Differential Expression of Zinc Transporters in Prostate Epithelia of Racial Groups Transporters in Prostate Epithelia of Racial Groups

Zinc (Zn) is essential for a very large number and variety of cellular functions but is also potentially toxic. Zn homeostasis is therefore dynamically maintained by a variety of transporters and other proteins distributed in distinct cellular and subcellular compartments. Zn transport is mediated by two major protein families: the Zip family, which mediates Zn influx, and the ZnTs which are primarily linked to Zn sequestration into intracellular compartments and are, thereby, involved in lowering cytoplasmic Zn free ion concentrations. In the prostate epithelial cell, the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of prostatic fluids. The loss of Zn accumulation is the most consistent and persistent characteristic of prostate malignancy. Currently, there are no direct methods to determine the relative Zn levels in various cell types of the prostate gland (i.e. stroma, glandular epithelia, acini, muscular, etc) and no reliable ways to compare the Zn in normal versus malignant areas of the gland. Here we report a new method to show a differential Zn staining method that correlates with various stages of prostate cancer development in situ and the expression of a human Zn transporter1-hZIP1 -in situ by in situ reverse transcriptase-polymerase chain reaction hybridization (ISRTPCR) that correlate with the relative Zn levels determined by the differential Zn staining method. By utilizing these methods we show for the first time that: 1) the relative Zn levels are very low in the malignant glands, 2) normal glands show high Zn levels in both glandular epithelia and in stromal tissues, 3) the Zn levels begin to decrease in pre-malignant glands and precedes the development of malignancy, 4) the expression of human Zn transporter1 (hZIP1) appears to correlate with the Zn levels in the prostate glands and may be the major Zn regulator in this organ. Fresh frozen sections from 42 male prostate biopsies with a clinical history of prostate cancer, and 6 from autopsy specimens with normal glands who died from automobile accidents, were processed for RT-in situ-PCR. Fresh frozen tissues were utilized to determine the relative intracellular Zn levels in various histological areas of the prostate glands. All prostate sections were from the peripheral zones of the glands. In this study, Figure 1A shows the high level of cellular Zn that characterizes the normal glandular epithelial cells (green color in Fig 1A). In contrast, the stroma exhibits relatively lower levels of zinc. Therefore, the in situ Zn staining utilizing two different color indicators with different affinity and intracellular threshold provides the differential Zn accumulation between normal glandular epithelium and stromal . The marked reduction of cellular Zn in the epithelium of the two high grade intraepithelial neoplasia are apparent in Figure 1 B and C. 4 Similar pattern is also seen in the patient with adenocarcinoma Gleason score 3+3 (moderately differentiated) in Figure 1D. Like the expression of hZIP1, the loss of Zn occurs early in malignancy. Due to the depletion of Zn in the malignant glands, the stromal Zn level gives the appearance of relatively higher Zn levels. Many studies have observed that Zn levels are greatly decreased in extracts of resected malignant tissue preparations. However, our present study provides the first in situ detection of the depleted cellular Zn levels in adenocarcinomatous glands as compared to the high Zn levels in normal glandular epithelium. Of note, the decrease in Zn level in the malignant glands is due to a decrease in the cellular accumulation of zinc. This suggests that the decrease in intracellular zinc, and not impaired secretion of Zn into the lumen (prostatic fluid), is principally responsible for the decrease in malignant tissue Zn level. Thus, the results of our study are consistent with previous studies. Correspondingly, in Figure 2 the relative expression of mRNA expression for hZIP1 were determined in the 42 prostate resections. The typical results represented in Figure 2 were consistently observed in the frozen sections of all 42 prostate resections. The results show that hZIP1gene expression is evident uniformly in the epithelium of the normal peripheral zone glands and is relatively low in the stroma (Fig 2A). hZIP1 expression is markedly down regulated to the extent of not being demonstrable in the two high grade adenocarcinomatous glands (red colors of the glandular epithelia in Fig 2B-C) and in moderately differentiated adenocarcinoma glands (Fig 2D); however, it is present in the stromal tissues but at much lower levels as compared to the normal control (Fig 2A). Figure 3 shows the hZIP1 expression profiles in the frozen sections of the normal glands adjacent to malignant glands in two of the patients (Fig 3A and B). As one can observe in Figures 3A and Figure 3B, on the right side of each slide there are normal appearing glands that exhibit relatively strong yellow/green staining for hZIP1 expression and as one moves toward the left the degree of expression of hZIP1 decreases as the tumor grade of adenocarcinomatous glands begin to increase. In the same patient (Figure 3B) as one moves further to the left (Figure 3C), one can easily recognize lower grade tumor and relatively higher degree of hZIP1 expression. In this section, one can also note the overall increase in the relative hZIP1 expression. Figure 1: Zinc Levels in Prostate Tissue Frozen Sections. Representative Zinc Levels in Prostate Sections, Figure 1: Fresh frozen tissues were utilized to determine the relative intracellular Zn levels in various histological areas of the prostate glands. All prostate sections were from the peripheral zones of the glands. High Zn is represented by Newport Green, yellow/green stain and low Zn is represented by TSQ red stain. A) Figure 1A shows normal prostate gland from a 42 year old subject. Of note, the high level of cellular Zn indicated by dark green staining with new port green Zn indicator dye that characterizes the normal glandular epithelial cells (black arrows, green color in Fig 1A). In contrast, the stroma exhibits relatively lower levels of zinc, indicated by less intense green color in the stroma (white arrows). Therefore, the in situ Zn staining utilizing two different color indicators with different affinity and intracellular threshold provides the differential Zn accumulation between normal glandular epithelium and stroma [18-19]. The marked reduction of cellular Zn in the epithelium of the two high grade intraepithelial neoplasia are shown in Figure 1 B and C. The malignant region of the peripheral zone shows a significant depletion of Zn in the malignant glandular epithelium as exhibited by the red staining (white arrows) in three patient’s resected tissues. Here, one notes relative depletion of Zn indicated by TSQ red Zn indicator dye and relatively higher levels of Zn in the