine whether MT-3 was absent in normal breast tissue, was overexpressed in breast cancers, and if MT-3 overexpression would be associated with disease outcome. A combination of immunohistochemistry and reverse-transcription polymerase chain reaction was used to demonstrate that the normal breast had no detectable expression of MT-3 mRNA or protein. Using immunohistochemistry, it was shown that MT-3 was overexpressed in 25 of 34 cases of breast cancer. In all cases of positive staining, MT-3 was diffusely localized to the cytoplasm. The tumors from these 34 cases were divided as to outcome based on known 5-year survival, with 20 patients being disease free at 5 years (good outcome) and the other 14 having recurring disease within 5 years (bad outcome). When analyzed for MT-3 staining, it was shown that there was a trend for increased MT-3 immunoreactivity in the group having bad outcomes. However, when the tumor subgrouping was further defined on the basis of carcinoma in situ (CIS), there was a marked significant difference in MT-3 staining between patients with good and bad outcomes. Limited to DCIS, MT-3 staining was significantly increased in patients with bad outcomes compared to those with good outcomes. Thus, these studies demonstrate that MT-3 is overexpressed in selected breast cancers and that overexpression is associated with tumors having a poor prognosis.

Expression of MT-3 mRNA in human kidney, proximal tubule cell cultures, and renal cell carcinoma

The human metallothionein 3 (MT-3) gene has recently been identified and characterized as a brain-specific MT having growth inhibitory activity for neuronal cells. One objective of the present study was to determine if MT-3 is brain-specific or also present in the renal system, a site for chronic toxicity due to heavy metal exposure. Using RT-PCR methodology, MT-3 mRNA was shown to be expressed in the human renal system at levels below mRNA for the beta-actin gene. MT-3 mRNA was shown to be expressed in all samples obtained from both the developing and adult renal systems, from 20 weeks of fetal age to 72 years. Cultures of human proximal tubule (HPT) cells were used to determine if MT-3 mRNA expression is influenced by metal exposure. Exposure of HPT cells to either Zn2+ or Cd2+ resulted in an early (within 24 h), but unsustained increase in MT-3 mRNA. The demonstration of MT-3 mRNA expression in the kidney indicates that MT-3 may play an important early role in the response of the cell to metal exposure. MT-3 mRNA expression was also examined in tissues and cells from three cases of renal cell carcinoma. MT-3 was found to be expressed in all three cases at levels similar to those found for normal kidney, providing evidence that MT-3 mRNA expression is not altered in this cancer.

Metallothionein isoform 1 and 2 gene expression in the human prostate: Downregulation of MT‐1X in advanced prostate cancer

Studies have shown an association of metallothionein (MT) overexpression with tumor type and grade. However, a family of genes underlies the expression of these proteins. The goals of this study were to define the expression of MT genes and protein in normal human prostate and to provide evidence that the expression of the MT isoforms is altered in prostate cancer.

Metallothionein isoform 3 expression in the human prostate and cancer-derived cell lines.

Expression of metallothionein isoform 3 (MT‐3) was initially reported to be confined to neural tissues. However, it was recently demonstrated that MT‐3 is expressed in epithelial cells of the human kidney. This motivated the current examination of the expression of MT‐3 in the human prostate.

Transient induction of metallothionein isoform 3 (MT-3), c-fos, c-jun and c-myc in human proximal tubule cells exposed to cadmium.

Cadmium (Cd(+2)) has been shown to transiently increase the expression of mRNA for the third isoform of the metallothionein (MT-3) gene family in cultured human proximal tubule (HPT) cells. The goal of the present study was to further define the expression of MT-3 in mortal (HPT) and immortal (HK-2) cultures of HPT cells when exposed to lethal and sub-lethal concentrations of Cd(+2) under both acute and chronic time periods of exposure. Expression of MT-3 mRNA and protein was determined in cultured HPT cells and HK-2 cells using reverse-transcription-polymerase chain reaction (RT-PCR) and immuno-blotting, and expression of c-fos, c-jun and c-myc mRNA by RT-PCR. The results confirmed that exposure of the HPT cells to Cd(+2) induced a transient increase in MT-3 mRNA and extended the induction to include a subsequent transient increase in the level of the MT-3 protein. The induction of MT-3 was rapid and returned to control values within 48 h of exposure despite the continued presence of lethal and sublethal concentrations of Cd(+2). It was also demonstrated that the pattern of expression of MT-3 mRNA was similar to that of the early response genes, c-fos, c-jun and c-myc. It was shown that the HK-2 cells did not express MT-3 when exposed to Cd(+2), but had similar expression of the c-fos, c-jun and c-myc genes. The results demonstrate that MT-3 expression is metal responsive in HPT cells.

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

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 by immortalized human proximal tubule cells (HK-2) following exposure to heat shock, sodium arsenite, or cadmium chloride

The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined in immortalized human proximal tubule cells (HK-2) exposed to heat shock, sodium arsenite, or cadmium chloride (CdCl2) under both acute and extended conditions of exposure. It was demonstrated that the HK-2 cells did not exhibit the classic heat-shock response when subjected to an acute physical (heat) or chemical stress (sodium arsenite or CdCl2). Heat stress, elevated temperature at 42.5°C for 1 h, caused a marked increase only in hsp 70 mRNA and protein, but not hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 µ M sodium arsenite for 4 h or CdCl2 for 4 h. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60, and hsp 70 mRNA and protein. It was shown that the basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated when compared to those found in unstressed HPT cells and that the basal levels were similar to those found in HPT cells under stress conditions. These results suggest that the failure of the HK-2 cells to in crease hsp 27 and hsp 60 levels in response to physical and chemical stress is because they already possess elevated basal levels of these proteins. This would indicate that one or more of the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70, stress response family members. Overall, the results suggest that although there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.

Expression of heat shock protein 60 in human proximal tubule cells exposed to heat, sodium arsenite and CdCl2

Abstract The expression of hsp 60 mRNA and protein were determined in human proximal tubule cells (HPT) exposed to lethal and sub-lethal concentrations of Cd 2+ under both acute and extended conditions of exposure. It was demonstrated that HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite). Heat stress, elevated temperature at 42.5°C for 1 h, caused an increase in both hsp 60 mRNA and protein following removal of the stress. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 μM sodium arsenite for 4 h. Acute exposure of HPT cells to 53.4 μM CdCl 2 for 4 h also resulted in an increase in hsp 60 mRNA and protein following removal of the metal. An extended exposure to Cd 2+ was modeled by treating the cells continuously with Cd 2+ at both lethal and sub-lethal levels over a 16-day time course. It was demonstrated that chronic exposure to Cd 2+ failed to increase either hsp 60 mRNA or protein expression in HPT cells, even at concentrations of Cd 2+ that were lethal to the cells during the time course. In fact, hsp 60 protein levels were decreased compared to controls at lethal levels of Cd 2+ exposure. These findings suggest that hsp 60 expression may have two distinct roles when the human proximal tubule cell is exposed to Cd 2+ . A protective role through hsp 60 induction when the proximal tubule cell is acutely exposed to Cd 2+ and a deleterious role when hsp 60 protein is down-regulated during extended exposure to Cd 2+ .

Tissue Culture of Human Renal Epithelial Cells Using a Defined Serum-Free Growth Formulation

Background: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory’s cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. Methods: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms’ tumors. Results: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms’ tumors. Conclusions: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.

Expression of heat shock protein 27 in developing and adult human kidney

The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state.