Sympathectomy decreases size and invasiveness of tongue cancer in rats.

The sympathetic nervous system plays a role in carcinogenesis wherein locally released sympathetic neurotransmitters affect proliferation, angiogenesis, vessel permeability, lymphocyte traffic and cytokine production. The present in vivo study was designed to investigate whether surgical sympathectomy, both unilateral and bilateral, had an effect on tumor growth, interstitial fluid pressure (IFP) and lymphatics in rat tongue cancer. We used 4-nitroquinoline-1-oxide (4-NQO) in drinking water for 19 weeks to induce tongue cancer in 20 Dark Agouti rats. After 11 weeks, one group underwent unilateral sympathectomy and another underwent bilateral sympathectomy, while the third group underwent sham surgery. By 19 weeks, tumors in the bilaterally sympathectomized (BL-SCGx) rats were significantly smaller (P<0.05), more diffuse in appearance and less invasive (P<0.05) compared with the large exophytic tumors in the sham-operated rats. The relative lymphatic area was significantly decreased (P<0.05) in tumors in the BL-SCGx rats compared with the sham group. Interestingly, the tumors in rats that underwent unilateral or bilateral sympathectomy had a significantly lower (P<0.05) IFP than those in sham rats. Lack of tyrosine hydroxylase (TH) immunoreactive nerves and few neuropeptide Y (NPY) positive fibers indicate absence of sympathetic nerve fibers in the bilateral sympathectomized group. The peritumoral lymph vessel area was correlated with the tumor size (P<0.001), depth of invasion (P<0.001), weight of rats (P<0.005) and IFP (P<0.05). In conclusion, the present study presents evidence that deprivation of sympathetic nerves decreases tumor growth in rat tongue, probably caused by decreasing IFP and lymph vessel area.

Gene expression profile in oral squamous cell carcinomas and matching normal oral mucosal tissues from black Africans and white Caucasians: the case of the Sudan vs. Norway.

Expression profile of 588 known genes relating to tumour biology, was examined between oral squamous cell carcinomas (OSCCs) and matching normal oral mucosal tissues (NOMTs) obtained from Sudanese (n=11) and Norwegian (n=11) patients. cDNA probes were synthesised from total RNA and hybridised with the Atlas human cancer cDNA expression array membranes. RT-PCR and immunohistochemistry were applied to confirm the expression pattern of a subset of the 588 genes. Differences in expression of the genes examined were found between the OSCCs and the NOMTs on the Atlas membranes. Several of these genes were either up- or down-regulated 1.6-fold or higher in the OSCCs compared to the NOMTs in the cases from the two populations. We found that 181 (31%) and 195 (33%) genes were either up-regulated or down-regulated in the OSCCs from the Sudan and Norway, respectively. From the total number of genes (n=376) found expressed in the OSCCs investigated from the two countries, 53 genes (14%) showed common expression profile [35 (66%) were up-regulated and 18 (34%) were down-regulated] and 70 genes (19%) showed opposite regulation status. Results of the RT-PCR and immunohistochemistry confirmed the hybridisation data. These findings may provide an OSCCs-specific gene expression profile in patients from the two countries, suggesting that alterations of 123 genes are common in these OSCCs regardless of ethnic differences or other socio-cultural risk factors between the patients from the two countries. The findings might further suggest that specific genes are frequently involved in these OSCCs, which may provide novel clues as diagnostic, prognostic biomarkers and/or targets for therapy. The Atlas human cancer cDNA expression array technique can be useful to examine and describe the expression profile of known genes frequently involved in OSCCs from different populations.

Apoptosis and expression of Bax and Bcl-2 in snuff- and non-snuff associated oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is etiologically linked to tobacco and alcohol consumption. A higher frequency of p53 gene mutations was reported in snuff (toombak)-associated OSCC from the Sudan versus those from non-users (Ibrahim et al., 1999, 10). MATERIALS AND METHODS: OSCC from Sudanese toombak users (n = 13) and non-users from the Sudan (n = 6) and Norway (n = 24) were analysed for bax, bcl-2 and Ki-67 immunohistochemically. Apoptosis was evaluated by the TUNEL method. The OSCC from the Sudan had previously been studied for p53 gene mutations. RESULTS: We found a higher apoptotic rate and a higher bax expression in OSCC from Norway compared with those from the Sudan (p < 0.05) irrespective of toombak use. No significant differences were detected in apoptosis, bax, bcl-2 and Ki-67 in OSCC from the Sudan in relation to toombak use or p53 gene status. CONCLUSION: In OSCC, apoptosis was associated with bax expression and was unaffected by p53 gene status or toombak use in OSCC from the Sudan.

