Abstract
Acetylcholinesterase (AChE) and human herpesvirus type 8 (HHV-8) antigens were studied in tissue sections from 56 squamous cell carcinomas (SCC) and five retinoblastomas (Rb). Approximately 62.5% of SCC and 80% of Rb showed positive staining for AChE. AChE staining in tumors was much higher than in normal control tissue. However, only 21.4% of SCC and 60% of Rb contained HHV-8 antigens. Of the 56 SCC, 17.9% were positive for both AChE and HHV-8 antigens, whereas 60% Rb were positive for both markers. The co-existence of AChE and HHV-8 antigens may play a role in the development of SCC and Rb. A possible mechanism for the development of these tumors is discussed.
Acetylcholinesterase (AChE) plays a key role in terminating neurotransmitter activity at cholinergic synapses. A single gene mapped at 7q22 chromosal position encodes AChE in muscles and nerves (1), homopoietic cells (2), embryonic tissues (3), germ cells (4) and a variety of tumors (5). A growing body of evidence suggests that this enzyme plays a very important role in tumorogenesis. The gene for AChE has been shown to be amplified, mutated and/or aberrantly expressed in a variety of human tumors (6). The aberrant expression of this gene can be manifested at the DNA, mRNA or protein level (6-8). These alterations and elevation in AChE gene products are seen in a variety of tumors despite the fact that AChE is not present in their normal counterparts. Currently it is considered to be a useful tumor marker (5-14). High AChE activity is reported in brain, breast, lung and ovarian tumors and leukemia (6-17). Neuroblastoma cells genetically engineered to overexpress AChE, developed tumors in vivo at a notably greater rate compared to transfection controls. Similar observations have been reported for glioma cell lines (18, 19). The AChE peptide contains S/T-P-X-Z motif which is one of the many substrates for cdc-2 kinases. Phosphorylation of retinoblastoma (Rb) protein by cdc-2 kinases may be the molecular mechanism linking AChE with tumor cell proliferation (6-16).
Human herpesvirus type 8 (HHV-8) is a lymphotropic and oncogenic virus (20-24). HHV-8 DNA sequences have been found in 50% small cell carcinoma (SCC) cases (22-26). HHV-8 expresses unique viral proteins such as kaposin and latent nuclear antigen (LNA-1) in malignant cells (21, 27). LNA-1 seems to act as a transcription cofactor which may target Rb protein to disrupt the E2F transcriptional regulatory pathway. On the other hand, kaposin may inactivate the P53 regulatory pathway (23, 27).
SCC and retinoblastoma are relatively common in Saudi Arabia and about 12% of the adult population is seropositive for HHV-8. Based on these observations, an attempt was made to determine the frequency of co-existence of AChE and HHV-8 antigens in 56 SCC and 5 retinoblastomas. A possible molecular mechanism involved in the induction of SCC and retinoblastoma in the presence of AChE and HHV-8 antigens is discussed.
Materials and Methods
Fresh SCC and retinoblastoma tissues were obtained at the time of surgery at the King Khaled Eye Specialist Hospital (KKESH), Riyadh, Saudi Arabia. Tumor tissues were collected randomly from patients with confirmed SCC or retinoblastoma. Normal retinal tissues were collected from healthy donors. Controls for HHV-8 antigens were cells from primary effusion lymphoma with HHV-8 infection (PEL) which were kindly provided by Dr. Ethel Cesarman of Cornell University, New York, USA. Tissues and cell pellets were fixed in 10% buffered formalin, embedded in paraffin and sectioned. Four micron sections were plated on polylysin-coated slides (2 sections/slide) and used for immunoperoxidase staining. One slide from each sample was stained with hematoxylin to study tissue morphology.
Immunoperoxidase staining. Deparaffinized tissue sections with appropriate positive and negative controls were incubated with 0.3% hydrogen peroxide and rinsed twice with phosphate-buffered saline (PBS). Slides were then treated with pepsin (13 mg/48 ml in 0.2 N HCl) for ten minutes at room temperature and washed twice in PBS. The sections were incubated with 10% goat serum (Chemicon Temecula; CA, USA) at 37°C for 30 minutes. Monoclonal antibodies to AChE (Chemicon) and HHV- LNA-1 (ABI, Columbia MD, USA) were diluted (1:100) and applied to tissue sections and incubated overnight at 37°C in a moist chamber. The sections were rinsed with PBS containing 0.2% Triton® X-100 and incubated for three hours at 37°C with a biotin-labeled secondary antibody (goat antimouse immunoglobulin). After rinsing with PBS with 0.2% Triton® X-100, the slides were then incubated for three hours at 37°C with the avidin-biotin-horseradish peroxidase complex and sites of peroxide activity were visualized after incubation with 3-amino-9-ethylcarbazole in 0.01% hydrogen peroxide (ABC Kits, Foster City, CA, USA). The slides were counterstained for two minutes in Mayer's hematoxylin, rinsed in water and then six drops of crystal mount (Fisher Scientific, Springfield, NJ, USA) were applied to each section and slides were then baked at 60°C for 30 minutes. The sections were mounted in Permount (Fisher Scientific) and examined (28).
Results
Fifty-six SCC tissues and five Retinoblastoma tissues were analyzed by using immunohistochemical staining for the presence or absence of AChE and HHV-8 antigens. Positive and negative controls were normal retinal sections incubated with and without AChE antibody respectively (Figure 1 a, b).
Approximately 62.5% of SCC gave clear and strong positive staining for AChE; the rest of the samples showed weak diffuse staining (Table I). Normal retina was used as positive control for the detection of AChE. Two representative photographs of positive staining in SCC and Rb are given in Figure 1 c and d along with the positive and negative controls.
