Transforming growth factor alpha in chemically transformed hamster oral keratinocytes

The cheek pouch of the Syrian hamster is an excellent tissue for the experimental induction of oral cancer by carcinogenic chemicals. Lysate prepared from a cell line (HCPC-1) derived from one of these hamster oral tumors greatly increased the growth of these oral tumor cells in vitro. We now show that the mitogenic substance, transforming growth factor alpha (TGF-alpha), is present in all of the chemically transformed hamster oral tumors examined (in vitro and in vivo). In no adult normal tissue of the Syrian hamster can we detect expression of TGF-alpha. TGF-alpha could be partly or wholly responsible for the mitogenic activity detected in the lysate of the chemically transformed hamster oral keratinocytes. Both normal and chemically transformed hamster oral keratinocytes express the receptor to epidermal growth factor. The consistent detection of TGF-alpha and epidermal growth factor receptor mRNAs in these hamster oral tumor cells suggests that an autocrine growth mechanism might be operative. This hamster cheek pouch oral cancer model can be used for the molecular analysis of how TGF-alpha and epidermal growth factor receptor might be involved in the malignant transformation of epithelial tissues.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Detection of transforming growth factor-alpha messenger RNA in normal and chemically transformed hamster oral epithelium by in situ hybridization

We have recently demonstrated the consistent detection of transforming growth factor alpha (TGF-alpha) in chemically transformed hamster oral tumors. By Northern blot analysis, no TGF-alpha mRNA can be detected in normal cheek pouch mucosa. The consistent expression of TGF-alpha associated with the malignant transformation in the well-defined hamster oral cancer model prompted us to hypothesize that the aberrant expression of this important cellular gene could be related to a specific stage of epithelial alteration. In situ hybridization was used to test this hypothesis. We now report that by in situ hybridization we can detect TGF-alpha mRNA in normal hamster oral epithelium and also at all stages of transformation. In all epithelium, labeling of TGF-alpha mRNA in the basal layer is more pronounced than that observed in the spinous and squamous layers. There is a significant increase of TGF-alpha mRNA labeling early in 7,12-dimethylbenz(a)anathracene-induced oral carcinogenesis. This increase is associated with morphological changes of epithelial hyperplasia or dysplasia. Although lesions exhibiting full-thickness epithelial dysplasia (carcinoma in situ) showed more labeling of TGF-alpha mRNA than do areas of lesser dysplasia, the transition to full-fledged papillary or invasive squamous cell carcinoma is not associated with further elevations of TGF-alpha expression.     

Activation of ras oncogenes in chemically transformed BALB/MK-2 mouse keratinocytes

BALB/MK-2 cells are an epidermal growth factor (EGF)-dependent cell line derived from BALB/c mouse epidermis that can undergo terminal differentiation under appropriate conditions. Previous studies have shown that transformation of these cells by retroviral oncogenes relieves the EGF requirement while blocking the terminal differentiation program. In this report we show that BALB/MK-2 cells are sensitive to transformation by the chemical carcinogens dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N'-nitroso-guanidine (MNNG). BALB/MK-2 cells transformed by these carcinogens proliferate in the absence of EGF and do not undergo terminal differentiation in response to calcium. However, the cells retain their anchorage growth dependence and are nontumorigenic in nude mice. NIH 3T3 transfection analysis showed that the endogenous Ha-ras gene had been activated in both DMBA- and MNNG-transformed cells and the Ki-ras gene had been activated in the MCA-transformed cells. Additionally, non-ras transforming activity was detected in some MNNG-transformed BALB/MK-2 cells. Thus, the BALB/MK-2 cell line provides a reproducible in vitro assay system for chemical transformation of epithelial cells and for identification of oncogene activations associated with changes in growth control and differentiation.     

Influences of chymase and angiotensin I-converting enzyme gene polymorphisms on gastric cancer risks in Japan.

