Increased nitric oxide levels and iNOS over-expression in oral squamous cell carcinoma

Inducible nitric oxide synthase (iNOS) is responsible for generating high levels of nitric oxide (NO) in tissues. Increased iNOS expression has been demonstrated in a number of carcinomas including head and neck squamous cell carcinoma (SCC). However, iNOS levels have not been evaluated specifically in oral cavity SCC, or in the precancerous lesions that progress to oral SCC. Also, NO levels have not been measured in oral precancerous or cancerous tissues. We therefore measured iNOS mRNA, iNOS protein and NO in oral SCC, oral dysplasias and normal oral epithelium. We used RT-PCR to quantify and compare iNOS mRNA levels in these oral tissue specimens. We found that iNOS mRNA was overexpressed in 41% of oral SCC but in only 8% of dysplasia specimens (P = 0.003). Immunohistochemistry was used to evaluate iNOS protein levels in oral SCC, oral dysplasias and normal oral epithelium. A significantly higher percentage of oral SCC specimens showed the highest level of iNOS staining relative to the oral dysplasias and normal oral epithelial samples (95% of oral SCC, 50% of dysplasias, and only 0% of normal epithelial controls, P < 0.0001). The positive staining for iNOS was limited to the SCC cells. Production of NO from iNOS was quantified using HPLC and found to be significantly higher in oral SCC (1.45 +/- 0.56 microg/ml) than normal epithelial controls (0.43 +/- 0.26 microg/ml) (P = 0.0013). We conclude that iNOS mRNA levels and NO production are significantly increased, in oral SCC compared to oral dysplasias and normal epithelial controls. These findings suggest that increased iNOS expression and the generation of high NO levels might have a role in oral SCC development.     

Differential expression of the non-receptor tyrosine kinase BRK in oral squamous cell carcinoma and normal oral epithelium

BRK is a non-receptor tyrosine kinase whose functional role is poorly understood. Although it is an epithelial specific kinase, its expression appears to be tissue specific. To date, little is known about BRK expression in human oral epithelium. We investigated expression of BRK in human oral squamous cell carcinomas (OSCC) and normal oral epithelium (NOE) using immunohistochemistry, laser confocal microscopy and Western blotting. The subcellular localization of BRK was identified by confocal microscopy and Western blotting of nuclear and cytoplasmic extracts from these cells. The results indicate that NOE express higher levels of BRK compared with OSCC cells. In NOE and moderately differentiated OSCC cells, BRK was localized in the nucleus and cytoplasm. However, in poorly differentiated OSCC cells, BRK was localized in perinuclear regions. These results suggest that BRK expression differs in normal and OSCC which may reflect a possible functional involvement in OSCC.     

p53 expression in oral cancer: observations of a South Indian study

Oral cancers constitute a significant proportion of hospital admissions among cancer patients in India. The aim of this study was to elucidate the role of tumour suppressor gene p53 in the pathogenesis of oral squamous cell carcinoma by immunohistochemistry. Though extensive studies on p53 alterations in oral cancers have been done in Western countries and some Asian countries, only a few studies have emerged from India, especially the Southern states. This study would therefore be helpful in providing an Indian perspective, with particular reference to South Indian states. A total of 110 cases of oral squamous cell carcinoma, 35 dysplastic lesions, 15 hyperplastic lesions, and 50 samples of normal mucosa were assessed for p53 expression. Out of 110 cases of oral carcinoma, 40 (36%) were p53 positive. Among 35 cases of dysplastic lesions studied, 6 (17%) showed p53 positive staining. None of the hyperplastic lesions and normal oral lesions showed any evidence of p53 positivity. However, in 9 out of 40 (23%) cases of positive infiltrating squamous cell carcinomas, the adjacent or overlying non-tumourous epithelium demonstrated focal areas of p53 positive staining in the basal and parabasal layers of the epithelium. In addition, in 7 out of 110 (6%) cases, cytoplasmic staining was observed. In these samples, nuclei were not stained. Our results indicate that p53 over-expression may be involved in only a certain proportion of oral carcinomas. The fact that 6% of the dysplastic lesions were p53 positive, and adjacent non-tumourous epithelium of 23% infiltrating squamous cell carcinomas showed positive staining for p53 in the progenitor compartment of the epithelium indicates that p53 immunoreactivity could be used to detect early tumours as well.     

