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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.28 n.2 Temuco jun. 2010

http://dx.doi.org/10.4067/S0717-95022010000200044 

Int. J. Morphol.,28(2):609-613, 2010.

Death-Associated Protein Kinase is Underexpressed in High-Grade Oral Squamous Cell Carcinoma

La Expresión de la Proteína Quinasa Asociada a Muerte Celular (DAP Quinasa) está Reducida en Carcinoma de Células Escamosas Oral de Alto Grado

*Ismário Silva de Meneses; *Rafael Reis de Souza; **Verónica de Lourdes Sierpe Jeraldo; **Danielle Rodrigues Ribeiro Cavalcante; **,***Francisco Prado Reis & **,***Ricardo Luiz Cavalcanti de Albuquerque Júnior

* School of Dentistry, University Tiradentes, Aracaju, Sergipe, Brazil

** Post-Graduating Program in Health and Environment, University Tiradentes, Aracaju, Sergipe, Brazil

***Laboratory of Morphology and Structural Biology, Science and Technology Institute, Tiradentes University, Aracaju, Sergipe, Brazil.

 

Correspondence to:


Summary: An immunohistochemical analysis of 40 cases of oral squamous cell carcinoma was performed to evaluate the relationship between the expression pattern of death-associated protein kinase (DAPk) positive cells with the histological malignancy grading of these lesions. According to our results, eleven cases (27.5%) were high-grade malignancy tumours and 29 (72.5%) were low-grade ones. We found that 92.86% of the low-grade tumours were positive to anti-DAP kinase antibody whereas only 7.14% of the high-grade tumours presented positivity, and this difference was statistically significant (p<0.01). Sixteen (55.2%) of the low-grade carcinomas exhibited moderate immunoreactivity whereas ten cases (34.5%) showed weak staining and three cases (10.3%) were negative tumours. Immunostaining was lacking in nine (81.8%) of the high-grade carcinomas and "weak" in the two (18.2%) remaining cases. Thus, DAPk expression is significantly decreased in high-grade oral carcinomas, and evidences indicate that it might be related to the severity of cytological atypia.

KEY WORDS: Immunohistochemistry; DAP kinase; Apoptosis; Squamous cell carcinoma.


RESUMEN: Fue realizado un análisis inmunohistoquímico de 40 casos de carcinoma oral de células escamosas para evaluar la relación entre el patrón de expresión de la proteína quinasa (DAPK) asociada a la muerte celular y la clasificación histológica de malignidad de estas lesiones. Según nuestros resultados, 11 casos (27,5%) eran tumores de alto grado de malignidad y 29 (72,5%) de bajo grado. Se encontró que 92,86% de los tumores de bajo grado de malignidad fueron eran positivos para anticuerpos anti-DAP-quinasa, mientras que sólo 7,14% de los tumores de alto grado presentaron positividad, esta diferencia fue estadísticamente significativa (p <0,01). En 16 casos (55,2%) los carcinomas de bajo grado de malignidad mostraron inmunorreactividad moderada mientras que 10 casos (34,5%) mostraron una tinción débil y 3 casos (10,3%) fueron negativos. La inmunotinción estuvo ausente en 9 (81,8%) de los carcinomas de alto grado y "débil" grado de malignidad en los 2 (18,2%) casos restantes. Así, la expresión DAPK se redujo significativamente en los carcinomas orales de alto grado y las evidencias indican que podría estar relacionado con la severidad de la atipia citológica.

Palabras clave: Inmunohistoquimica; DAP kinasa; Apoptosis; Carcinoma de células escamosas.


Introduction

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour occurring in the oral cavity. In the majority of the cases, it is diagnosed at an advanced stage and consequently present with a poor prognosis (Biazevic et al., 2006; Borges et al., 2008). In the last few years relevant advancements in the diagnosis and treatment of oral cancer has been observed. Nevertheless, despite this recent improvement, there are still many difficulties in evaluating the prognosis of OSCC. Thus, various studies attempting to establish the role of protooncogenes, antioncogenes and apoptosis-regulating genes in the tumoral progression have recently been performed in order to find more significant information to evaluate and predict the biological behavior of this neoplasm (Albuquerque et al., 2003).