BACKGROUNDS AND AIMS: The renin-angiotensin system plays an important role in homeostasis. Angiotensin II, which is generated by chymase and angiotensin I-converting enzyme (ACE), controls blood pressure as well as angiogenesis and cell proliferation. The aim of this study was to clarify the association of the chymase gene (CMA/B) and ACE polymorphisms with susceptibility to gastric cancer and peptic ulcer. METHODS: We assessed CMA/B A/G and ACE insertion/deletion (I/D) polymorphisms in H. pylori-positive gastric cancers (n = 119), gastric ulcers (n = 127), and duodenal ulcers (n = 105), and controls (n = 294) consisting of H. pylori-positive gastritis alone (n = 162) and H. pylori-negative subjects (n = 132) by PCR methods. RESULTS: In CMA/B polymorphism, the age- and sex-adjusted odds ratios (OR) of A/A and A/G genotypes relative to the G/G genotype for gastric cancer risk were 7.115 (95% confidence interval, 1.818-27.845) and 1.956 (95% confidence interval, 1.137-3.366), respectively. There was an increased risk for gastric ulcer in the A/A genotype (OR, 3.450; 1.086-10.960). However, there was no association between ACE polymorphism and susceptibility to gastric cancer and peptic ulcer. In allele combination analysis of CMA/B and ACE polymorphisms, the A/I allele combinations (CMA/B G/A or A/A and ACE I/I genotype) significantly increased the risk of gastric cancer development (OR, 4.749, 2.050-11.001) compared with the G/I allele combinations (CMA/B G/G and ACE I/I genotype). CONCLUSIONS: The CMA/B polymorphism was associated with an increased risk for gastric cancer and gastric ulcer development. The genotyping test of the renin-angiotensin system could be useful for the screening of individuals with higher risks of gastric cancer and gastric ulcer.     

Genetic variation in angiotensin I-converting enzyme (ACE) and breast cancer risk: the multiethnic cohort

The A-240T and I/D polymorphisms in the angiotensin I-converting enzyme (ACE) gene are markers of circulating ACE levels and have been associated with numerous cardiovascular disease outcomes. More recently, the low-activity A and I alleles at these polymorphic sites have been inversely related with breast cancer risk. We assessed the relationship between the A-240T and I/D ACE variants and breast cancer risk in a case-control analysis (n = 1263 cases with invasive breast cancer and 2269 controls) among African-American, Japanese, Latina, and white women in the Multiethnic Cohort Study. Odds ratios and 95% confidence intervals are presented adjusted for established breast cancer risk factors. Among all women combined, we observed no significant association between the A-240T polymorphism and breast cancer risk. For the I/D polymorphism, contrary to expectation, women with the I/I genotype had a marginally significant increase in breast cancer risk (versus DD genotype: odds ratio, 1.30; 95% confidence interval, 1.05-1.61), although associations were not entirely consistent across ethnic groups. These data do not support the hypothesis that women with lower ACE levels, as predicted by the low-activity A and I ACE alleles, are at reduced risk of breast cancer. Overall, these results suggest that the A-240T and I/D ACE polymorphisms are not likely to be strong predictors of breast cancer risk.     

Epidemiology of smokeless tobacco use: a national study

The prevalence and patterns of smokeless tobacco use and its correlates were assessed in the National Institute on Drug Abuse National Household Survey of residents 12 years of age and older. Overall, 11% of the general population have "ever tried" chewing tobacco, snuff, or other smokeless tobacco. Of these, 5% were former users and 3% used smokeless tobacco almost daily in the past year. Rates of its use differed significantly by sex, age group, race, region, and metropolitan area size. Although females were far less likely to try it, those who did were as likely as males to be daily users. Smokeless tobacco users were also more likely to use alcohol, cigarettes, and marijuana. In general, those who used smokeless tobacco almost daily were more likely to report poor health and hospitalization for illness or injury in the past year, even when other substance use was controlled. Smokeless tobacco users also were more likely to report symptoms of depression. Finally, some substituted smokeless tobacco for cigarettes, but youths (12-17 yr old) were more likely than older tobacco users to use both forms of tobacco regularly.     

