Showing papers in "Molecular Carcinogenesis in 2005"

Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature.

Abstract: The gene encoding human 8-oxoguanine glycosylase 1 (hOGG1) is involved in DNA base excision repair. The encoded DNA glycosylase excises 7,8-dihydro-8-oxoguanine (8-OHdG), a highly mutagenic base produced in DNA as a result of exposure to reactive oxygen species (ROS). Polymorphisms in this gene may alter glycosylase function and an individual's ability to repair damaged DNA, possibly resulting in genetic instability that can foster carcinogenesis. In order to elucidate the possible impact of polymorphisms in hOGG1, we performed a literature review of both functional and epidemiologic studies that assessed the effects of these polymorphisms on repair function, levels of oxidative DNA damage, or associations with cancer risk. Fourteen functional studies and 19 epidemiologic studies of breast, colon, esophageal, head and neck, lung, nasopharyngeal, orolaryngeal, prostate, squamous cell carcinoma of the head and neck (SCCHN), and stomach cancers were identified. Although the larger functional studies suggest reduced repair function with variant alleles in hOGG1, the evidence is generally inconclusive. There is some epidemiologic evidence that risk for esophageal, lung, nasopharyngeal, orolaryngeal, and prostate is related to hOGG1 genotype, whereas risk of breast cancer does not appear related. In studies that explored potential interactions with environmental factors, cancer risk for hOGG1 genotypes differed depending on exposure, especially for colon cancer. In summary, there is limited evidence that polymorphisms in hOGG1 affect repair function and carcinogenesis. Larger, well-designed functional and epidemiologic studies are needed to clarify these relationships, especially with respect to interactions with other DNA repair enzymes and interactions with environmental factors that increase carcinogenic load.

Proprotein convertases: “Master switches” in the regulation of tumor growth and progression

Abstract: Proprotein convertases (PCs) are a group of Ca2+-dependent serine proteases that have homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This group is comprised of less than a dozen members, known as furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, PC7/PC8/LPC, SKI/S1P, and NARC-1/PCSK9. Four PCs (Furin, PACE4, PC5, and PC7) have been localized to several different tissues and epithelial or nervous system tumors. PCs activate their cognate substrates by limited proteolysis at the consensus sequence RXR/KR downward arrow. Many PC substrates are well known cancer-associated proteins such as growth factors, growth factor receptors, integrins, and matrix metalloproteases (MMPs). For example, IGF-1 and its receptor, TGF-beta, VEGF-C, and MT-MMPs have direct roles in tumor progression and metastasis. Furin, a well-studied member of the PC family, has been associated with enhanced invasion and proliferation in head and neck, breast, and lung cancer. Conversely, inhibition of PC activity by PDX or several PC pro-segments, resulted in reduced processing of these key cancer-related substrates in human squamous cell carcinomas (SCC), colon adenocarcinoma, and astrocytoma cell lines. In parallel to these changes in cell proliferation and invasiveness as well as metastatic ability were markedly impaired. By controlling the maturation/activation of key cancer-associated proteins, PCs act as "master switches" at different levels during tumor development and progression. The manifold effects of PCs, influencing tumor cell proliferation, motility, adhesiveness, and invasiveness, should be exploited by further developing competitive/inhibitory therapeutic strategies that would be able to neutralize simultaneously the most salient cancer cell properties.

Nucleotide excision repair as a marker for susceptibility to tobacco-related cancers: a review of molecular epidemiological studies.

Abstract: DNA repair is a complicated biological process consisting of several distinct pathways that play a central role in maintaining genomic stability. Research on DNA repair and cancer risk is a vital, emerging field that recently has seen rapid advances facilitated by the completion of the Human Genome Project. In this review, we described phenotypic and genotypic markers of nucleotide excision repair (NER) that have been used in molecular epidemiology studies. We summarized the population-based studies to date that have examined the association between DNA repair capacity phenotype and genetic polymorphisms of the NER genes and risk of tobacco-related cancers, including cancers of the lung, head and neck, prostate, bladder, breast, and esophagus. We also included studies of melanoma and nonmelanoma skin cancers because individuals with defective NER, such as patients with xeroderma pigmentosum (XP) are highly susceptible to ultraviolet light (UV)-induced melanoma and nonmelanoma skin cancers. The published data provide emerging evidence that DNA repair capacity may contribute to genetic susceptibility to cancers in the general population. However, many of the studies are limited in terms of the size of the study populations. Furthermore, all published findings are still considered preliminary, the assays used in the studies have yet to be validated, and the results need to be confirmed. Large and well-designed population-based studies are warranted to assess gene-gene and gene-environment interactions and to ultimately determine, which biomarkers of DNA repair capacity are useful for screening high-risk populations for primary prevention and early detection of tobacco-related cancers.

