Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with decreased risk for basal cell carcinoma.

The incidence of basal cell carcinoma (BCC) is significantly reduced in individuals treated with inhibitors of angiotensin I-converting enzyme (ACE) that produces angiotensin II. The objective of this study was to investigate the possible association of a functional polymorphism in the ACE gene, which affects its transcription, with risk for BCC. In DNA samples of 92 patients with BCC and 103 healthy controls of Greek origin and comparable age and gender, we studied the ACE gene insertion/deletion (I/D) polymorphism. Fisher's exact test was used for comparison of allele and genotype frequencies between the control and patients' groups. The detected low expression I allele frequency in the group of BCC patients was significantly decreased compared to controls (15.8 vs. 31.1 %, respectively; P = 0.001). ID heterozygotes exhibited 3.06 times lower BCC risk, compared with DD homozygotes (P = 0.001; OR = 0.327, 95 % CI = 0.174-0.615). The protective role of I allele was particularly prominent in women (P = 0.007, OR = 0.299, 95 % CI = 0.125-0.716), while for men it exhibited a marginal level (P = 0.041). These findings indicate that the low expression ACE I allele carriers have a decreased risk for BCC. The protective effect of the ID genotype against BCC may be explained by a possible underlying mechanism involving the effect of produced angiotensin II levels on its receptors due to putatively different binding affinity.

A common 9 bp deletion in the ataxia-telangiectasia-mutated gene is not associated with oral cancer.

BACKGROUND: The ataxia-telangiectasia-mutated gene (ATM) product is a well-characterized tumour suppressor that plays a key role in the maintenance of genomic stability. Given the fact that the loss of heterozygosity at the ATM locus is common in head and neck tumours, we investigated the possible association of 7636del9, which is the most frequent ATM deletion, with risk for oral cancer. PATIENTS AND METHODS: The 7636del9 9nt deletion was investigated in DNA samples of 67 German and Greek patients with oral cancer and 57 healthy controls of equivalent ethnicity, age and gender, by polymerase chain reaction (PCR) followed by electrophoretic analysis. RESULTS: The anticipated deleted sequence was not detected in any of the DNA samples of oral cancer patients or controls. CONCLUSION: The findings of the present study indicated no association of the most common mutation in the ATM gene with risk for oral cancer.

Diabetes enhances the expression of H-ras and suppresses the expression of EGFR leading to increased cell proliferation.

EGFR kinase activity triggers numerous signaling pathways, such as the Ras/Raf/MAPK cascade, leading to the activation of various mitogen activated protein kinases, which are implicated in cell proliferation through induction of several genes, including c-fos. The possible effect of diabetes on the expression of the oncogenes EGFR, H-ras and c-fos was investigated in an experimental model of chemically induced oral oncogenesis in normal and diabetic (type I) Sprague-Dawley rats. Thirteen diabetic and twelve normal rats developed cancer after 4NQO treatment, while six diabetic and six normal animals were used as controls. The biopsies were classified pathologically (ranging from dysplasia to moderately differentiated oral squamous cell carcinoma) and were studied immunohistochemically. Several representative histological regions from each biopsy were analysed in regard to EGFR, H-ras and c-fos expression, and a comparison between normal and diabetic rats was effected. A trend of decreased EGFR expression in diabetic compared to normal rats was revealed throughout oncogenesis, which was significant in the stage of dysplasia (P<0.05). On the contrary, a trend of increased H-ras expression was observed in diabetic compared to normal rats during oncogenesis, which rose significantly in early invasion and well differentiated OSCC (P<0.001 and P<0.01 respectively). Finally, no statistical differences concerning c-fos expression were detected between diabetic and normal animals. In conclusion, it seems that diabetes reduces the expression of EGFR and initiates the Ras/Raf/MAPK signal transduction pathway by enhancing activation of other signalling molecules, such as the diabetes-associated Insulin Receptor Substrate-1, leading to increased cell proliferation without c-fos involvement.

H-ras and c-fos exhibit similar expression patterns during most stages of oral oncogenesis.