Approximately 21.4% of the 56 samples were positive for HHV-8 antigen LNA-1 (Table I). Positive controls for HHV-8 were cells from primary effusion lymphoma with HHV-8 infection (PEL). When incubated with HHV-8 antibody, these cells showed bright nuclear staining. Representative photographs are shown in Figure 2 a and b. Only 17.9% of the 56 samples contained both AChE and HHV-8 antigen (Table I).
Five retinoblastomas were also analyzed for the presence or absence of AChE and HHV-8 antigen. Approximately 80% of Rb were positive for AChE and 60% stained positively for HHV-8 antigen (Table I); however, 60% of retinoblastomas were positive for both AChE and HHV-8 antigen (Table I).
Discussion
All SCC studied in this study were positive for AChE; however, only 62.5% cases showed very strong AChE staining. Similar results were reported previously in ovarian, brain, lung and breast tumors and leukemia (6-17).
Distribution of AChE and HHV-8 antigens in squamous cell carcinomas (SCC) and retinoblastomas (Rb).
Approximately 80% of retinoblastomas analyzed also showed positive staining for AChE. HHV-8 antigen LNA-1 was present in 21.4% of SCC and 60% of retinoblastomas. Only 17.9% of SCC and 60% retinoblastoma analyzed contained both AChE and HHV-8 antigen.
To our knowledge, no reports are available showing HHV-8 and/or AChE presence in retinoblastomas; however, 50% of SCC studied previously by other investigators contained HHV-8 antigen (21, 22, 27).
Our observations suggest that HHV-8 may possibly be involved in the development of these tumors through inactivation of tumor suppressor genes such as P53 and Rb protein and activation of AChE.
The regulatory pathways existing in normal and tumor cells are outlined in Figure 3.
Mutational deregulation of the cell cycle is a hallmark of cancer. The protein product of the retinoblastoma gene (Rb) is the prototype tumor suppressor gene by virtue of its central role in regulating the cell cycle. P53 is a key regulator of cell cycle and apoptosis control, and is also mutated in more than half of human cancer cases.
These two pathways form an interconnected tumor suppressor network. In most types of cancer, the Rb pathway is disrupted such that Rb is inactivated, either by mutation of the Rb gene or by functional inactivation of the Rb gene by hyperphosphorylation of the protein. Cellular P53 levels are very low in normal cells, due to an inhibitory interaction with HDM2 that targets P53 for degradation. In cancer cells, P53 undergoes post-translational modifications which abolish its ability to bind to HDM2 and be inhibited by it (29-31).
In normal cells, P53 and Rb proteins (Rb bound to E2F) are active and reduce the cell cycle rate which suppresses tumor development. In tumors, the P53 and Rb genes are inactive either because of mutations or because P53 is hydrolyzed and the Rb-E2F complex is broken due to hyperphosphorylation or interaction with certain proteins from SV40 large T antigen, adenovirus E1A, HPV, E6 and E7 proteins, HHV-8 kaposin and LNA-1 proteins and P33 of Bacculovirus (32-36). Certain cellular proteins such as AChE may also affect the cdc kinase activities and reverse the regulatory pathway which exists in normal cells (6, 16).
Immunohistochemical staining for AChE (×40 magnification). a, Normal retina (negative control), retinal sections stained in the absence of AChE antibody. b, Normal retina (positive control), retinal sections stained in the presence of AChE antibody. c, SCC tissue section positively stained in the presence of AChE antibody. d, Rb tissue section positively stained in the presence of AChE antibody.
Immunohistochemical staining for HHV-8 antigen (×40 magnification). a, PEL (negative control) stained in the absence of HHV-8 antibody. b, PEL (positive control) stained in the presence of HHV-8 antibody. c, SCC tissue section stained in the presence of HHV-8 antibody. d. Rb tissue section stained in the presence of HHV-8 antibody.
Regulatory pathways in normal and tumor cells.
As has been reported earlier (21-27), along with AChE, the HHV-8 kaposin protein may inactivate P53 and/or LNA-1 may break up the Rb-E2F complex, allowing the development of tumors through the regulatory pathways shown in Figure 3.
In our study, although the results are preliminary, they do indicate that the increased levels of AChE in SCC and retinoblastoma may activate cdc kinases, which in turn may increase the phosphorylation of Rb protein and hence activate the cell cycle rate via interruption of P53 regulatory pathway (tumor cells, Figure 3 a). In some tumors, the presence of HHV-8 LNA-1, however, suggests that HHV-8 may interrupt the Rb-E2F regulatory pathway in normal cells allowing them to become cancerous cells (tumor cells, Figure 3 b). Similar mechanisms have been proposed and reported in the development of other tumors (6-16, 21-23, 27).
Since AChE appears to play developmentally important roles in multiple cell types, the amplification and overexpression of the AChE gene may also affect the progression of various tumors. If the mechanism is confirmed, AChE may be an excellent target for tumor treatment using specific inhibitors of AChE and cdc kinases, as well as antisense mRNA treatment strategy (37-44).
Acknowledgements
The positive controls for HHV-8 were kindly provided by Dr. Ethel Cesarman of Cornell University, New York, U.S.A. Funds for this project were provided by the Research Department at KKESH. The excellent technical assistance of Khitam El-Hati is gratefully acknowledged. We also thank Rasha Al-Shubaian for her help in the preparation of this manuscript.
- Received January 27, 2009.
- Revision received June 4, 2009.
- Accepted July 2, 2009.
- Copyright © 2009 The Author(s). Published by the International Institute of Anticancer Research.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.