The feasibility of monitoring NF‐κB associated cytokines: TNF‐α, IL‐1α, IL‐6, and IL‐8 in whole saliva for the malignant transformation of oral lichen planus

Abstract: Previous investigations have demonstrated that immune activation and chronic inflammation may be one of the causes of oncogenesis. A previous study from our lab has shown significant increases of NF-kappaB dependent cytokines, TNF-alpha, IL-1alpha, IL-6, and IL-8 in different oral fluids from oral lichen planus (OLP) patients. The aim of this analysis was to explore the potential of detecting these cytokines in whole unstimulated saliva (WUS) in monitoring the malignant transformation of OLP. Thirteen patients with OLP (with epithelial dysplasia), 13 cases with oral squamous cell carcinoma (OSCC), and 13 age-sex matched controls were enrolled in the study. The WUS samples were collected and the level of TNF-alpha, IL-1alpha, IL-6, and IL-8 in WUS was determined by ELISA. In moderate and severe dysplasia, the level of each cytokine was significantly higher than in control. In moderate dysplasia, TNF-alpha and IL-1alpha were significantly increased at a level without difference from OSCC, but IL-6 and IL-8 was detected at a concentration significantly lower than OSCC. In severe dysplasia, the level of TNF-alpha was also not significantly different from that of OSCC, and the level of IL-1alpha, IL-6, and IL-8 was still significantly lower than that of OSCC. The level of four cytokines between smokers and non-smokers in each group did not show a significant difference. These results indicate that the change of NF-kappaB dependent cytokines in WUS may in part reflect the malignant transformation of OLP and the analysis of these cytokines and may provide a useful, non-invasive surrogate endpoint for monitoring malignant transformation as well as the therapeutic response of OLP. This is the first in vivo study utilizing saliva to confirm preclinical data that NF-kappaB is upregulated in oral carcinogenesis.

Identification of differentially expressed genes in oral squamous cell carcinoma

Abstract: Rapid advances in multimodality therapy have not significantly improved the overall 5-yr survival of oral cancer patients in the past two decades, thereby underscoring the need for molecular therapeutics. The development of new treatment strategies for more effective management of oral cancer requires identification of novel biological targets. Therefore, the aim of this study was to identify novel genes associated with oral tumorigenesis by comparing gene expression profile of oral squamous cell carcinomas (OSCCs) and matched nonmalignant oral epithelial tissues with differential display. Of the 180 differentially expressed cDNAs isolated, reamplified, and cloned into pGEMT-Easy Vector, 26 cDNAs were confirmed to be upregulated in OSCCs by reverse Northern blot analysis. The differentially expressed genes included components of immune system, signaling pathways, angiogenesis, cell structure, proliferation, apoptosis, cell-adhesion, and cellular metabolism. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of 15 OSCCs and matched nonmalignant oral tissues provided the first evidence that 14-3-3-zeta, melanoma metastasizing clone D (MEMD), KIAA0471, sperm protein 17 (SP17), TC21, and anti-TNF alpha antibody are upregulated in OSCCs. Immunohistochemical analysis confirmed overexpression of 14-3-3-zeta and TC21 protein, a member of the Ras family, in OSCCs as compared to histologically normal oral tissues validating the differential display analysis. Identification of six novel differentially expressed genes in oral tumors adds to the repertoire of genes associated with oral carcinogenesis and provides candidate potential biological targets for diagnosis and/or therapy. Further characterization of the 14 unknown differentially expressed cDNAs identified in this study may provide significant clues for understanding the molecular mechanisms underlying oral tumorigenesis.

Microsatellite instability and protein expression of the DNA mismatch repair gene, hMLH1, of lung cancer in chromate-exposed workers