The effect of surgical wounding on tumour development.

For more than a century, a role for wound healing in the outgrowth of tumours has been implied based on observations in both experimental and clinical studies. Wound healing can be divided into stages of inflammatory, proliferative, repair and remodelling processes. Through proper regulation of activation of epithelial, endothelial and inflammatory cells, platelets and fibroblasts, and the production of growth factors, wounds heal and the various cell types resume their normal function. In tumour growth, similar processes of cell activation and growth factor production are observed. These processes are, however, differently regulated leading to ongoing cellular activation. In recent years, growth factors such as EGF, TGF-alpha and TGF-beta, bFGF, IGF I and II, and PDGF have been identified to play a role in the different stages of wound healing. In addition, some of these factors have now been identified as also being involved in the outgrowth of tumours. In this review, cell types involved in wound healing and tumour growth, as well as the growth factors and cytokines they produce and the role of the extracellular matrix, extensively present in both conditions, are being discussed. A better understanding of the time interval during which the sequelae of events in wound healing occur in relation to the time interval of tumour recurrence may be the basis for defining new therapeutic strategies that can interfere with tumour outgrowth without affecting wound healing processes. These new therapeutic approaches may be of importance especially after surgery or other invasive (diagnostic) procedures in cancer patients.     

The value of epidermal growth-factor receptor (egfr) as a tumor-marker for human oral premalignant and malignant lesions

A previous study demonstrated increased epidermal growth factor receptor (EGFR) in oral dysplasia while another showed decreased EGFR in oral dysplasia. The present study examined immunohistochemical expression of EGFR in 33 dysplastic oral lesions as well as in 9 normal oral mucosa specimens, 12 hyperplastic oral lesions and 10 oral squamous cell carcinomas (SCCs). There were no significant differences in EGFR staining either in intensity or in the epithelial layers stained among the normal oral epithelium, hyperplastic and dysplastic lesions. In addition, no significant difference was noted between keratinized and non-keratinized specimens and among lesions from different sites. Oral SCCs demonstrated significantly stronger staining than the normal oral mucosa, hyperplastic and dysplastic lesions (p=0.0011). At this time, the conflicting data on the EGFR expression in oral dysplastic lesions indicate that this receptor is not a good marker for oral dysplasia. Because most of the available data (including our results) show that the majority of oral SCC overexpress EGFR, this receptor may be useful in the diagnosis and treatment of some oral cancers.     

Role of EGFR family receptors in proliferation of squamous carcinoma cells induced by wound healing fluids of head and neck cancer patients

BackgroundMounting evidence suggests that recurrence of resected head and neck squamous cell carcinomas (HNSCCs) is due to the outgrowth of unrecognized residual tumor cells as well as to the premalignant and/or precursor-field epithelial cells. We studied the impact of processes triggered by HNSCC surgery in stimulating both residual tumor cells [demonstrated to overexpress epidermal growth factor receptor (EGFR)], and premalignant cells surrounding the resected lesion.Patients and methodsEGFR expression/activation by immunohistochemistry/biochemistry and gene status by FISH were investigated in 23 primary HNSCCs and surrounding tissues. The ability to induce cell proliferation of wound healing drainages collected from 12 relapsed and 11 not relapsed patients was evaluated by a colorimetric assay in squamous cell carcinoma cell lines A431 (carrying EGFR amplification) and CAL27 (carrying three EGFR copies) in the presence/absence of EGFR therapeutic inhibitors.ResultsPrimary tumors showed intermediate/high EGFR expression (91%), EGFR phosphorylation and EGFR-positive FISH (35%). Normal, metaplastic and dysplastic epithelium surrounding the resected tumor displayed EGFR overexpression. EGFR activation and gene amplification were observed in normal and dysplastic epithelium, respectively. Each tested wound healing drainage induced the cells to proliferate and the proliferation was significantly higher in relapsed compared with not relapsed HNSCC patients (P = 0.02 and P = 0.03). Anti-EGFR treatments inhibited the drainage-induced proliferation, with the highest inhibitory efficiency by cetuximab on A431 cells, while CAL27 cell growth was more efficiently inhibited by tyrosine kinase inhibitors.ConclusionsSurgery could favor the proliferation of cells showing EGFR overexpression/activation/amplification such as residual tumor cells and/or precursor-field epithelial cells already present after surgery. Treatment with anti-EGFR reagents inhibits wound-induced stimulation, according to the EGFR family status.     