Mutations of the p53 gene in oral squamous-cell carcinomas from Sudanese dippers of nitrosamine-rich toombak and non-snuff-dippers from the Sudan and Scandinavia.

Using PCR-SSCP/DNA sequencing methods, we analyzed 14 oral squamous-cell carcinomas (OSCCs) and 8 pre-malignant oral lesions from different Sudanese patients for prevalence of mutations in exons 5 to 9 of the p53 gene in relation to toombak-dipping status. OSCCs (14 from Sudan, 28 from Scandinavia), and 3 pre-malignant oral lesions from Sudanese non-dippers were used as controls. A statistically significant increased incidence in mutations of the p53 gene was found in OSCCs from toombak dippers (93%; 13/14), as compared with those from non-dippers in Sudan (57%; 8/14) and in Scandinavia (61%; 17/28) respectively. In OSCCs from dippers, mutations were found in exons 5 to 9, while in those from non-dippers they were found in exons 5, 7, 8, 9, and no mutations were found in exon 8 in any of the OSCCs from Sudan. Certain types of mutations, however, were similar with respect to exposure to toombak. OSCCs from dippers showed 15 transversions, 9 transitions, 3 insertions and one deletion, compared with 7 transversions, 2 transitions and one deletion found in OSCCs from Sudanese non-dippers, and 9 transversions, 17 transitions and 2 insertions found in those from non-dippers in Scandinavia. No mutations were found in any of the non-malignant oral lesions in relation to dipping or non-dipping status. These findings suggest that (i) the use of toombak plays a significant role in induction of increased p53 gene mutations, (ii) mutations observed were similar to those induced by tobacco-specific N-nitrosamines (TSNAs) in experimental animal models and those already reported in toombak dippers, (iii) types of mutations associated with TSNAs were similar in the exposed and the control groups, (iv) a novel mutation in exon 6 was found in the OSCCs from toombak dippers, (v) the p53 exons 5 (codon 130), 6 (codons 190, 216) and 7 (codons 229, 249, 252) mutations are probable hot spots for toombak-related OSCCs. Further studies are necessary to validate the increased incidence and exon locations of the p53-gene mutations as a biomarker of malignant transformation in populations in which the oral use of tobacco is habitual.

Expression of biomarkers (p53, transforming growth factor alpha, epidermal growth factor receptor, c-erbB-2/neu and the proliferative cell nuclear antigen) in oropharyngeal squamous cell carcinomas.

Using immunohistochemistry, expression of p53, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), c-erbB-2/neu and proliferating cell nuclear antigen (PCNA) was examined in 26 fresh frozen tissue specimens of oropharyngeal squamous cell carcinomas (SCCs). p53 gene mutations were examined by polymerase chain reaction (PCR)/DNA sequencing methods in 22 carcinomas. The findings were examined for correlations with patients' clinicopathological parameters. Expressions of p53 and PCNA were also examined in 21 formalin-fixed corresponding tissues. Of the fresh frozen tissue specimens, 77% (20/26) showed expression and 68% (15/22) showed mutations (substitutions) of the p53, with significant clustering of the mutations in exons 5 (8/22; 36%), 7 (4/22; 18%) and 8 (5/22; 23%). No mutations were found in exon 6. There was a discordance between expression of p53 protein and mutations of the gene. Parallel to expression and mutations of the p53 found in most of the specimens, expression of TGF-alpha, EGFR, c-erbB-2/neu and PCNA was found in 88% (22/25), 92% (23/25), 58% (14/24) and 91% (21/23) of the specimens, respectively. For the formalin-fixed tissue specimens, 62% (13/21) and 90% (19/21) expressed p53 and PCNA, respectively. Examining for correlations with patients' clinicopathological parameters, expression of p53, TGF-alpha, EGFR and c-erB-2/neu seemed to negatively correlate with the increase of the tumour grade. The present work suggests that: (1) lack of negative growth regulation due to inactivation of the p53 gene together with activation of other proto-oncogenes are necessary genetic events in the carcinogenesis of oropharyngeal SCCs; (2) in oropharyngeal SCCs, p53 gene mutations were clustered in exons 5 (codons 130-186), 7 (codons 230-248) and 8 (codons 271-282) which perhaps suggests that tobacco carcinogens probably affect the mutational hot spots of the p53 gene at codons 157, 175, 186, 248, 273 and 282; and (3) fresh frozen and formalin-fixed tissue specimens give similar results when an immunohistochemical method is applied. The importance of p53, TGF-alpha, EGFR, c-erbB-2/neu and PCNA as biomarkers in oropharyngeal SCCs deserves particular attention because it might offer further understanding of the development of these carcinomas.