BACKGROUND: H-ras and c-fos oncogenes interact in signalling pathways but their level and time course of expression during oral cancer development are unclear. The present study used an animal model for the simultaneous investigation of H-Ras and c-Fos expression in sequential stages of oral oncogenesis. MATERIALS AND METHODS: Three experimental groups of Syrian golden hamsters (A, B and C; 10 animals each) and one control group (7 animals) were used. The buccal pouches of hamsters in groups A, B and C were treated with 0.5% of the carcinogen 9,10-dimethyl-1,2-benzanthracene and were excised at 10, 14 and 19 weeks, respectively. The biopsies, which included tissue stages ranging from normal oral mucosa to moderately differentiated carcinoma, were studied immunohistochemically. RESULTS: A reduction in both H-Ras and c-Fos expression was observed from group A to B and from hyperplasias to early tumour stages, while a simultaneous increase was noted from group B to C and from well-differentiated to moderately-differentiated carcinomas. The H-ras/c-fos expression ratio had a value of approximately (1.09 +/- 0.21) in five out of seven studied tissue stages. CONCLUSION: H-Ras and c-Fos exhibit a similar expression pattern throughout most stages of oral carcinogenesis, an observation supported by the known molecular pathway connecting H-ras signalling with subsequent c-fos gene transcription.

The interleukin-10 (-1082A/G) polymorphism is strongly associated with increased risk for oral squamous cell carcinoma.

BACKGROUND: Increased levels of interleukin-10 (IL-10) have been observed in patients with oral cancer, possibly as a result of suppression of the immune response. Based on this, the -1082A/G polymorphism, which influences IL-10 gene expression level, was investigated in regard to its possible association with risk for oral cancer. PATIENTS AND METHODS: The polymorphism was examined in DNA samples of 144 patients with oral squamous cell carcinoma and 141 healthy controls of equivalent gender, age and ethnicity. RESULTS: The detected allele frequencies for the high expression G allele were significantly higher in patients compared to controls (34.7% versus 21.3%, respectively, p=0.0004), as well as in patients that were smokers but not those that were heavy alcohol consumers. This highly significant difference in G allele frequency was mainly due to the increase of AG heterozygotes in patients compared to controls (OR 3.05, 95% CI 1.84-5.05). CONCLUSION: These findings suggest that the high expression G allele of the -1082A/G polymorphism of the inflammation and angiogenesis-related IL-10 is strongly associated with increased risk for oral cancer.

Gene expression polymorphisms of interleukins-1 beta, -4, -6, -8, -10, and tumor necrosis factors-alpha, -beta: regression analysis of their effect upon oral squamous cell carcinoma.

PURPOSE: Functional DNA polymorphisms affecting gene expression and serum or saliva levels of interleukins IL-1 beta,-4,-6,-8,-10 and tumor necrosis factors TNF-alpha,-beta have been associated with increased risk for the development of oral squamous cell carcinoma (OSCC). The present retrospective case-control study examines possible interactions between seven cytokine genotype polymorphisms and their combinatory effect in predicting the occurrence of OSCC in Caucasians. METHODS: Three hundred and thirty Greeks and Germans were studied, consisting of 162 OSCC cases and 168 healthy controls of comparable age, gender, and ethnicity. A series of multivariate logistic regression models, adjusted for age and gender, was constructed in order to assess the contribution of homozygous or heterozygous variant genotypes of polymorphisms IL-1 beta (+3953C/T), IL-4 (-590C/T), IL-6 (-174G/C), IL-8 (-251A/T), IL-10 (-1082A/G), TNF-alpha (-308G/A) and TNF-beta (+252G/A) upon overall, early and advanced stages of OSCC development. RESULTS: The contribution of TNF-alpha and IL-6 was consistent and robust in almost all models constructed. Furthermore, when the mode of inheritance of each variant allele was taken into account in a "biological" multivariate logistic regression model, four polymorphisms emerged as primary predictors for overall stages of OSCC: TNF-alpha (OR = 15.27; 95% CI = 7.30-31.96), IL-6 (OR = 8.33; 95% CI = 3.95-17.58), IL-8 (OR = 3.54; 95% CI = 1.69-7.43) and IL-10 (OR = 2.65; 95% CI = 1.28-5.46). Finally, IL-1 beta, IL-4 and TNF-beta polymorphisms were not primary predictors of OSCC development in all constructed models. CONCLUSIONS: This study revealed the highly significant contributions of two out of seven studied cytokines (IL-6 and TNF-alpha) in the occurrence of OSCC. Based on these findings and previous reports, possible stoichiometrical interactions of cytokines leading to OSCC development are discussed.

