ARTICLE |
Correspondence to: George G. Chen, Department of Surgery, Prince of Wales Hospital, U. of Hong Kong, Shatin, N.T. Hong Kong. E-mail: gchen@cuhk.edu.hk
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The interaction between the HPV (human papilloma virus) 16 E7 and other cell growth factors, such as p53 and NFB in laryngeal cancer is not clearly understood. The aim of this study was to examine the expression of these three proteins in tumor and non-tumor laryngeal tissues from patients with laryngeal squamous cell carcinoma. These three proteins were dominantly expressed in the nucleus and their levels were higher in the tumor tissue than in the non-tumor tissue, although the comparison between the tumor and non-tumor tissues of p53 staining did not reach significance. The intensity of the nuclear stain of E7 and p53 was stronger than that of p65, a subunit of NF
B. Correlation analysis revealed that there was a positive relationship between the level of HPV16 E7 and the expression of p65. The correlation between E7 and p53 was also significant, although to a lesser degree. The finding of nuclear localization of p65 suggests that NF
B is constantly activated in the laryngeal cancer cells, whereas the sequestration of p53 in the nucleus may represent a mutated form of p53, which is probably inactivated by HPV16 oncoproteins. In conclusion, this study suggests that the nuclear localization of NF
B and p53 may play a role in the development of human laryngeal squamous cell carcinoma infected with HPV16. (J Histochem Cytochem 51:533539, 2003)
Key Words:
laryngeal squamous cell, carcinoma, HPV16 E7, NFB, p53
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HUMAN PAPILLOMA VIRUS (HPV) is a small double-stranded DNA virus that is capable of infecting cutaneous and mucosal epithelium, resulting in a variety of pathological lesions. Laryngeal cancer is one such disease whose pathogenesis is related to HPV infection. Among the more than 100 different types of HPV identified, HPV16 is the most common high-risk virus. Its contribution to neoplastic progression is predominantly through the action of the viral oncoproteins E6 and E7. Expression of HPV16 E6 and E7 proteins is sufficient for the immortalization of primary human epithelial cells and induces histological abnormalities reminiscent of premalignant HPV-associated squamous intraepithelial lesions (
The E7 of HPV16 is a 98-amino acid oncoprotein localized predominantly in the nucleus. In cooperation with the ras oncogene, it is able to induce full transformation of rodent and human primary cells, which are carcinogenetic when injected into mice (B is one such molecule, with the ability to inhibit cell death and to maintain and promote the growth of cells (
B. In addition, the involvement of Bcl-2 family members in alteration of apoptosis in human laryngeal cancer may also provide a hint of an association of NF
B with the pathogenesis of laryngeal cancer (
B induces the expression of anti-apoptotic Bcl-2 (
B and the level of p53. Overexpression of p53 inhibits NF
B activity, and a high level of NF
B suppresses p53 transactivation (
B, information about this aspect in human laryngeal cancer is still lacking. The aim of the present study is to examine the expression of these three proteins in tumor tissue obtained from patients with laryngeal cancer and in corresponding non-tumor tissue from the same subjects.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Laryngeal tissue samples were collected from the operating theater as soon as the laryngectomy specimen was removed from the operative field. They were then snap-frozen and stored in liquid nitrogen for future immunohistochemical (IHC) analysis. Eighteen patients with laryngeal squamous cell carcinoma were analyzed and all patients were from the Chinese population in Hong Kong. In addition to tumor tissues, their corresponding non-tumor laryngeal tissues were obtained as controls. All tumor and non-tumor tissue specimens were confirmed by pathological examination. We obtained informed consent from all patients for subsequent use of their resected tissues. Anti-HPV16 E7, p53 and p65 (NFB) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and all were polyclonal antibodies. According to the information provided by the manufacturer, HPV16 E7 was not reactive with E7 from other types of HPV and the p53 antibody would recognize both wild-type and mutant p53 proteins.