Expression of keratin 13, 14 and 19 in oral squamous cell carcinomas from Sudanese snuff dippers: lack of association with human papillomavirus infection.

In stratified squamous epithelia, altered expression of keratins (Ks) is one possible marker of malignant potential. In the epithelium of the uterine cervix, presence of human papillomaviruses (HPVs) is increasingly regarded as a marker of risk for cervical cancer. However, a similar role in oral cancer and precancer remains controversial. To address these questions, formalin-fixed, paraffin-embedded oral carcinomas from Sudanese snuff dippers (n=14) and oral carcinomas from Sudanese (n=14), Swedish (n=19) and Norwegian (n=41) non-snuff dippers were examined by immunohistochemistry for expression of K types 13, 14 and 19 using monoclonal antibodies. HPV infection was searched for in all the carcinomas by in situ hybridization (ISH) using the cocktail HPV OmniProbe and the ViraType probe. Carcinomas from Sudanese (snuff dippers/non-snuff dippers) were also examined for HPV infection by polymerase chain reaction (PCR) using the general HPV primers GP5+/GP6+. For the oral carcinomas from snuff dippers, moderate to intense expression of K13 (71%; 10/14), K14 (86%; 12/14) and K19 (93%; 13/14) was found. For the oral carcinomas from non-snuff dippers, weak to moderate expression of K13 (64%; 47/74), K14 (43%; 32/74) and K19 (45%; 33/74) was found. HPV DNA was not detected in any of the carcinomas from three countries when examined by ISH. The Sudanese (from snuff dippers/non-snuff dippers) oral carcinomas were also negative for HPV DNA with the PCR. The present study shows that (i) there is a high level of expression of K13, K14 and K19 in oral carcinomas from snuff dippers compared to those from non-snuff dippers, (ii) this high level of expression may arise from dysregulation of keratinocyte proliferation and maturation caused by damaging effects of snuff, (iii) the HPV genome is not found in Sudanese (snuff dippers/non-snuff dippers), Swedish or Norwegian oral carcinomas, and (iv) this may suggest that these viruses do not play a prominent role in the aetiology of oral carcinomas from these countries.

Expression of keratin 13, 14 and 19 in oral hyperplastic and dysplastic lesions from Sudanese and Swedish snuff-dippers: association with human papillomavirus infection.

Changes in the expression of keratins (Ks), indicating disturbed tissue differentiation, is one possible marker of malignant potential in stratified squamous epithelia. The presence of human papillomaviruses (HPVs) in the epithelium of the uterine cervix is increasingly regarded as a marker of risk for cervical cancer: However, a similar role in oral cancer and precancer remains controversial. To address these questions, potentially malignant oral mucosal lesions from Sudanese (9 hyperplasias/40 dysplasias) and Swedish (15 hyperplasias) snuff-dippers were examined by immunohistochemistry for expression of K types 13, 14 and 19 using monoclonal antibodies directed against each. HPV infection was searched for by in situ hybridization (ISH) using the cocktail HPV OmniProbe and the ViraType probe. For the Sudanese lesions, moderate to intense expression of both K13 (basal, basal/intermediate, basal/intermediate/superficial and intermediate/superficial cell layers) and K14 (basal, basal/intermediate cell layers) was found in 49/49 (100%). For the Swedish lesions, weak to moderate expression of K13 (basal, basal/intermediate cell layers) was found in 12/15 (80%) and 10/15 (67%), respectively. In the Sudanese lesions, expression of K13 showed a distinct pattern through the oral mucosa and its verrucous projections, with an increase towards the superficial cell layers of dysplastic, but not hyperplastic epithelium. K19 was expressed in the basal cell layer in 16/49 (33%) of the Sudanese lesions, while all the Swedish lesions were negative. HPV was found in only 2 Sudanese cases, both of which harboured both type 6 and type 11: both these cases demonstrated mild epithelial dysplasia, The present study shows that a) there is a high prevalence of expression of both K13 and K14 in oral lesions from Sudanese toombak dippers indicating dysregulation of keratinocyte maturation b) one-third of the Sudanese oral lesions expressed K19, regarded as a basal keratin representing epithelial dedifferentiation, which may prove to be a valuable risk marker in follow-up studies c) HPV genome is found infrequently in oral lesions from Sudanese toombak-dippers, suggesting that these viruses may not play a prominent role in the early stages of carcinogenesis in these subjects. These markers were less often expressed in the Swedish lesions, consistent with their much lower rate of malignant transformation.