Enhancement of erbB2 and erbB3 expression during oral oncogenesis in diabetic rats.

PURPOSE: The expression of erbB2 and erbB3 receptors was investigated in an experimental model of chemically induced oral carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. METHODS: Thirteen diabetic and twelve normal rats developed precancerous and cancerous lesions after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. Sections of biopsies from all animals were classified histologically in the following categories: normal mucosa, hyperplasia, dysplasia, early invasion, well- and moderately-differentiated squamous cell carcinoma. Each section was studied immunohistochemically using monoclonal antibodies against erbB2 and erbB3 proteins and six representative histological regions in each section were analysed. RESULTS: The erbB2 was expressed at very low levels in normal rats, while in diabetic animals its expression was significantly increased during early invasion (P = 0.04). The erbB3 expression was significantly elevated in well-differentiated carcinoma in normal animals (P = 0.01), while in diabetic animals it was significantly increased during oral mucosal hyperplasia and dysplasia (P = 0.03 and 0.0007, respectively). The comparison of erbB2 expression between diabetic and normal rats revealed significant differences in all stages except for the tumor stage of moderately differentiated carcinoma (P = 0.01, 0.00001, 0.00001, 0.003, and 0.00001). In regard to erbB3 expression, significant differences between diabetic and normal rats existed only in normal, non-cancerous and precancerous stages (P = 0.007, 0.0001, 0.0003). CONCLUSIONS: It seems that diabetes enhances the expression of both erbB2 and erbB3 in certain stages of oral oncogenesis possibly resulting in promotion of cell proliferation and inhibition of apoptosis.

Increased risk for oral cancer is associated with coagulation factor XIII but not with factor XII.

In light of recently found contribution of factors associated with thrombosis and inflammation to carcinogenesis, we investigated the possible association of coagulation factors XII and XIII with increased risk for oral cancer. In DNA samples of patients with oral squamous cell carcinoma and healthy controls of comparable ethnicity, age and sex, we studied the C46T polymorphism in FXII gene which affects gene transcription and the V34L polymorphism in FXIII gene which affects enzyme activity resulting in alteration of the fibrin network structure. No significant differences were observed in genotype and mutant allele frequencies of the FXII C46T polymorphism between patients and healthy controls. On the contrary, the obtained data for FXIII V34L polymorphism revealed a significant frequency increase of the L allele, which results in thinner fibrin network, in the whole group of patients compared to controls (33.1 versus 22.2% respectively, Fischer value P=0.006). In addition, LL homozygotes had a 3-fold greater risk for developing oral cancer (OR 2.893, 95% CI 1.056-7.890), while in VL heterozygotes a 2-fold greater risk was observed (OR 1.868, 95% CI 1.126-3.101). Significantly increased frequency of L allele was also observed in sub-groups of patients without family history of thrombophilia or cancer, with and without tobacco abuse and with alcohol abuse (P<0.05). Interestingly, in comparison to controls only patients with early cancer stages I and II had significantly increased L alleles and not patients with advanced stages III and IV. These findings suggest that the presence of L allele is strongly associated with oral cancer generation but not with its progression and metastasis. In the presence of L allele, the fibrin network is composed of thinner fibers, is less porous and facilitates tumor stroma formation and therefore tumor cell proliferation. Nevertheless, this thinner and less porous fibrin network inhibits cell migration.

Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway.