Tissues stored in liquid nitrogen were thawed at room temperature (RT). After being thawed, they were fixed in a 10% neutral buffered formalin solution, underwent tissue processing, and were embedded in paraffin. Formalin fixation before embedding was less than 30 hr throughout, which is important for preserving the antigenic determinants analyzed in this study. Tissues were sectioned at 4-µm thickness. Immunostaining was performed on paraffin sections according to the standard procedure of an ABC kit from Vector Laboratories (Burlingame, CA). In brief, the staining methods were as follows. Tissue sections were deparaffinized and rehydrated through three changes of xylene and graded alcohol. After tissue sections were boiled in citrate-based antigen unmasking solution for 1 min and cooled in Milli-Q water, the endogenous peroxidase activity in the tissue sections was quenched with a 3% hydrogen peroxide solution for 5 min. A 1.5% normal blocking serum, supplemented with avidin solution (Avidin/Biotin blocking kit; Vector Laboratories) was used to block tissue sections for 30 min. Preparations were then incubated with a primary antibody overnight at 4C. The primary antibodies were prepared in 1.5% normal blocking serum supplemented with biotin solution from the Avidin/Biotin blocking kit and were used at a working dilution of 1:200. After tissue sections were washed with PBS, a biotinyl-labeled secondary antibody, horse anti-goat IgG, was applied for 30 min. After tissue sections were washed with PBS, ABC reagent (Avidin/Biotin kit; Vector Laboratories) conjugated with horseradish peroxidase was applied for 30 min. The staining of HPV16 E7, p53, and p65 antigen was visualized by either DAB or NovaRED substrate (Vector Laboratories). The reaction was stopped by rinsing the sections in tapwater. All incubations were done in a humidified environment at RT except where indicated in the text. Finally, sections were counterstained with Vector Gill's hematoxylin. After dehydration through graded alcohol and clearing with xylene, sections were mounted with DPX permanent mountant. Negative controls were prepared by using non-immune serum instead of the primary antibody. The specificity of the antibodies used was verified by corresponding blocking peptides, which were from the same source (Santa Cruz Biotechnology) as the antibodies. In some experiments, the relevant blocking peptides that specifically block the primary antibody were used to show the specificity of the antibody. The intensity of the antigen staining was scored according to the standard described in Table 1.
|
To detect apoptotic cells, in situ labeling of the 3'-end of the DNA fragments generated by apoptosis-associated endonucleases was performed using the DeadEnd apoptosis detection kit (TUNEL assay) from Promega (Madison, WI). Briefly, the sections, after being dewaxed in xylene and rehydrated in ethanol, were incubated with 20 µg/ml proteinase K at RT for 15 min. The slides were then incubated with a terminal transferase enzyme and biotinylated nucleotide mix at 37C for 60 min to allow the end-labeling reaction to occur. Endogenous peroxidase activity was blocked by treating the slides with 0.3% hydrogen peroxide in PBS, pH 7.2. Horseradish peroxidase-labeled streptavidin solution was then applied on the slides and incubated for 30 min at RT. After incubation the color was developed with the peroxidase substrate, hydrogen peroxide, and the stable chromogen diaminobenzidine. The slides were then mounted and examined with a light microscope. For control purposes, tissues treated with DNase I were used as a positive control. DNase I is an agent that can cause fragmentation of the chromosomal DNA, an early sign of apoptotic cells. Cells were defined as apoptotic if the entire nuclear area of the cell labeled positively. Apoptotic bodies were defined as small positively labeled globular bodies in the cytoplasm of the cells, which could be detected either singly or in groups. To estimate the apoptotic index that represented the percentage of apoptotic cells in a given area, apoptotic cells and bodies were counted in 10 high-power fields and this figure was divided by the number of cells in the same high-power fields.
All values were expressed as mean ± SEM. The relationships among variables were analyzed with the Wilcoxon test using the software StatView (Abacus Concept; Berkeley, CA). A p value of less than 0.05 was taken as statistically significant. Spearman correlation and linear regression were applied to determine the relationships among different markers.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of E7, p65, and p53
The E7 protein was detected in 59% of tumor tissues and was almost exclusively located in the nucleus (Fig 1A and Fig 2), with only two tissues being positive in both the nucleus and the cytoplasm. The level of nuclear E7 was significantly higher in the tumor tissue than in the non-tumor tissue (Fig 1A, Fig 1B, and Fig 3). The p65 protein was mainly detected in the nucleus (82% of the cases) in both tumor and non-tumor tissues (Fig 1C and Fig 2). As for the E7 protein, the expression of nuclear p65 protein was much higher in the tumor tissue than in the non-tumor tissue (Fig 1C, Fig 1D, and Fig 3). Eighty-two percent of cases were positive for p53 protein, which was found in the nucleus in most cases (Fig 1E and Fig 2). Its level in the nucleus was increased in the tissue samples from the tumor area compared with that from the non-tumor region (Fig 1E, Fig 1F, and Fig 3). However, the difference did not reach significance, and this was probably due to the large variation of its distribution among the tissue samples tested. When we compared the staining intensity of these three proteins in the nucleus, we found that the expression of both E7 and p53 was at a similar level and that both of them were much higher than p65 (Fig 1A1C and Fig 3). Our co-IHC staining of the E7 protein and the p53 proteins demonstrated that both could be co-localized in the nucleus, and in some cases they appeared to overlap in a given place in the nucleus, suggesting that they might bind each other (Fig 1G). A similar result was obtained for E7 and p65 (Fig 1H).
