Proliferative potential in nasopharyngeal carcinoma: correlations with metallothionein expression and tissue zinc levels

A. Jayasurya, B.H. Bay4, W.M. Yap2, N.G. Tan3 and B.K.H. Tan1

Anatomy Department and
1 Pharmacology Department, National University of Singapore, Kent Ridge, S119260,
2 Pathology Department, Tan Tock Seng Hospital, Moulmein Road, S308433,
3 Otolaryngology Department, Singapore General Hospital, Outram Road, S169608, Singapore


    Abstract
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 Abstract
 Introduction
 References
 
Metallothionein (MT) is a cysteine-rich protein with pleiotropic functions and a high binding affinity for heavy metals. The present study was designed to examine the relationship between MT expression and tissue zinc levels in conjunction with cell proliferation in nasopharyngeal cancer (NPC). Proliferative activity in NPC was quantified by Ki67 immunolabelling and MT expression was determined by immunohistochemistry. Total zinc and subcellular zinc fractions were analysed by flame atomic absorption spectrometry. MT immunostaining was observed in the nuclei of NPC cells, with the percentage MT immunopositivity ranging from 3.0 to 59.7%. Thirteen tumours displayed weak MT staining and the remaining 11 showed moderate to strong immunostaining. There was a significant positive correlation between MT and Ki67 positivity (P = 0.0127). Tissue zinc levels were higher in NPC as compared with benign nasopharyngeal tissues (4.800 ± 0.4610 versus 2.889 ± 0.4045 µg/g dry wt tissue, respectively; P = 0.0122). Nuclear zinc levels in NPC were significantly higher than levels in membrane and cytosolic fractions (mean zinc levels 1.4840 ± 0.1489, 0.6286 ± 0.0789 and 0.3014 ± 0.0250 µg/mg protein, respectively). A linear relationship was also observed between nuclear zinc levels and MT immunostaining (P = 0.0024) as well as with Ki67 immunopositivity (P = 0.0123). Our results show that MT and zinc are correlated with proliferative activity in NPC, providing further insights into the biology of this enigmatic and aggressive tumour.

Abbreviations: EGF, epidermal growth factor; MT, metallothionein; NPC, nasopharyngeal carcinoma.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Metallothioneins (MTs) are a family of intracellular cysteine-rich proteins involved in a variety of cellular processes (13). The pleiotropic physiological roles of MT include metalloregulatory functions in cell growth, differentiation, repair and protection against oxidative stress-induced injury and apoptosis (1,3,4). Cell proliferation is believed to be a putative function of MT as there have been reports indicating MT to be a cell cycle-dependent protein. MT-positive nuclei were observed to be present specifically in the S to G2M phases of the cell cycle of regenerating hepatocytes (5). A 2- to 3-fold increase in MT expression (quantitated by enzyme-linked immunosorbent assay) was observed in proliferating human colonic HT-29 cells (6).

The proliferative potential of MT has also been linked to zinc, a trace element which participates in the activity of a number of DNA and RNA polymerases (2,7,8). MT has a high binding affinity for zinc and Zn-MT has been hypothesized to play important roles in transmission and expression of genetic information, in response to signals for cell activation (2,8). Zinc ions are sequestered in the metal thiolate clusters of the metalloprotein (7). In this study, our aim was to investigate the relationship of MT and zinc with proliferative activity in undifferentiated nasopharyngeal carcinoma (NPC), a cancer which is common among the Chinese population in South East Asia (4).

Post-nasal biopsy specimens from 30 patients who had ear, nose and throat symptoms or neck masses were included in this study. Twenty-four specimens were histologically confirmed to be undifferentiated NPC (WHO type 3), three specimens showed normal histology of the nasopharynx without evidence of NPC and the remaining three specimens were found to have benign lymphoid hyperplasia. Undifferentiated carcinoma is the most common histological type of NPC occurring in Singapore (9). All the tissues were fixed in 10% formalin and embedded in paraffin. Nine nasopharyngeal tumour biopsies and six benign biopsies (comprising the three lymphoid hyperplasia specimens and three normal biopsies without evidence of NPC) were also collected in 1.15% potassium chloride for elemental analysis.