The Death-associated protein kinase (DAP kinase) is a 160kDa cytoskeletal-associated calcium/calmodulin-dependent serine/threonine kinase which plays an important role in programmed cell death (Chen et al., 2006; Zalckvar et al., 2009). Overexpression of DAP kinase has been demonstrated to induce apoptosis in various cell line (Yamamoto et al., 2002; Pelled et al., 2002; Pulling et al., 2004). On the other hand, research has demonstrated that DAP kinase protein expression is frequently reduced or absent in tumour cell lines (Raveh & Kimchi, 2001; Bai et al., 2004). In addition, low expression of this protein, particularly in response to hypermethilation, has been associated to high invasiveness or metastatic potential of malignat neoplastic cells (Simpson et al., 2002; Lehmann et al., 2002; González-Gómez et al., 2003; Voso et al., 2004; Lévy et al., 2004).

There are only few reports looking at the possible role of DAP kinase in the pathogenesis of oral or head and neck cancer (Rosas et al., 2001; Ogi et al., 2002). Thus the goal of this study is to investigate the relation between the immunohistochemical expression of DAP kinase and the histological grading of malignancy in squamous cell carcinoma of the oral mucosa.

Material and method

Forty cases of OSCC were retrieved from the files of the Oral Pathology Service of the School of Dentistry of the University Tiradentes (Aracaju, Brazil). The tumors were graded as high or low score according to the criteria of histological malignancy grading established by Bryne et al. (1989). For immunohistochemical reactions, 3mm thick sections were prepared from formalin-fixed paraffin-embedded surgical specimens. Immunohistochemical staining was carried out using the Labelling Streptoavidin Biotin method (LSAB) (Large Volume Detection System antipolyvalent, Dako, LSAB2 kit peroxidase, Dako Corporation, Capinteria, CA, USA). The sections were deparaffinized in toluene and submitted to antigen recovery by immersion of the slides in citrate buffer, 0.01M, pH 6.0 treated in microwave oven (three cycles for five minutes each at 650W). After rehydratation in phosphate buffered saline (PBS, pH 7.2), the sections were incubated with the primary antibody anti ­ DAP kinase (clone V55, Abgent, CA, USA, 1:50, 18h), followed by biotinylated antirabbit immunoglobulins (Dakopatts) and finally with streptoavidin ­ peroxidase conjugate (Detection Kit ­ peroxidase/DAB,Dakopatts). The last two incubations were performed for 30 min at room temperature. The reaction was revealed with 0.03% diaminobenzidine (Sigma) in buffered solution for 12 min and then the sections were counterstained with Meyer's haematoxilin. Non-specific reactivity of the antibodies was checked by omitting the primary antibody.

Two observers evaluated all slides independently, and the immunostaining pattern of the antibody anti-DAP kinase was analyzed by light microscopy, considering the following parameters: (+++), when more than 50% of the tumour cells were labeled, (++) when 10 to 50% were positive; and (+) when less than 10% of positive cells could be seen. Carcinomas lacking immunostained cells were considered negative (-). After the immunohistochemical analysis, these data were correlated with the malignancy histological grading of the tumours. Significance was statistically determined using c2 statistics. A p-value < 0.05 was regarded as significant.

Results

Morphological analysis showed that eleven cases (27.5%) were high-grade malignancy tumours and twenty-nine (72.5%) were low-grade malignancy tumors. Positive cytoplasmatic immunolabelling could be verified in twenty-eight cases (70.0%). Two (7.14%) of the twenty-eight DAP kinase-positive carcinomas were high-grade tumors whereas twenty-six (92.8%) were low-grade tumours. On the other hands, nine (75%) of the twelve DAP kinase-negative carcinomas were high-grade tumours and three of them low-grade lesions (Fig. 1).

Fig. 1. Expression of death-associated protein kinase correlated with the
histological malignancy grading of oral squamous cell carcinoma low and high grade.

Sixteen (55.2%) of the low-grade carcinomas exhibited moderate immunoreactivity whereas ten cases (34.5%) showed weak staining. Three cases (10.3%) were negative tumours. Immunostaining was lacking in nine (81.8%) of the high-grade carcinomas and weak in the two remaining cases (18.2%) (Table I, Fig. 2). Our results indicated an inverse relationship between DAP kinase immunoreactivity and the histological grading of tumoral malignancy (p<0.01). Positive staining was found in isolated cells or small cell groups within larger tumoral islands and sheets. Immunoreactivity showed to be more intense in cells adjacent to keratinization areas (diskeratotic cells and keratin pearls) but extensively absent in areas of severe cytological atypia.