|
|
|
The Relationships Among Expression of E7, p65, and p53
To evaluate whether there was any link between the viral protein E7 and cell growth factors (p65 and p53), we performed Spearman correlation analysis, a nonparametric method, to quantitate the degree of linear association between two variables. We found a positive correlation between E7 and p65 in the tumor tissue (Fig 4A). To a lesser degree, there was also a correlation between E7 and p53 (Fig 4B). However, the data did not reveal a significant correlation between p53 and p65 (Fig 4C).
|
Apoptotic Index
Apoptotic cells, as detected by the DeadEnd apoptosis detection kit from Promega, were very rare in both tumor and non-tumor tissues (data not shown). There was also no significant connection between the apoptotic index and the viral protein E7 or cell growth factors (p65 and p53) detected in the present study. A significant number of cells became apoptotic in the positive control tissues that were treated with DNase I, which is known to cause fragmentation of the chromosomal DNA.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increasing evidence has suggested that NFB plays a role in the development of human cancers (
B in human laryngeal cancer is not clear, although there are limited publications addressing the association between HPV and NF
B in human cancer.
induced activation and nuclear localization of p65, a subunit of NF
B, in squamous cell carcinoma cell lines of human pharyngeal origin and that such an activation of NF
B prevented cell death.
B activities in studies of HPV6- and HPV11-related laryngeal papilloma tissue. To our knowledge, there is no report on the relationship and interaction between HPV16 E7 and NF
B in human laryngeal cancer tissues. In our present study, we demonstrated that the expression of p65 was significantly increased in the tumor tissue and that the expression was mainly confined to the nucleus, in which the E7 protein was preferentially housed. Overlapping of both proteins in the same nuclear location was found on many occasions, which probably suggests a potential interaction between them. In the inactivated condition, NF
B is located in the cytosol as a three-subunit complex consisting of two prototypical subunits of p50 and p65 and an inhibitory subunit called IkB (
B has been activated. It has been shown that the E7 protein accumulates mainly in the nucleus (
B promote the survival of cells and stimulate cell growth, the overlapping expression of NF
B and HPV16 E7 in the nucleus found in this study may indicate occurrence of cross-talk between them. E7 does not directly target various tumor-promoting genes but it does alter proliferation and cell growth via degrading tumor suppressor Rb, abrogating surviving protein p21, and enhancing anti-apoptotic Bcl-2 (
B is known to be a cellular signal in the Rb, p21, and Bcl-2 pathways. NF
B is required to induce Rb hyperphosphorylation and thus promote progression of G1 to 5. (
B in their nuclei can upregulate p21, probably via increasing the transcription (
B level is suggested to deter the binding of NF
B subunits to the regulatory sites in the Bcl-2 promoter region and to subsequently result in downregulation of Bcl-2 (
B may create a more favorable environment for the persistent HPV infection and subsequent oncogenesis (
B, which is in favor of our result that there is a positive correlation between the level of E7 and the expression of NF
B in the nucleus.