For immunohistochemistry, 4 µm sections from paraffin-embedded tissue specimens were mounted on 3-aminopropyltriethoxy silane (Sigma-3648). After dewaxing and dehydration, sections were incubated with 0.5% hydrogen peroxide in methanol for 30 min (to inhibit the activity of endogenous peroxidase). For MT immunostaining, sections were incubated in 4% normal goat serum for 1 h at room temperature, followed by overnight incubation at 4°C using a 1:600 dilution of the primary monoclonal E9 antibody (Dako, Copenhagen, Denmark), an epitope directed against the MTI and MTII isoforms (4). As a negative control, the primary antibody was omitted. A known MT-positive ovarian cancer specimen was used as a positive control (10). Visualization was by the standard avidin–biotin complex technique (Dako). The sections were then counterstained with Harris haematoxylin. For Ki67 immunolabelling there was a pretreatment step where sections were boiled in citrate buffer (pH 6) at 92°C for 20 min, before incubation with polyclonal anti-Ki67 antibody (Dako, Glostrup, Denmark) at 1:100 dilution for 1 h at room temperature. The rest of the staining protocol was the same as for MT immunohistochemistry except that the sections were counterstained with methyl green. The numbers of MT and Ki67 immunopositive cells were counted under a light microscope (Zeiss Axioplan) using a 40x objective. A total of 10 high power fields were randomly chosen.

For zinc analysis, tissues were homogenized in 1.15% potassium chloride and freeze-dried. After adding 2.5 ml of 10 mM nitric acid to the homogenate, the solution was mixed thoroughly in a vortex and allowed to stand for 5 h. The solution was then centrifuged at 18 000 r.p.m. for 30 min and the supernatant collected for estimation of total tissue zinc levels by flame atomic absorption spectrometry. For subcellular fractionation, tissues were processed in 4 ml of cold homogenizing medium (0.25 M sucrose, 5 mM MgCl2, 10 mM Tris–HCl, pH 7.4) as previously described (11). The subcellular fractions were separated by differential centrifugation. The homogenate was centrifuged at 1000 g for 10 min to collect cell nuclei and the remaining supernatant was further centrifuged at 10 000 g for 1 h to pellet plasma membranes representing crude lysosomal, mitochondrial and microsomal fractions. To purify the nuclear pellet, centrifugation was carried out at 60 000 g for 1 h through 2.2 M sucrose, 1 mM MgCl2 and 10 mM Tris–HCl, pH 7.4. All three subcellular fractions were then analysed for zinc after digestion with 10 mM nitric acid for 5 h. Protein was estimated by the Bio-Rad Protein Assay.

The Graphpad Prism software package was used for statistical analysis. Pearson's correlation was used to analyse the relationship between percentage Ki67 expression and zinc levels and MT expression. Mean values were analysed using Student's t-test.

All the nasopharyngeal tumour tissues showed positive immunoreactivity for MT expression. The percentage MT immunopositive cells ranged from 3.0 to 59.7%. The mean percentage MT staining was 14.82 ± 3.52%. MT staining was observed to be essentially in the nucleus (Figure 1AGo). Six tumours showed strong immunostaining (25%), five had moderate staining (21%) and 13 had weak staining (54%). The epithelial lining of normal nasopharyngeal tissue and lymphoid hyperplasia displayed positive staining for MT in the basal epithelial cells.



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Fig. 1. Light micrographs of NPC tissues incubated with anti-MT antibody (A) and anti-Ki67 antibody (B). x630.

 
Nuclear immunopositivity for Ki67-labelled cells in NPC is shown in Figure 1BGo. The positive fraction ranged from 4.7 to 32.32% with a mean of 18.03 ± 1.897%. A significant positive correlation was observed between Ki67 and MT expression (P = 0.0127; Table IGo). There was also a significant difference between Ki67 expression and intensity of MT staining (P = 0.04). Mean percentage Ki67-positive cells in weakly MT stained tumours was 14.56 ± 2.12%, as compared with 22.14 ± 2.93% for moderately to strongly stained NPC.