An updated meta-analysis on association between angiotensin I-converting enzyme gene insertion/deletion polymorphism and cancer risk

The Alu repetitive sequence insertion/deletion (I/D, rs4646994) polymorphism in the angiotensin I-converting enzyme (ACE) gene may alter cancer susceptibility, but results of current studies are inconclusive. To derive a more precise estimation of the relationship between the ACE I/D polymorphism and cancer risk, we performed an updated meta-analysis of all eligible studies. All studies published up to July 2013 concerning the association between the ACE I/D polymorphism and cancer risk were identified by systematically searching PubMed, EMBASE, Wanfang, CNKI, and Cqvip databases. The odds ratios (ORs) with 95 % confidence intervals (CIs) were pooled using the fixed/random-effects model in Review Manager 5.1 and STATA 12.0. A total of 46 case–control studies including 7,025 cases and 34,911 controls were identified and evaluated. Overall, we did not observe a direct association between the ACE I/D polymorphism and general cancer risk (DD?+?DI vs. II OR?=?0.95, 95 %CI?=?0.84–1.07, P?=?0.40). In the subgroup analysis by cancer type, a significant increased susceptibility of prostate cancer was found for variant homozygotes (DD vs. II?+?ID OR?=?2.15, 95 %CI?=?1.01–4.55, P?=?0.05). Additionally, no significant association was observed in other subgroup analyses according to ethnicity, control source, sample size and quality control of genotyping. In summary, our results suggested that the ACE I/D polymorphism might not be a common risk factor for overall cancer susceptibility, but might contribute to the susceptibility of prostate cancer. More studies with larger sample sizes are required in the future.     

Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco

Nuclear Factor-kappaB (NF-kappaB) activation and COX-2 overexpression have been reported in head and neck cancer, but the relationship between these proteins remains to be investigated. To determine the relationship between NF-kappaB and COX-2 in Smokeless Tobacco (ST) associated oral tumorigenesis, we performed immunohistochemistry in serial sections from 107 OSCCs, 78 oral precancerous lesions (OPLs) (58 hyperplasias, 20 dysplasias) and 15 histologically normal oral tissues and correlated with clinicopathological data. Significant increase in NF-kappaB and COX-2 immunopositivity was observed from normal oral mucosa to OPLs to OSCCs (p = 0.009 and p = 0.002 respectively). Upregulation of NF-kappaB and COX-2 was observed as early as in hyperplasia [p = 0.006; OR = 6.1 and p = 0.003; OR = 7.6, respectively]. Expression of both proteins was found to be significantly associated in OPLs (p = 0.000; OR = 12.6) and OSCCs (p = 0.001; OR = 4.0). Intriguingly, khaini consumption correlated with NF-kappaB immunopositivity in OPLs (p = 0.05, OR = 3.8) and OSCCs (p = 0.01, OR = 3.4) and with COX-2 expression in OPLs (p = 0.03; OR = 4.3). In vitro experimental system of ST associated oral carcinogenesis was used to demonstrate ST (khaini) and NNK mediated activation of NF-kappaB and COX-2, supporting the clinical data. In conclusion, this study demonstrates correlation between over expression of NF-kappaB and COX-2 in early precancerous stages of development of oral cancer and sustained elevation down the tumorigenic pathway, underscoring their potential as targets for early intervention. In vitro studies demonstrated that NNK may be one of the carcinogenic components of ST (khaini) inducing activation of NF-kappaB and COX-2 in oral precancer and cancer cells, suggesting plausible role in ST-induced oral carcinogenesis.     

Keratin expression of both chemically and virally transformed human epidermal keratinocytes during the process of neoplastic conversion

Human epidermal cells, despite being 'immortalized' or 'transformed' by combinations of either oncogenic virus (SV40, adenovirus 12 or Kirsten murine sarcoma virus) or chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide) exhibited similar keratins (although quantitatively reduced) to that of control cells when grown in vitro. However, athymic nude mouse tumors derived from such cells exhibited suppression of the 52, 56 and 58 kd (basic type II) keratins and a predominance of small-sized (40-48 or 50 kd) (acidic type I) keratins. The synthesis of these specific keratins was resumed following re-establishment of cell lines in culture. These results suggest that the changes in keratin protein profiles frequently exhibited by human carcinomas represent a component of the pleomorphic transformed phenotype which can be uncoupled from neoplastic growth.     