The Swedish snus and the Sudanese toombak: are they different?

In Sweden, snuff (locally known as snus), was introduced since the year 1637. Presently, Sweden has the highest per capita consumption and sale figures of snuff in the world, and the habit is becoming increasingly popular. Snus is manufactured into a dry form used in the nasal cavity and a moist form used in the oral cavity. Snus manufactured for oral use is a moist ground tobacco of Dark Kentucky or Virginia species mixed with an aqueous solution of water and other blending ingredients. This form of snuff is found in two types: (1) loose and (2) portion-bag-packed. These are the most widely used. The loose moist form (1-2 g a quid) is the most popular type consumed by 73% of the males, followed by the portion-bag-packed form (0.5-1 g a quid), consumed by 13% of the males, while 14% of the males are mixed users. The majority of snus users place the quid in the vestibular area of the upper lip, and the prevalence among persons 15 years of age or older in 15.9% among males and 0.2% among females. The pH of snus has declined from a previous range of 8-9 to a range of 7.8-8.5, moisture content ranges 35-60% and nicotine content is in the order of 5-11 mg/g dry wt tobacco-specific N-nitrosamines (TSNAs) in micrograms (N'-nitrosonornicotine: NNN 5-9; 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone: NNK 1-2; N'-nitrosoanatabine: NAT 2-5). In the Sudan, snuff, locally known as toombak, was introduced approximately 400 years ago. It is always processed into a loose moist form, and its use is widespread in the country. Tobacco used for manufacture of toombak is of the species Nicotiana rustica, and the fermented ground powder is mixed with an aqueous solution of sodium bicarbonate. The resultant product is moist, with a strong aroma, highly addictive and its use is widespread particularly among males. Its pH range is 8-11, moisture content ranges 6-60% and nicotine content is from 8 to 102 mg/g dry wt, and TSNAs contents in micrograms (NNN 420-1 550; NNK 620-7 870; NAT 20-290). Snus and toombak dippers develop a clinically and histologically characteristic lesion at the site of dipping. Probably due to control of the TSNAs in snus, this type of snuff is associated with a lower risk of cancer of the oral cavity (relative risk: RR 5-6-fold), whereas the risk for cancer of the oral cavity among toombak users was high (RR 7.3-73.0-fold). In conclusion, the two snuff products significantly differ in many aspects. Most notable differences are tobacco species, fermentation and ageing, nicotine and TSNAs content, pH, expression of the p53 tumour suppressor gene, and keratin types 13, 14, and 19. It was, therefore, the object of the present study to highlight the oral health hazards of toombak, and to compare it with snus regarding the aforementioned differences.

Immunohistochemical detection of p53 in archival formalin-fixed tissues of lip and intraoral squamous cell carcinomas from Norway.

We investigated the expression of p53 in 82 formalin-fixed, paraffin-embedded archival tissue specimens of lip and intraoral squamous cell carcinomas (SCCs) from the period 1930-1995, by immunohistochemistry using three monoclonal antibodies (MAbs DO-7, DO-1 and 1801). Before incubation, sections were pretreated with 0.1% Protease enzyme at 37 degrees C for 10 min followed by 5 + 5 min microwave oven heating at 700 W and 425 W, respectively. Formalin-fixed tissues of 10 carcinomas of the uterine cervix positive for p53 were used as controls. With one or more of the three MAbs, p53 was expressed in 73% of the 82 SCCs examined. With only protease enzyme pretreatment or microwave oven heating, p53 was expressed in 9/82 and 12/82 of the SCCs, respectively. Of the 82 SCCs, 60%, 45% and 23% expressed p53 with DO-7, DO-1 and 1801, respectively. The kappa coefficient indicated poor agreement between these results for the antibodies, and for lip and intraoral SCCs, except for p53 expression in intraoral SCCs demonstrated by DO-1/1801, which showed fair agreement. The present study suggests that combined protease pretreatment and microwave oven heating of tissue sections improved unmasking of p53 antigenic sites in archival material stored for up to 65 years.