PURPOSE: The expression of oncogenic protein c-jun was investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. MATERIAL AND METHODS: Thirteen diabetic and twelve normal rats developed cancer after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. The biopsies were classified pathologically from oral mucosal dysplasia to moderately differentiated oral squamous cell carcinoma (OSCC) and studied immunohistochemically using monoclonal antibody against c-jun protein. RESULTS: Higher c-jun levels were observed in non-cancerous and precancerous stages of normal rats compared with diabetic rats, while in different tumour stages, the expression of c-jun was practically identical for both groups. CONCLUSION: It seems that diabetes does not affect the c-jun N-terminal kinase (JNK)/c-jun pathway.

Expression of cell cycle regulator p16 is not affected by diabetes during oral oncogenesis.

BACKGROUND: The tumor suppressor protein p16 plays a vital role in the regulation of the cell cycle. The expression of p16 was investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. MATERIALS AND METHODS: Tissue sections ranging from normal oral mucosa to moderately differentiated oral squamous cell carcinoma (OSCC) were studied immunohistochemically. RESULTS: In normal rats p16 expression increased gradually during oral oncogenesis, but a significant increase was observed only in moderately differentiated OSCC (p=0.038). On the contrary, in diabetic rats the detected gradual increase was significant in hyperplasia, dysplasia, early invasion and well-differentiated OSCC (p<0.001). Nevertheless, there was no significant difference in p16 expression during oral oncogenesis between normal and diabetic animals. CONCLUSION: It seems that the expression of cell cycle regulator p16 is not affected by diabetes in the studied animal model of oral oncogenesis.

EGFR and c-Jun exhibit the same pattern of expression and increase gradually during the progress of oral oncogenesis.

BACKGROUND: Epidermal growth factor receptor (EGFR) and c-Jun oncogenes are implicated in the same pathway of signal transduction affecting cell differentiation. In order to investigate their possible correlation with sequential histological stages of OSCC formation, we established an experimental model of induced oral carcinogenesis in Syrian golden hamsters. MATERIALS AND METHODS: Thirty-seven animals were divided into one control group (n=7) and three experimental groups (n = 10 each), which were treated with a carcinogen and sacrificed at 10, 14 and 19 weeks after treatment. Tumour sections were studied using monoclonal antibodies against EGFR and c-Jun proteins. RESULTS: The same pattern of expression was observed for both oncogenes, with a significant gradual increase of positively stained cells throughout oral carcinogenesis. CONCLUSION: Since EGFR and c-Jun are implicated in the same molecular pathway of signal transduction, it may be assumed that an increase in EGFR levels leads to increased activation of phospholipase Cy signal transduction cascade, which in turn activates c-Jun protein. Therefore, c-Jun expression in oral cancer seems to be increased through the EGFR-PLCy-Raf-MEK-ERK pathway and not the H-ras-Raf-MEK-ERK/JNK pathway.

Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with increased risk for oral cancer.

INTRODUCTION: In light to recently found contribution of factors associated with thrombosis and inflammation to carcinogenesis, we investigated the possible association of angiotensin I- converting enzyme (ACE) with increased risk for oral cancer. MATERIALS AND METHODS: In DNA samples of 160 patients with oral squamous cell carcinoma and 153 healthy controls of comparable ethnicity, age and sex, we studied the insertion/deletion (I/D) polymorphism in the ACE gene, which affects its transcription. RESULTS: The I allele frequencies were significantly increased in patients compared to controls, 40.6% versus 27.5% (p < 0.001), respectively. The II homozygotes had a three-fold greater risk for developing oral cancer (odds ratio 3.17, 95% C.I. 1.32-7.61). A significant increase of I alleles was observed in patients regardless their smoking or alcohol consumption habits, early or advanced stage of cancer, presence or absence of a family history for cancer or thrombophilia (Fischer values p < 0.05). DISCUSSION: These findings suggest that the I/D polymorphism, by affecting the ACE gene expression, is associated with the progress of oral oncogenesis.

Diabetes increases both N-ras and ets-1 expression during rat oral oncogenesis resulting in enhanced cell proliferation and metastatic potential.