Establishment and characterization of a cell line from smokeless tobacco associated oral squamous cell carcinoma

A cell line, AMOS-III has been established from the surgically resected specimen of an untreated primary human oral squamous cell carcinoma of the floor of mouth from a chronic smokeless tobacco consumer. Immunocytochemical analysis showed epithelial specific antigen, cytokeratins 5, 10, 13 and 16 and integrin ₆ markers in AMOS-III cells, confirming the epithelial lineage of the cell line. Analyses of morphology, ultrastructure, karyotype, anchorage independent growth and immunocytochemical properties of the cell line demonstrated the transformed phenotype of epithelial cells. AMOS-III cells have doubling time of 42–44 h. Giemsa-banding patterns of chromosomes confirmed the human origin of the AMOS-III cells. Molecular analysis of cancer-related gene products, p53 and p21^cip1/waf1 showed the presence of wild type p21^cip1/waf1 and truncated p53 proteins. The molecular mechanism underlying the action of retinoids in preventing the occurrence of second primary tumors in oral cancer patients remain to be clearly defined. Treatment of AMOS-III cells with all-trans retinoic acid (ATRA) at 10⁻⁴ μM resulted in 81% cell death. ATRA treatment resulted in enhanced expression of p21^cip1/waf1, nuclear translocation of retinoic acid receptors and apoptotic cell death. Thus, this cell line provides an in vitro model for elucidating the mechanism involving p53 inactivation and p21^cip1/waf1 overexpression in smokeless tobacco-induced oral cancer. Furthermore, the ATRA responsiveness of the cell line underscores its potential utility in identifying the retinoid responsive molecular targets in oral cancer cells.     

The prevalence of oral lesions in smokeless tobacco users and an evaluation of risk factors.

BACKGROUND. The widespread use of smokeless tobacco (ST) has prompted concern in regard to the development of oral lesions in long-term users. METHODS. For inclusion in the current study, a subject must have used an ST product, either snuff or chewing tobacco, for at least 6 months. The subjects were recruited by advertising, and none was referred for the evaluation of an oral lesion. The following were performed on all subjects: assessment of exposure to ST, cigarettes, and alcohol; examination of the oral cavity; a biopsy, if an oral lesion was found; and analysis of a blood sample for beta-carotene. The dietary intake of most of the subjects was analyzed. RESULTS. Of the 347 ST users, all of whom were white male subjects, 45 (13.0%) had an oral lesion. Thirty-five of the lesions were hyperkeratosis and 10 were epithelial dysplasia. CONCLUSIONS. Snuff exposure was associated significantly with the presence of an oral lesion (P < 0.0001). A decreased vitamin C intake also was found among the ST users with oral lesions (P < 0.01). The ST users with epithelial dysplasia, as compared with those with hyperkeratotic lesions, were slightly older, had a lower intake of vitamin C (P < 0.05), and were more likely to have used chewing tobacco than snuff.     

Renin-angiotensin system inhibitors, angiotensin I-converting enzyme gene insertion/deletion polymorphism, and cancer: the Rotterdam Study