Expression of c-erbB proto-oncogene family members in squamous cell carcinoma of the head and neck.

The exact role of oncogenes and proto-oncogenes in the development of squamous cell carcinoma of the head and neck (SCCHN) is still debatable. The expression of the c-erbB-2, c-erbB-3 and c-erbB-4 members of the epidermal growth factor receptor family was examined in 16 fresh frozen tissue specimens of SCCHN using avidin-biotin complex immunohistochemistry, with monoclonal and/or polyclonal antibodies directed against each. Eight fresh frozen tissue specimens of normal oral mucosa were included as controls. Of the SCCHN examined, mixed membrane/cytoplasmic staining (moderate to intense) of c-erbB-2 was found in 14/16 cases (88%). When present in the specimen, immunopositive staining of c-erbB-2 was seen in some of the oral surface epithelial cell layers (basal, intermediate and/or superficial) as well as the tumour islands. Weak cytoplasmic staining of c-erbB-3 and c-erbB-4 was found in 13/16 (81%) and 11/16 (69%) cases respectively. When present in the specimen, c-erbB-3 and cerbB-4 immunopositive staining was seen in some of the oral surface epithelial cell layers (basal, intermediate and/or superficial) as well as the tumour islands. For the positive carcinomas for c-erbB-2, c-erbB-3 and c-erbB-4, the epithelium located near the carcinomas showed weak mixed membrane/cytoplasmic staining of c-erbB-2 in 5/14 cases (36%), weak cytoplasmic staining of c-erbB-3 in 7/13 cases (54%) and of c-erbB-4 in 3/11 cases (27%). All the normal control oral mucosa showed the same pattern of staining for c-erbB-2, c-erbB-3 and c-erbB-4 found in the epithelium located near the carcinomas. Only expression of c-erbB-2 was found to correlate with the increase in the tumour stage, while co-expression of c-erbB-2, c-erbB-3 and c-erbB-4 was found to correlate with the patient survival time in 25% of the carcinomas examined. The present study shows that a) expression of c-erbB-2, but not c-erbB-3 and c-erbB-4 correlates with the increase of the tumour stage b) co-expression of c-erbB-2, c-erbB-3 and c-erbB4 correlates with decreased survival time in some of the cases of SCCHN, but not the majority c) co-expression of the c-erbB family in normal oral mucosa as well as in the carcinoma may question whether the increased tendency for development of the disease is due to co-expression of c-erbB proto-oncogenes in head and neck lesions.

Immunohistochemical detection of p53 in non-malignant and malignant oral lesions associated with snuff dipping in the Sudan and Sweden.

Immunohistochemistry was used to examine the expression of p53 in pre-malignant oral lesions and oral squamous-cell carcinomas (SCCs) from Swedish and Sudanese snuff-dippers, as well as in pre-malignant oral lesions and oral SCCs from non-snuff-dippers from the Sudan, Sweden and Norway. Of the 14 SCCs from Sudanese snuff-dippers, 21% (3/14) expressed p53. Of the 14, 60 and 41 SCCs from non-snuff-dippers from the Sudan, Sweden and Norway, 64% (9/14), 65% (39/60) and 68% (28/41) expressed p53, respectively. A statistically significant difference in expression of p53 was found in SCCs from Sudanese snuff-dippers compared to those from non-snuff-dippers from all/or any of the 3 countries. None of the suspected pre-malignant oral lesions from Sudanese snuff dippers or non-snuff-dippers expressed p53. Only 2 out of the 15 oral fibro-epithelial hyperplastic lesions from Swedish snuff-dippers expressed p53. Some of the oral epithelial dysplastic lesions, as well as the carcinoma in situ lesions from Norwegian non-snuff-dippers, expressed p53, while the oral fibro-epithelial hyperplastic lesions did not. The low relative frequency of p53 expression found in oral SCCs from snuff-dippers compared to those from non-snuff-dippers might suggest differences in mechanisms of oncogenic action induced by snuff. Alternatively, the pathogenesis of malignant oral lesions from snuff-dippers may follow a p53-independent pathway. In view of the unusually high levels of the tobacco-specific nitrosamines (TSNA) found in the type of snuff used in the Sudan, investigations of p53 mutations or oncogenes are needed.