BACKGROUND: The expression of N-ras and ets-1 proteins was investigated in an experimental model of chemically-induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. MATERIALS AND METHODS: Tissue sections ranging from normal mucosa to moderately-differentiated oral squamous cell carcinoma were studied using monoclonal antibodies against N-ras and ets-1 proteins. RESULTS: In diabetic rats, N-ras expression increased with tumor advancement, while in normal rats N-ras was not detected in initial stages of oral oncogenesis and increased only in well-differentiated OSCC. The same pattern of elevated ets-1 expression was observed both in diabetic and normal rats, but in cancerous stages this expression was higher in diabetic than in normal rats. CONCLUSION: It seems that diabetes may contribute to increased cell proliferation due to N-ras constitutive activation, as well as to enhanced invasion and metastatic potential by increasing ets-1 levels.

Diabetes does not influence oral oncogenesis through fibroblast growth factor receptors.

BACKGROUND: Increased expression of fibroblast growth factors and their receptors (FGFRs) has recently been described in oral squamous cell carcinoma. In addition, we have previously described a molecular basis for an association between oral cancer and diabetes. The expression of FGFR-2 and FGFR-3 investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) rats. MATERIALS AND METHODS: Tissue sections ranging from normal mucosa to moderately-differentiated oral squamous cell carcinoma were studied using monoclonal antibodies against FGFR-2 and FGFR-3 proteins. RESULTS: A similar pattern of elevated FGFR-2 and FGFR-3 expression was observed in the initial stages of oncogenesis for both diabetic and non-diabetic animals. In the last stages of oral oncogenesis, the expression of both proteins remained relatively stable. CONCLUSION: It seems that diabetes does not affect the FGFR-2 and FGFR-3 pattern of expression throughout the various stages of oral oncogenesis.

Coagulation-related factors, thrombomodulin and protein Z, are not associated with risk for oral cancer.

BACKGROUND: The link between thrombosis and cancer has been well established. Levels of protein Z and thrombomodulin indirectly regulate thrombin productionl and therefore may affect cancer susceptibility. PATIENTS AND METHODS: The functional polymorphisms -13A/G and -33G/A in protein Z and thrombomodulin genes (respectively) influence transcription. The two polymorphisms were investigated in 160 oral cancer patients and 168 controls of equivalent age, gender and ethnicity using restriction fragment length polymorphism typing. RESULTS: The frequency of the -13G allele, which results in lower expression of protein Z gene, was not significantly elevated in patients compared to controls (8.1% and 6.3%, respectively; odds ratio 1.35, 95% confidence interval 0.72-2.56). No carriers of the thrombomodulin low expression -33A allele were identified, underscoring the rarity of this allele in Caucasians. CONCLUSION: Inherited predisposition affecting protein Z or thrombomodulin levels does not modulate susceptibility to oral cancer. Any possible contribution of thrombin to oral oncogenesis may involve other factors.

The 1040C/T polymorphism influencing thermal stability and activity of thrombin activatable fibrinolysis inhibitor is associated with risk for oral cancer.

No studies thus far have investigated the contribution of thrombin activatable fibrinolysis inhibitor (TAFI) to oral oncogenesis. We studied the activity-related 1040C/T polymorphism in 150 patients with oral cancer and 138 healthy controls matched by age, gender, and ethnicity. The increased-activity T allele frequency was significantly reduced in patients compared with controls (28.7% vs. 37.0%, P < 0.05). T/T homozygotes had about half the probability of developing oral cancer (O.R. 0.39, 95%C.I. 0.13-1.14), while no significant difference was observed in C/T heterozygotes. The observed prophylactic effect of increased TAFI activity might result from reduction of plasmin and inhibition of extracellular matrix dissolution.

Diabetes alters expression of p53 and c-myc in different stages of oral oncogenesis.