Lectin-binding sites in preneoplastic and neoplastic lesions of human and rodent respiratory tracts

The localization of fluorescein-labeled lectins, i.e., concanavalin A (Con A), Ricinus communis-120 (RCA), and wheat-germ agglutinin (WGA), were studied histologically in F344 rat epithelial lesions produced in the course of chemical carcinogenesis. WGA could not be demonstrated in these lesions. Although all lesions showed positive-binding sites when high concentrations of either Con A or RCA were used, a dilution study showed that the epithelial lesions had different affinities for lectins. With both Con A and RCA, dysplastic and neoplastic lesions showed the strongest intensity of fluorescence and squamous metaplasia showed the weakest. Normal and hyperplastic epithelia showed intermediate intensity. In the dilution study, RCA showed eight times more affinity and Con A showed two times more affinity for dysplastic and neoplastic epithelia than for normal or hyperplastic epithelium. Similar affinity patterns were observed in human lesions and tumors. With Con A, 58% of tumors showed much stronger fluorescence than did normal epithelium, and 44% of the tumors showed positive fluorescence with RCA. Although both lectins exhibited a stronger affinity for all the dysplastic-neoplastic lesions than for normal or hyperplastic epithelium, RCA proved to be the most adequate marker for preneoplastic lesions.     

Loss of epithelial cell surface carbohydrates during experimental oral carcinogenesis in the rat

Cell surface glycoconjugates were investigated in a rat model of oral chemical carcinogenesis. The lectins Griffonia simplicifolia (GS-I-B4; specific for alpha-D-galactosyl end groups) and Ulex europeus (UEA-I; specific for alpha-L-fucosyl groups) were examined microspectrofluorimetrically in the oral epithelium of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) and compared with those treated with solvent alone. After labelling with GS-I-B4, the fluorescent intensity of the basal and parabasal epithelial cells was significantly less after 9 months of 4NQO treatment and in overt squamous cell carcinomas compared to controls. The fluorescent activity of the spinous epithelial cells in the non-invasive tissues treated with 4NQO and in the well differentiated (sites of keratin elaboration) malignant epithelium of squamous cell carcinomas was unchanged after labelling with UEA-I. UEA-I failed to stain undifferentiated (areas lacking keratin) malignant epithelium. The findings indicate that alpha-D-galactosyl residues are diminished on the membranes of premalignant and malignant rat epithelial cells. The expression of alpha-L-fucosyl groups, however, remains unchanged in premalignant rat oral epithelium and is closely correlated to the presence of keratin in the malignant epithelium of squamous cell carcinomas.     

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.     

Expression of the CA1 determinant by carcinomas and by non-malignant epithelial cells in oral lesions

The expression of the Ca antigen was investigated in 5 groups of oral lesions comprising 7 squamous cell carcinomas, 2 pre-invasive carcinomas, 7 lesions of types believed to predispose to carcinoma, 19 lesions of types that do not predispose to carcinoma and 5 biopsies of normal oral mucosa. Using an indirect immunoperoxidase method, the neoplastic epithelium reacted positively with the Ca1 antibody in only 4 out of 7 oral squamous cell carcinomas and the reaction varied between the specimens as to the intensity and number of positively stained cells. Several benign oral lesions specifically bound the Ca1 antibody in areas of epithelium showing infiltration with inflammatory cells. These lesions comprised 5 fibrous epulides, 1 pyogenic granuloma, 1 denture-induced hyperplasia and 1 non-diagnostic ulcer. We conclude that the Ca1 antibody is not sufficiently specific for the carcinoma to be of value in the diagnosis of malignant and premalignant lesions of the oral mucosa.     