The frequency of HPV16 found in our samples was 59%. In Europe and North America, the rate of HPV infection in laryngeal or pharyngeal squamous cell carcinoma varies between 22% and 83% (
Extensive studies have been conducted on the interaction between HPV16 E6 and p53 and the effect of HPV16 E7 on Rb. However, publications of the relationship between E7 and p53 are limited. The p53 protein was detected in 82% of cases, and in the majority it was located in the nucleus, where the E7 protein was also found. The p53 nuclear import or retention is essential for its normal function in growth inhibition or induction of apoptosis (
Both p53 and NFB are well known for their roles in the control of cell proliferation and growth. Co-localization of p53 and HVP16 E7 in the nucleus may reflect an interaction between them, which may lead to the mutation of p53 and the sequestration of p53 in an area closed to the E7 protein. The nuclear localization of NF
B (p65) is a clear sign indicating that NF
B is constantly activated and such an activated state of NF
B may serve as a signal to relay the effect of the E7 oncogenic protein to other downstream molecules such as Rb, p21, and Bcl-2, whose levels are known to be abnormal in human laryngeal cancer (
![]() |
Footnotes |
---|
1 Abstract was presented at the 6th Joint Meeting of the Histochemical Society and the Japan Society of Histochemistry and Cytochemistry, University of Washington, Seattle, WA, July 1821, 2002.
Received for publication September 3, 2002; accepted November 19, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bosari S, Viale G, Roncalli M, Graziani D, Borsani G, Lee AK, Coggi G (1995) p53 gene mutations, p53 protein accumulation and compartmentalization in colorectal adenocarcinoma. Am J Pathol 147:790-798[Abstract]
Boyer SN, Wazer DE, Band V (1996) E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 56:4620-4624[Abstract]
Bren GD, Solan NJ, Miyoshi H, Pennington KN, Pobst LJ, Paya CV (2001) Transcription of the RelB gene is regulated by NF-kappaB. Oncogene 20:7722-7733[Medline]
Chen S, Fribley A, Wang CY (2002) Potentiation of tumor necrosis factor-mediated apoptosis of oral squamous cell carcinoma cells by adenovirus-mediated gene transfer of NF-kappaB inhibitor. J Dent Res 81:98-102
Dickens P, Srivastava G, Loke SL, Larkin S (1991) Human papillomavirus 6, 11, and 16 in laryngeal papillomas. J Pathol 165:243-246[Medline]
Duffey DC, Chen Z, Dong G, Ondrey FG, Wolf JS, Brown K, Siebenlist U et al. (1999) Expression of a dominant-negative mutant inhibitor-kappaBalpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo. Cancer Res 59:3468-3474
Duffey DC, CrowlBancroft CV, Chen Z, Ondrey FG, NejadSattari M, Dong G, Van Waes C (2000) Inhibition of transcription factor nuclear factor-kappaB by a mutant inhibitor-kappaBalpha attenuates resistance of human head and neck squamous cell carcinoma to TNF-alpha caspase-mediated cell death. Br J Cancer 83:1367-1374[Medline]
Feuillard J, Schuhmacher M, Kohanna S, AssoBonnet M, Ledeur F, JoubertCaron R, Bissieres P et al. (2000) Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes. Blood 95:2068-2075
Fujikawa K, Furuse M, Uwabe K, Maki H, Yoshie O (1994) Nuclear localization and transforming activity of human papillomavirus type 16 E7-beta-galactosidase fusion protein: characterization of the nuclear localization sequence. Virology 204:789-793[Medline]
Funk JO, Waga S, Harry JB, Espling E, Stillman B, Galloway DA (1997) Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 11:2090-2100
Gilmore TD, Koedood M, Piffat KA, White DW (1996) Rel/NF-kappaB/IkappaB proteins and cancer. Oncogene 13:1367-1378[Medline]
Greenfield I, Nickerson J, Penman S, Stanley M (1991) Human papillomavirus 16 E7 protein is associated with the nuclear matrix. Proc Natl Acad Sci USA 88:11217-11221[Abstract]
Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS, Jr (1999) NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 19:5785-5799
Hirvikoski P, Kellokoski JK, Kumpulainen EJ, Virtaniemi JA, Johansson RT, Kosma VM (1999) Downregulation of p21/WAF1 is related to advanced and dedifferentiated laryngeal squamous cell carcinoma. J Clin Pathol 52:440-444[Abstract]