Fig. 2. (a) Low-grade squamous cells carcinoma presenting islands of keratinizing
well-differentiated epithelial cells (HE, 400x). (b) High-grade squamous cells carcinoma
exhibiting small nests of poorly differentiated pleomorphic epithelial cells (HE, 400x). (c)
Strong immunohistochemical positivity for DAP kinase in the cytoplasm of tumoral cells in
a low-grade squamous cells carcinoma (LSAB, 400x). (d) Lack of immunostaining in tumoral
cells of a high-grade squamous cells carcinoma (LSAB, 400x).

 

Discussion

Various studies have demonstrated that DAPk gene transcription is involved in apoptosis phenomenon and therefore may play a relevant role in suppressing oncogenic transformation (Raveh & Kimchi, 2001; González-Gómez et al.; Bai et al.). DAPk protein downregulates the expression of integrins, and consequently blocks cellular adhesion, thereby activating a particular pathway of apoptosis known as anoikis (Wang et al., 2002). Nevertheless, the capacity to promote apoptosis through DAPk pathway is frequently lost in anoikis-resistent cells, resulting in anchorage independence, a hallmark of malignant tumour cells closely associated to metastasis (Frisch & Screaton, 2001). Loss of DAP kinase RNAm and protein expression was found in human cancer, often as a result of silencing by DNA hypermethylation (Lehmann et al.; Simpson et al.; Pulling et al.). In addition, DAPk appears to be underexpressed in metastatic cell lines (Lehmann et al.; Simpson et al.; Pulling et al.).

We investigated the relationship between the immunohistochemical expression of DAPk and the histological grading of malignancy in forty cases of squamous cell carcinoma of the oral mucosa. According to our results, the intensity of the immunohistochemical expression of DAPk is inversely proportional to the histological malignancy grading of oral squamous cell carcinoma. This theory is supported by the fact that immunoreactivity was more intense in cells adjacent to keratinizatinized areas, hallmarks of well-differentiated tumors, but lacked in areas of severe cytological atypia. Likewise, loss of DAPk expression has been reported in a wide range of tumour types (Raveh & Kimchi; Lehmann et al.; Simpson et al.; Bai et al.). Surprisingly, three cases of low-grade tumours failed in expressing DAPk. However, it must be emphasized that all those tumors presented marked cytological atypia and minimum keratinization, despite they were classified as low-grade tumors. Thus, these findings seem to point at a link between the phenomenon of "dedifferentiation" and loss of DAPk expression in OSCC. Nevertheless, the mechanism responsible for silencing DAPk gene is not completely clear yet. It has been suggested that hypermethylation of CpG islands (segments of DNA within the gene promoter characterized by extensive repetition of residues of cytosine followed by residues of guanine) might suppress gene transcription (Lehmann et al.; González-Gómez et al.; Pulling et al.), but its role in the carcinogenesis of oral carcinoma is still unknown. In addition, the relationship between loss of DAK expression and worse overall survival prognosis was recently demonstrated in breast cancer (Lévy et al.) but further investigations are necessary to assess the role of DAPk in evaluating the survival rates and prognosis of carcinomas of the oral mucosa.

In conclusion, we have found that DAPk expression decreased significantly in high-grade oral carcinomas, which means that this expression might correlate with the histological malignancy grading of these tumour.

 

References

Albuquerque, R. L. Jr.; Miguel, M.C.; Costa, A. L. & Souza, L. B. Correlation of C-erbB-2 S-100 expression with the malignancy grading and anatomical site in oral squamous cell carcinoma. Int. J. Exp. Path., 84:259-65, 2003.         [ Links ]

Bai, T.; Tanaka, T.; Yukawa, K.; Maeda, M. & Umesaki, N. Reduced expression of death associated protein kinase in human uterine and ovarian carcinoma cells. Oncol. Rep., 11:661-5, 2004.         [ Links ]

Biazevic, M. G.; Castellanos, R. A.; Antunes, J. L. & Michel-Crosato, E. Trends in oral cancer mortality in the city of São Paulo, Brazil, 1980-2002. Cad. Saúde Pública, 22:2105-14, 2006.         [ Links ]

Borges, F. T.; Garbin, C. A.; Carvalhosa, A. A.; Castro, P. H. & Hidalgo, L. R. Oral cancer epidemiology in a public laboratory in Mato Grosso State, Brazil. Cad. Saúde Pública, 24:1977-82, 2008.         [ Links ]

Bryne, M.; Koppang, H. S.; Lilleng, R.; Stene, T.; Bang, G. & Dabelsteen, E. New malignancy grading is a better prognostic indicator than Broders' grading in oral squamous cell carcinomas. J. Oral Pathol. Med., 18:432-7, 1989.         [ Links ]