Expression of histocompatibility antigens and characterization of mononuclear cell infiltrates in normal and neoplastic colorectal tissues of humans

Serial frozen sections were prepared from 22 colorectal carcinomas. Additional samples were obtained from the adjacent normal bowel in 10 patients, from 6 concomitant adenomas in 5 patients, and from another 4 isolated adenomas. Mononuclear cell infiltrates were stained by the indirect immunoperoxidase technique with the use of a panel of 6 mouse monoclonal antibodies to human leukocyte antigens. The degree of infiltration was graded from 4 (heavy) to 0 (nil). The colorectal carcinomas and adjacent normal bowel showed an equal degree of leukocyte infiltration (HLe-1), graded 3-4 in 8 cases and 2-3 in the other 2 cases. In 7 carcinomas cytotoxic-suppressor T-lymphocytes (UCHT-4) graded 2-3 predominated over helper T-cells (OKT-4) graded 0-1. By contrast, in the adjacent normal bowel cytotoxic and helper cells were present in equal numbers. Among the adenomas leukocyte infiltration was grade 4 in 9 and grade 3 in 1. In 9 of the 10 adenomas cytotoxic cells graded 2 predominated over helper cells graded 0-1. The number of helper cells was equivalent among 6 concomitant adenomas and carcinomas from 5 patients. Adenomatous epithelial cells expressed class II major histocompatibility complex antigens (OKIa-1). However, carcinomatous or normal epithelium showed only faint staining with OKIa-1. The similarity in cell infiltration is consistent with an adenoma-carcinoma sequence. The predominance of cytotoxic cells in carcinomas that expressed class I major histocompatibility complex supports the association between lymphocyte infiltration and a favorable prognosis.     

Differential expression of heat shock protein 27 in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma

The aim of this study was to evaluate HSP27 expression in fetal, normal and inflamed oral mucosal epithelium and in oral premalignant epithelial lesions and in their ensuing invasive cancers. In developing human oral epithelia, immunoreactions for HSP27 were moderately observed in suprabasal keratinocytes of palate and tongue. Normal oral epithelium had an intense suprabasal positivity. In inflamed oral mucosa, HSP27 staining was stronger in basal and suprabasal keratinocytes than in normal epithelium. Most oral premalignant lesions showed no (5 cases, 29%) or low (8 cases, 46.4%) staining. In OSCC both low and high HSP27 levels of expression were observed. HSP27 immunolabelling was down-regulated in poorly differentiated areas and up-regulated in highly differentiated ones. These findings indicated that HSP27 expression seems to protect cells from apoptosis during inflammation, while the down-regulation in dysplasia could impair the protective mechanism against mutagenesis induced by environmental factors and thus enhancing the transformation of oral epithelial dysplasia into OSCCs.     

Loss of expression of TGF-beta1, TbetaRI, and TbetaRII correlates with differentiation in human oral squamous cell carcinomas

Despite advances in biological and molecular characteristics, the prognosis of oral squamous cell carcinomas is still very unfavourable and is based on the classical clinicopathological parameters. However, tumors with similar clinicopathological characteristics may differ dramatically in their clinical outcome. Thus, the identification of novel prognostic factors is necessary to improve prognostic and therapeutic approaches. Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor of epithelial cell proliferation, thus, inactivation of TGF-beta1 signalling may play a role in cancer. The expression levels of TGF-beta1 and its type I and type II receptors (TbetaRI and TbetaRII) were assessed by immunohistochemical and Western blot analyses in 22 oral squamous cell carcinoma lesions, in their normal adjacent mucosa and in the squamous carcinoma cell lines FaDu and CAL27. Immunohistochemistry on 22 oral carcinomas and case-matched normal oral mucosae demonstrated that TGF-beta1, TbetaRI, and TbetaRII were intensively and homogeneously expressed in all normal epithelia. In contrast, TGF-beta1 and its receptors were significantly reduced in poorly (G3) differentiated tumors as compared to moderately (G2) and well differentiated (G1) lesions (p=2.8 x 10(-3), p=1.3 x 10(-3), p=2.8 x 10(-3) and p=1.3 x 10(-3), respectively). The progressive reduction of the expression levels was confirmed by Western blotting. The oral squamous carcinoma cell lines Cal27 and FaDu demonstrated a reduced and a lack of TbetaRI expression, respectively. A significant decrease of TbetaRII expression, as compared to Cal27 cells, was shown in FaDu cells. Thus, the decreased expression of TbetaRII combined with the absence of TbetaRI could account for the resistance of FaDu cells to the growth-inhibiting effect of TGF-beta1. TGF-beta1 and TGF-beta1 receptor expression significantly decreased as tumors became less differentiated and thus more aggressive, suggesting a functional role of these molecules in oral tumor progression.     