Huang SM, McCance DJ (2002) Down regulation of the interleukin-8 promoter by human papillomavirus type 16 E6 and E7 through effects on CREB binding protein/p300 and P/CAF. J Virol 76:8710-8721
Huxford T, Malek S, Ghosh G (1999) Structure and mechanism in NF-kappa B/I kappa B signaling. Cold Spring Harbor Symp Quant Biol 64:533-540[Medline]
Jackel MC, Sellmann L, Dorudian MA, Youssef S, Fuzesi L (2000) Prognostic significance of p53/bcl-2 co-expression in patients with laryngeal squamous cell carcinoma. Laryngoscope 110:1339-1345[Medline]
Jung M, Dritschilo A (2001) NF-kappa B signaling pathway as a target for human tumor radiosensitization. Semin Radiat Oncol 11:346-351[Medline]
Knippschild U, Milne D, Campbell L, Meek D (1996) p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage. Oncogene 13:1387-1393[Medline]
Kurland JF, Kodym R, Story MD, Spurgers KB, McDonnell TJ, Meyn RE (2001) NF-kappaB1 (p50) homodimers contribute to transcription of the bcl-2 oncogene. J Biol Chem 276:45380-45386
Lee KC, Crowe AJ, Barton MC (1999) p53-mediated repression of alpha-fetoprotein gene expression by specific DNA binding. Mol Cell Biol 19:1279-1288
Ma XL, Ueno K, Pan ZM, Hi SZ, Ohyama M, Eizuru Y (1998) Human papillomavirus DNA sequences and p53 over-expression in laryngeal squamous cell carcinomas in Northeast China. J Med Virol 54:186-191[Medline]
Mansur CP, Androphy EJ (1993) Cellular transformation by papillomavirus oncoproteins. Biochim Biophys Acta 1155:323-345[Medline]
Massimi P, Banks L (1997) Repression of p53 transcriptional activity by the HPV E7 proteins. Virology 227:255-259[Medline]
Mizokami H, Sawatsubashi M, Tokunaga O, Shin T (1999) Loss of retinoblastoma protein expression in laryngeal squamous cell carcinoma. Mod Pathol 12:47-53[Medline]
Pei XF, Sherman L, Sun YH, Schlegel R (1998) HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium. Carcinogenesis 19:1481-1486[Abstract]
Pennington KN, Taylor JA, Bren GD, Paya CV (2001) IkappaB kinase-dependent chronic activation of NF-kappaB is necessary for p21(WAF1/Cip1) inhibition of differentiation-induced apoptosis of monocytes. Mol Cell Biol 21:1930-1941
Phelps WC, Yee CL, Munger K, Howley PM (1988) The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A. Cell 53:539-547[Medline]
Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129-1136[Medline]
Seavey SE, Holubar M, Saucedo LJ, Perry ME (1999) The E7 oncoprotein of human papillomavirus type 16 stabilizes p53 through a mechanism independent of p19(ARF). J Virol 73:7590-7598
Shao J, Fujiwara T, Kadowaki Y, Fukazawa T, Waku T, Itoshima T, Yamatsuji T et al. (2000) Overexpression of the wild-type p53 gene inhibits NF-kappaB activity and synergizes with aspirin to induce apoptosis in human colon cancer cells. Oncogene 19:726-736[Medline]
Smith EM, Summersgill KF, Allen J, Hoffman HT, McCulloch T, Turek LP, Haugen TH (2000) Human papillomavirus and risk of laryngeal cancer. Ann Otol Rhinol Laryngol 109:1069-1076[Medline]
Stewart ZA, Pietenpol JA (2001) p53 signaling and cell cycle checkpoints. Chem Res Toxicol 14:243-263[Medline]
Suzuki T, Tsutsumi K, Nakajima T, Hara F, Kikuchi K, Yasumoto S (1996) Spontaneous mutation of p53 gene in human papillomavirus type 16 E7-immortalized human laryngeal epithelial cells. Acta Otolaryngol Suppl 522:94-98[Medline]
Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, Mizuno T et al. (1999) Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkappaB activation in primary hippocampal neurons. J Biol Chem 274:8531-8538
Tamatani M, Mitsuda N, Matsuzaki H, Okado H, Miyake S, Vitek MP, Yamaguchi A et al. (2000) A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-kappaB and Bcl-2. J Neurochem 75:683-693[Medline]
Vancurova I, Wu R, Miskolci V, Sun S (2002) Increased p50/p50 NF-kappaB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue. J Virol 76:1533-1536
Webster GA, Perkins ND (1999) Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol 19:3485-3495
Whisler LC, Wood NB, Caldarelli DD, Hutchinson JC, Panje WR, Friedman M, Preisler HD et al. (1998) Regulators of proliferation and apoptosis in carcinoma of the larynx. Laryngoscope 108:630-638[Medline]