Chen, R. H.; Wang, W. J.; & Kuo, J. C. The tumor suppressor DAP-kinase links cell adhesion and cytoskeleton reorganization to cell death regulation. J. Biomed. Sci., 13:193-9, 2006.         [ Links ]

Frisch, S. M. & Screaton, R. A. Anoikis mechanisms. Curr. Opin. Cell Biol., 13:555-62, 2001.         [ Links ]

González-Gómez, P.; Bello, M. J.; Alonso, M. E.; Lomas, J.; Arjona, D.; Amiñoso, C.; De Campos, J. M.; Isla, A.; Gutierrez, M. & Rey. J. A. Frequent death-associated protein-kinase promoter hypermethylation in brain metastases of solid tumors. Oncol. Rep., 10:1031-3, 2003.         [ Links ]

Lehmann, U.; Celikkaya, G.; Hasemeier, B.; Länger, F. & Kreipe, H. Promoter Hypermethylation of the Death ­ Associated Protein Kinase Gene in Breast Cancer is Associated with the Invasive Lobular Subtype. Cancer Res., 62:6634-8, 2002.         [ Links ]

Lévy, D.; Plu-Bureau, G.; Decroix, Y.; Hugol, D.; Rostène, W.; Kimchi, A. & Gompel, A. Death ­ Associated Protein Kinase Loss of Expression is a New Marker for Breast Cancer Prognosis. Clin. Cancer Res., 10:3124-30, 2004.         [ Links ]

Ogi, K.; Toyota, M.; Ohe-Toyota, M.; Tanaka, N.; Noguchi, M.; Sonoda, T.; Kohama, G. & Tokino, T. Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma. Clin. Cancer Res., 8:3164-71, 2002.         [ Links ]

Pelled, D.; Raveh, T.; Riebeling, C.; Fridkin, M.; Berissi, H.; Futerman, A. H. & Kimchi, A. Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons. J. Biol. Chem., 277:1957-61, 2002.         [ Links ]

Pulling, L. C.; Vuillemenot, B. R.; Hutt, J. A.; Devereux, T. R. & Belinsky, S. A. Aberrant promoter hypermethylation of the death-associated protein kinase gene is early and frequent in murine lung tumors induced by cigarette smoke and tobacco carcinogens. Cancer Res., 64:3844-8, 2004.         [ Links ]

Raveh, T. & Kimchi, A. DAP kinase ­ a proapoptotic gene that functions as a tumor suppressor. Exp. Cell Res., 264:185-92, 2001.         [ Links ]

Rosas, S. L. B.; Koch, W.; da Costa Carvalho, M. G.; Wu, L.; Califano, J.; Westra, W.; Jen, J. & Sidransky, D. Promoter Hypermethylation Patterns of p16, MGMT and DAP-kinase in Tumors and Saliva of Head and Neck Cancer Patients. Cancer Res., 61:939-42, 2001.         [ Links ]

Simpson, D. J.; Clayton, R. N. & Farrel, W. E. Preferential loss of Death Associated Protein Kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion. Oncogene, 21:1217-24, 2002.         [ Links ]

Voso, M. T.; Scardocci, A.; Guidi, F.; Zini, G.; Di Mario, A.; Pagano, L.; Hohaus, S. & Leone, G. Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes. Blood, 103:698-700, 2004.         [ Links ]

Wang, W. J.; Kuo, J. C.; Yao, C. C. & Chen, R. H. DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. J. Cell Biol., 159:169-79, 2002.         [ Links ]

Yamamoto, M.; Hioki, T.; Ishii, T.; Nakajima-Iijima, S. & Uchino, S. DAP kinase activity is critical for C(2)-ceramide-induced apoptosis in PC12 cells. Eur. J. Biochem., 269:139-47, 2002.         [ Links ]

Zalckvar, E.; Berissi, H.; Mizrachy, L.; Idelchuk, Y.; Koren, I.; Eisenstein, M.; Sabanay, H.; Pinkas-Kramarski, R. & Kimchi, A. DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy. EMBO Rep., 10:285-92, 2009.         [ Links ]

Correspondence to:

Ismário Silva de Meneses
School of DentistryUniversity Tiradentes
Aracaju, Sergipe
BRAZIL

Email: ismariok@hotmail.com

Received: 22-02-2010
Accepted:15-04-2010

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