BACKGROUND: Angiotensin I-converting enzyme (ACE) inhibitors, angiotensin II antagonists, and the ACE insertion/deletion (I/D) gene polymorphism all influence serum angiotensin II action. Because angiotensin II levels have been associated with cancer, the objective of the current epidemiologic study was to investigate whether renin-angiotensin system inhibitors and/or ACE genotypes were associated with an altered risk of colorectal, lung, breast, and prostate cancer. METHODS: Data were obtained from the Rotterdam Study, a population-based, prospective cohort study with 7983 participants. Participants who had a history of 1 of the cancers of interest (n = 216) or who had a medication history <6 months (n = 88) were excluded, leaving 7679 participants, of whom the ACE genotypes could be assessed in 6670 individuals. The mean follow-up was 9.6 years, during which 730 incident cancers occurred. The effect of medication, ACE I/D genotypes, and their interaction on cancer risk and progression was studied by using Cox proportional hazard models. RESULTS: Carriers of the high-activity genotype DD had an increased risk of breast cancer compared with low-activity II/ID genotype carriers (hazard ratio [HR], 1.47; 95% confidence interval [95% CI], 1.05-2.04), but no association was demonstrated for other cancers. DD carriers who were exposed to long-term and high-dose medication were at lower risk for cancer (HR, 0.28; 95% CI, 0.10-0.79). Short-term, high-dose users were at risk for colorectal cancer progression in the II/ID stratum (HR, 3.83; 95% CI, 1.67-8.79). CONCLUSIONS: Renin-angiotensin system-inhibiting drugs seemed to protect against cancer in individuals with the DD genotype, which was associated with high levels of ACE.     

Bimodal initiation of smokeless tobacco usage: implications for cancer education

One hundred fifty (150) randomly chosen adult male subjects (average age 37) drawn from a university general dental clinic were classified by self-report into three equal categories of fifty smokeless tobacco users, fifty cigarette smokers (non-smokeless tobacco users), and fifty non-tobacco users. According to questionnaire results, a bimodal curve was exhibited among smokeless tobacco users with the two peaks of initiation at ages 12 and 18 compared to a unimodal age of initiation for cigarette smokers at age 16. Two different cohorts of smokeless tobacco users based on age of initiation were observed with differences reported in level of education, urban versus rural origin, average length of time in using smokeless tobacco, and the type of smokeless tobacco product used. Young initiators appeared to be primarily from rural communities having a strong parental or familial influence when initiating smokeless tobacco usage, whereas older initiators appeared to be primarily from more urbanized regions with peer influences as the most significant for initiating smokeless tobacco usage. Other characteristics associated with one or the other cohort were also reported, pointing to different educational measures that may be necessary for prevention and intervention, based on the age of initiating smokeless tobacco usage, as well as place of residence.     

Use of smokeless tobacco among male adolescents: concurrent and prospective relationships

The concurrent and prospective correlates of the use of smokeless tobacco among 846 adolescent males were examined. There were 6- and 12-month follow-ups to the initial questionnaire. Substantial levels of tobacco, alcohol, and marijuana use were reported, with 21% reporting use of more than one drug in the last week. Daily smokeless tobacco users were more likely to initiate use of cigarettes, marijuana, and alcohol than were other males. In addition, the "having used" smokeless tobacco variable was related to increased use of cigarettes, marijuana, and alcohol at follow-up. Discriminant analysis of concurrent data identified peer use of smokeless tobacco and experience with cigarette smoking as the primary discriminating factors between males who had tried it and those who had not. Peer use of smokeless tobacco also discriminated between daily users and those who had tried it but had not gone on to become daily users. Thus peer influence seems to be an important factor not just in trial of smokeless tobacco but also in the development of a daily use pattern. Beginning use of smokeless tobacco was related to offers to use alcohol, cigarettes, and marijuana; peer use was the best predictor of continued daily use.     

Prevalence and predictors of smokeless tobacco use: Iowa's program against smoking

Data from surveys of adolescents were analyzed so that we could determine the prevalence of smokeless tobacco use and identify and compare the concurrent correlates of its use and cigarette smoking. Panel data from seventh through eleventh and eighth through twelfth graders between 1980 and 1984 and cross-sectional data from seventh graders in 1980 and 1985 suggest that boys are more likely to use SLT than are girls and that the boys' use has increased with age and over time. Concurrent correlates of seventh-grade boys' and girls' SLT use and cigarette smoking were identified with discriminant analyses. Predictors of smokeless tobacco use were compared with those of weekly smoking for boys and girls separately. Predictors of use by boys were also compared with those by girls. Trying alcohol and the other form of tobacco were the only predictors that related to the use of either form by boys and girls. Differences among other predictors were noted and their implications for prevention are discussed.     

Chronic exposure to chewing tobacco selects for overexpression of stearoyl-CoA desaturase in normal oral keratinocytes

Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.