Steroid hormone receptor expression and proliferation in rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet. Herein, the expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ER beta) and progesterone receptor (PR) was examined in mammary gland carcinomas induced by PhIP in female Sprague-Dawley rats. Quantitative real-time polymerase chain reaction demonstrated that ER alpha, ER beta and PR were statistically elevated by 3-, 4- and 8-fold in carcinomas compared with normal mammary glands. By immunohistochemistry, carcinomas showed statistically higher nuclear expression of all three steroid receptors with the majority of carcinomas showing at least 10% of epithelial cells stained for ER alpha (49/55, 89%), ER beta (41/55, 75%) and PR (48/55, 87%). Furthermore, the level of expression of the three steroid hormone receptors was positively correlated with each other across the bank of carcinomas (Spearman analysis, P < 0.05). The expression of ER alpha in carcinomas was associated with tumor grade, extent of nuclear pleomorphism and cellular proliferation as measured by proliferating cell nuclear antigen (PCNA) and phospho-Rb immunostaining (Spearman analysis, P < 0.05). Confocal microscopy was used to measure the percentage of epithelial cells showing nuclear colocalization of receptors, PCNA, and cyclin D1. Colocalization of the receptors, and the colocalization of the receptors with PCNA and cyclin D1 was strikingly higher in carcinomas than in the normal mammary gland. In carcinoma cells, 37% of ER alpha positive epithelial cells were colocalized with PCNA in contrast to just 0.25% of cells in the normal mammary gland. The findings from this study indicate that ER alpha, ER beta and PR were co-upregulated and nuclear localized in epithelial cells from rat mammary carcinomas compared with normal mammary glands, and that the co-upregulation was positively correlated with proliferation and cell cycle progression in carcinomas.     

Increase of GKLF messenger RNA and protein expression during progression of breast cancer

Genetic alterations found in carcinomas can alter specific regulatory pathways and provide a selective growth advantage by activation of transforming oncogenes. A subset of these genes, including wild-type alleles of GLI or c-MYC, and activated alleles of RAS or beta-catenin, exhibit transforming activity when expressed in diploid epithelial RK3E cells in vitro. By in vitro transformation of these cells, the zinc finger protein GKLF/KLF-4 was recently identified as a novel oncogene. Although GKLF is normally expressed in superficial, differentiating epithelial cells of the skin, oral mucosa, and gut, expression is consistently up-regulated in dysplastic epithelium and in squamous cell carcinoma of the oral cavity. In the current study, we used in situ hybridization, Northern blot analysis, and immunohistochemistry to detect GKLF at various stages of tumor progression in the breast, prostate, and colon. Overall, expression of GKLF mRNA was detected by in situ hybridization in 21 of 31 cases (68%) of carcinoma of the breast. Low-level expression of GKLF mRNA was observed in morphologically normal (uninvolved) breast epithelium adjacent to tumor cells. Increased expression was observed in neoplastic cells compared with adjacent uninvolved epithelium for 14 of 19 cases examined (74%). Ductal carcinoma in situ exhibited similar expression as invasive carcinoma, suggesting that GKLF is activated prior to invasion through the basement membrane. Expression as determined by Northern blot was increased in most breast tumor cell lines and in immortalized human mammary epithelial cells when these were compared with finite-life span human mammary epithelial cells. Alteration of GKLF expression was confirmed by the use of a novel monoclonal antibody that detected the protein in normal and neoplastic tissues in a distribution consistent with localization of the mRNA. In contrast to most breast tumors, expression of GKLF in tumor cells of colorectal or prostatic carcinomas was reduced or unaltered compared with normal epithelium. The results demonstrate that GKLF expression in epithelial compartments is altered in a tissue-type specific fashion during tumor progression, and suggest that increased expression of GKLF mRNA and protein may contribute to the malignant phenotype of breast tumors.     

ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer

ADAMTSL3/punctin-2 is a secreted glycoprotein that resembles the ADAMTS proteases. Recently, identification of frequent ADAMTSL3 mutations in colorectal cancer suggested it might have a regulatory role in cellular homeostasis in colorectal epithelium or in pathways to colorectal malignancy. Here, we used in situ hybridization to validate ADAMTSL3 antibodies for IHC of a variety of normal and malignant tissues, including colon cancer. Quantitative real-time PCR (RTQ-PCR) was used to compare mRNA expression levels in colon carcinoma (n = 10) and adjacent normal colon. ADAMTSL3 is expressed in epithelial cells of the colon, fallopian tube, skin, breast, prostate, epididymis, liver, pancreatic islets and bile ducts, as well as by vascular endothelial cells, smooth muscle cells, fibroblasts, cortical and ganglionic neurons and cardiac myocytes. Malignant epithelial cells in colon cancer, as well as breast, prostate, renal and skin tumors expressed ADAMTSL3. Normal colon showed stronger immunostaining of surface than basal crypt epithelium and staining of a variety of cells within the lamina propria and submucosa. Colon carcinomas demonstrated weaker staining in tumor cells than normal colon epithelium and weak stromal staining. RTQ-PCR comparison of ADAMTSL3 mRNA in colon carcinoma and adjacent normal colon demonstrated a statistically significant reduction in the tumors, possibly reflecting their decreased stromal content and lack of complete differentiation of tumor samples. The major findings of these studies are that ADAMTSL3 is expressed in numerous tissues, suggesting a broader regulatory role than in colorectal epithelium alone, and that colorectal cancer has both structural mutations as well as decreased expression of ADAMTSL3.     

Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.

PURPOSE: The loss of epithelial phenotypes in the process of carcinoma progression correlates with clinical outcome, and genetic/epigenetic changes accumulate aggressive clones toward uncurable disease. IkappaB kinase-alpha (IKKalpha) has a decisive role in the development of the skin and establishes keratinocyte phenotypes. We assessed clinical implications of IKKalpha expression in oral carcinomas and epigenetic aberrations for the loss of expression. EXPERIMENTAL DESIGN: We examined IKKalpha expression in oral carcinomas by immunostaining (n = 64) and genetic instability by microsatellite PCR (n = 46). Promoter methylation status was analyzed by bisulfite-modified sequence (n = 11). RESULTS: IKKalpha was expressed in the nucleus of basal cells of normal oral epithelium, but not or marginally detected in 32.8% of carcinomas. The immunoreactivity was significantly decreased in less differentiated carcinomas (P < 0.05) and correlated to long-term survival of patients (P < 0.01) with an independent prognostic value (P < 0.05). Although allelic/biallelic loss of the gene was limited to four cases, we detected microsatellite instability in 63.0% cases in which the immunoreactivities were decreased and the promoter was hypermethylated. CONCLUSION: These results showed that oral carcinomas exhibiting genetic instability and promoter hypermethylation down-regulate expression of IKK and suggest that the epigenetic loss of the expression closely associates with disease progression toward unfavorable prognosis.     

Heterogeneous nuclear ribonucleoprotein G shows tumor suppressive effect against oral squamous cell carcinoma cells.

PURPOSE: Heterogeneous nuclear ribonucleoproteins (hnRNP) are nucleic acid binding proteins involved in RNA processing. We found that hnRNP G is expressed in normal human oral epithelial cells while frequently not found in the cells derived from human oral squamous cell carcinomas (HOSCC). The current study was designed to test the hypothesis that hnRNP G is a tumor suppressor. EXPERIMENTAL DESIGN: We investigated the expression levels of hnRNP G protein in normal, precancerous, and malignant oral tissues by in situ immunohistochemistry. In addition, wild-type or mutant hnRNP G was ectopically overexpressed in HOSCC cells and their effects on cellular replication kinetics, colonogenic efficiency, anchorage-independent growth, and in vivo tumorigenicity were determined. RESULTS: In situ immunohistochemical staining showed robust presence of hnRNP G in the basal cell layers of normal oral epithelium but the level of its staining was markedly reduced in dysplastic or cancerous tissues. Ectopic expression of wild-type hnRNP G in cancer cells lacking hnRNP G expression or containing mutant hnRNP G resulted in severe retardation of proliferation, reduction of colonogenic efficiency, loss of anchorage-independent growth, and reduction of in vivo tumorigenicity in immunocompromised mice. In addition, hnRNP G overexpression led to up-regulation of the expression of TXNIP, a cell cycle inhibitory gene, and significantly reduced the expression of the genes that promote cellular proliferation, such as EGR1, JUND, JUNB, FOS, FOSL1, ROS, and KIT. CONCLUSIONS: These results indicate that hnRNP G is a tumor suppressor against HOSCC but its mechanisms of action remain to be further investigated.