BACKGROUND: The expression of tumour suppressor p53 and oncogene c-myc was investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. MATERIALS AND METHODS: Tissue sections ranging from normal mucosa to moderately differentiated oral squamous cell carcinoma were studied using monoclonal antibodies against mutant p53 and c-myc proteins. RESULTS: From hyperplasia to later stages of oral oncogenesis, mutant p53 expression was at higher levels in diabetic rats in comparison to normal animals, although the pattern of expression was similar. In contrast, c-myc expression was significantly higher in diabetic than in normal rats only in normal mucosa and hyperplasia. CONCLUSION: It seems that diabetes contributed to increased accumulation of mutations in the p53 gene, contributing to increased proliferation of tumour cells during oral oncogenesis. Additionally, diabetes appeared to enhance c-myc expression only in the initial stages of oral oncogenesis.

Loss of tumour suppressor p16 expression in initial stages of oral oncogenesis.

BACKGROUND: The p16 tumour suppressor gene is known to be involved in regulation of the cell cycle. p16 expression in sequential histological stages of oral squamous cell carcinoma (OSCC) formation was investigated using an experimental model of induced oral carcinogenesis in Syrian golden hamsters. MATERIALS AND METHODS: Thirty-seven animals were divided into one control group (N = 7) and three experimental groups (N = 10 each) which were treated with a carcinogen and sacrificed at 10, 14 and 19 weeks after treatment. Tumour sections were studied immunohistochemically using monoclonal antibodies against p16 protein. RESULTS: p16 was found significantly increased in hyperplasia, sharply decreased in dysplasia and in the subsequent stages of oral carcinogenesis. CONCLUSION: Inactivation of p16 occurs at the early stage of oral mucosal dysplasia in the multistep process of oral tumourigenesis. Therefore, p16 may be considered as a useful prognostic marker for the progression of oral cancer.

Association of matrix metalloproteinase-1 (-1607 1G/2G) polymorphism with increased risk for oral squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the possible relation of matrix metalloproteinase-1 (MMP-1) to increased risk for oral cancer, in light of recently found contribution of angiogenesis and thrombosis-related factors to the development of malignancies. MATERIALS AND METHODS: The 1G/2G polymorphism in the MMP-1 gene, which influences its expression, was examined in 156 patients with oral squamous cell carcinoma and 141 healthy controls of comparable ethnicity (Greeks and Germans), gender and age. RESULTS: In comparison to controls, the detected 2G allele frequency was significantly lower in the patient group and in subgroups with early cancer stages, with positive family history of thrombophilia, with tobacco abuse and without alcohol abuse (p < 0.05). These findings were mainly due to a significant decrease in 2G/2G homozygotes despite a small increase in 1G/2G heterozygotes in the above groups. CONCLUSION: These findings suggest a significant involvement of the MMP-1 -1607 1G/2G polymorphism in the increasing risk for oral cancer in the 1G allele European carriers.

Expression of ets-1 is not affected by N-ras or H-ras during oral oncogenesis.

PURPOSE: To determine whether ras-activated cascades lead to activation of ets-1 expression in sequential histological stages of oral oncogenesis in an experimental animal model. METHODS: Thirty-seven Syrian golden hamsters were divided into three experimental groups (A, B, C) and one control group. The hamsters' buccal pouches in experimental groups were treated with 0.5% 9, 10-dimethyl-1, 2-benzanthracene (DMBA) for 14 weeks and were excised at 10, 14, 19 weeks, respectively. The biopsies were classified pathologically (normal mucosa, hyperkeratosis, hyperplasia, dysplasia, early invasion, well and moderately differentiated carcinoma) and studied immunohistochemically. The two-tailed Student's t test was performed for each animal group and for each histological category. RESULTS: The ets-1 expression increased in early stages of oral tumor formation and invasion. The expression of N-ras gradually decreased during oral oncogenesis, as previously observed with H-ras. CONCLUSIONS: Neither N-ras nor H-ras affects ets-1 expression in contrast to other types of cancer in which N-ras and ets-1 are implicated in the same signalling pathway. Therefore, the existing pathway implicating these proteins might be somehow altered in oral cancer. It seems that ets-1 is a good prognostic marker for invasiveness and progression of oral cancer.