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Table I. Pearson's correlation coefficients between Ki67 immunolabelling and MT expression and zinc levels
 
Total zinc levels estimated in tumour tissues ranged from 2.96 to 6.12 µg/g dry wt tissue. Mean tissue zinc level in cancer tissues was significantly higher than in benign nasopharyngeal tissues (4.800 ± 0.4610 versus 2.889 ± 0.4045 µg/g dry wt tissue respectively; P = 0.0122). The mean zinc levels for the nuclear, membrane and cytosolic fractions in nasopharyngeal tumour tissues were 1.4840 ± 0.1489, 0.6286 ± 0.0789 and 0.3014 ± 0.0250 µg/mg protein, respectively. A significant positive correlation was noted between Ki67 labelling and total zinc content and nuclear zinc levels (Table IGo). In addition, a linear relationship was also observed between MT immunostaining and nuclear zinc level (Figure 2Go).



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Fig. 2. The relationship between MT immunohistochemical expression and nuclear zinc levels is shown together with the regression line and correlation coefficient.

 
The role of MT in relation to tumour development and progression has emerged as an important field of study in recent years (12,13). Cell proliferation is an important aspect of tumour growth, the other being apoptosis. In this study, we have shown that proliferative activity in NPC, a rapidly growing tumour (14) is directly proportional to MT expression. This is in accord with the findings of Hishikawa et al. (15) of a positive correlation between MT expression in squamous cell carcinoma of the oesophagus and staining for proliferative cell nuclear antigen. McCluggage and colleagues have also observed high MT expression in cervical lesions with high proliferative activity, namely Grade III cervical intra-epithelial neoplasms and invasive cervical squamous carcinoma (16). Bay and co-workers have reported higher levels of MT expression in the proliferative multiple layered epithelium of malignant serous and mucinous ovarian tumours compared with their benign counterparts (which have single epithelial layers) (10,17). The association of MT with proliferation has also been reported in breast cancer (18,19), colon carcinoma (18), renal cancer (20) and liver tumours (21). Besides proliferation, assessment of MT expression is known to provide prognostic information (22) and to be a marker of chemoresistance in tumours (23,24).

The mean tissue zinc level in NPC tissue was 1.7 times higher than that in benign nasopharyngeal tissue, concurring with the finding that zinc levels are generally higher in cancerous tissues (11,25,26). Total and nuclear zinc levels correlated with proliferative activity, indicating that zinc has a growth promoting effect on NPC. Another possible effect of zinc in the progression of NPC, a highly aggressive tumour, is that zinc has the capability to block apoptosis. Zinc supplementation is known to inhibit apoptotic death induced by diverse physical, chemical and immunological stimuli (27). The anti-apoptotic action of zinc has been attributed to its antioxidant properties (28), inhibition of Ca2+/Mg2+-dependent endonucleases (29) and, more recently, inhibition of interleukin 1ß converting enzyme/CED-3 proteases by modification of protein phosphatase or kinase activity (30).

The reason why we found most of the cellular zinc compartmentalized in the nucleus (62% compared with 26% in the membrane and 12% in the cytosolic fractions) could be that zinc not only regulates DNA and RNA synthesis, but is also found as finger domains in at least 300 DNA-binding proteins (31). The presence of nuclear zinc and the positive correlation of MT with nuclear zinc and proliferative activity may explain why MT, which is reported to be mainly located in the cytosol (32), is present predominantly in NPC cell nuclei. Preferential localization of MT has been previously postulated to be due to factors like tissue metal concentration, developmental stage of the tissues and enhanced cell proliferation (11,21,33). The nuclear MT phenotype has also been observed in human bladder tumour T24 cells in the exponential growth phase (34).

We have shown in this study that MT and zinc are correlated with cell proliferation in NPC. Thus far, factors known to influence cell proliferation in NPC include co-existent Epstein–Barr virus and Herpes virus infection (35), involvement of acidic fibroblast growth factor and epidermal growth factor (EGF) in autocrine growth stimulation (36) and overexpression of EGF receptors (37). This study represents one of the few attempts to study the concurrent relationship of MT and zinc with proliferative activity in human cancer tissues. Analysis of factors influencing tumour growth can provide insights into cancer cell biology that could potentiate the design of more efficacious cancer therapy (37).


    Notes
 
4 To whom correspondence should be addressed Email: antbaybh{at}nus.edu.sg Back


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Received May 2, 2000; revised June 26, 2000; accepted June 30, 2000.