Abstract: Our previous studies of lung cancer in chromate-exposed workers (chromate lung cancer) have revealed that the frequency of replication error (RER) in chromate lung cancer is very high. We examined whether the RER phenotype of chromate lung cancer is due to an abnormality of DNA mismatch repair protein. We investigated the expression of a DNA mismatch repair gene, hMLH1, and hMSH2 proteins using immunohistochemistry and microsatellite instability (MSI) in 35 chromate lung cancers and 26 nonchromate lung cancers. Lung cancer without MSI or with MSI at one locus was defined as “RER(−),” lung cancer with MSI at two loci was defined as “RER(+),” and lung cancer with MSI at three or more loci was defined as “RER(++).” The repression rate of hMLH1 and hMSH2 proteins in chromate lung cancer was significantly more than that of nonchromate lung cancer (hMLH1: 56% vs. 20%, P = 0.006, hMSH2: 74% vs. 23%, P < 0.0001). In chromate lung cancer, the repression rate for hMLH1 was 43% in RER(−), 40% in RER(+), and 90% in the RER(++) group. The repression rate of hMLH1 protein in the RER(++) group was significantly higher than that in the RER(−) and RER(+) groups (P = 0.039). The inactivation of hMLH1 expression strongly correlated with the microsatellite high instability phenotype in chromate lung cancer. The genetic instability of chromate lung cancer is due to the repression of hMLH1 protein. © 2004 Wiley-Liss, Inc.

Osteopontin induces multiple changes in gene expression that reflect the six "hallmarks of cancer" in a model of breast cancer progression.

Abstract: Tumor progression is a multistep process, which enables cells to evolve from benign to malignant tumors. This progression has been suggested to depend on six essential characteristics identified as the "hallmarks of cancer," which include: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Osteopontin (OPN) is an integrin-binding protein that has been shown to be associated with the progression of several cancer types, and to play an important functional role in various aspects of malignancy, particularly tissue invasion and metastasis. Here we studied genes regulated by OPN in a model of human breast cancer using oligonucleotide microarray technology by comparing the gene-expression profiles of 21NT mammary carcinoma cells transfected to overexpress OPN versus mock-transfected control cells. From over 12,000 human genes, we identified 99 known human genes differentially regulated by OPN whose expression changed by at least 1.5-fold and showed statistically significant differences in mean expression levels between groups. Functional classification of these genes into the hallmarks of cancer categories showed that OPN can affect the expression of genes involved in all six categories in this model. Furthermore, we were able to validate the expression of 18/19 selected candidate genes by quantitative real-time PCR, further supporting our microarray findings. This study provides the first evidence that OPN can lead to numerous gene expression changes that influence multiple aspects of tumor progression and malignant growth.

Hibiscus polyphenol-rich extract induces apoptosis in human gastric carcinoma cells via p53 phosphorylation and p38 MAPK/FasL cascade pathway.

Abstract: In view of the continuing need for effective anticancer agents, and the association of diet with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Polyphenols had been demonstrated previously to possess antioxidative and antitumor promoting effects. In this study, investigations were conducted to examine the mechanism of the anticancer activity of H. sabdariffa L., Hibiscus polyphenol-rich extracts (HPE). Using HPLC assay, HPE was demonstrated to contain various polyphenols. HPE induced cell death of eight kinds of cell lines in a concentration-dependent manner. Among them human gastric carcinoma (AGS) cells were the most susceptible to HPE (0.95 mg/mL HPE inhibited its growth by 50%). Our results revealed that AGS cells underwent DNA fragmentation, and had an increase in the distribution of hypodiploid phase (apoptotic peak, 52.36%) after a 24-h treatment with HPE (2.0 mg/mL). This effect of HPE in AGS cells might be mediated via p53 signaling and p38 MAPK/FasL cascade pathway, as demonstrated by an increase in the phosphorylation of p53 and the usage of a specific p38 inhibitor, SB203580. Thus, our data present the first evidence of HPE as an apoptosis inducer in AGS cells and these findings may open interesting perspectives to the strategy in human gastric cancer treatment.

Biological and chemical inhibitors of NF-κB sensitize SiHa cells to cisplatin-induced apoptosis

Abstract: Cisplatin, a chemotherapeutic agent, is known to induce apoptosis of cancer cells. We examined the role of NF-kappaB during cisplatin-induced apoptosis in two human cervical cancer cell lines, HeLa and SiHa, known to differ in their response to cisplatin treatment. We found that SiHa cells were relatively more resistant than HeLa cells to the cytotoxic effects induced by cisplatin as measured by MTT assays. HeLa cells were more sensitive to the apoptotic effects induced by cisplatin as shown by increases in annexin staining, DNA fragmentation, and loss of mitochondrial membrane potential. Similarly the activities of caspases 3, 8, and 9 and cleavage of PARP induced by cisplatin were more in HeLa than SiHa cells. Cisplatin induced NF-kappaB DNA binding activity in HeLa and SiHa cells but not in primary cervical cells and the active DNA binding complex in SiHa cells consists of p50 and RelA heterodimers. However, when NF-kappaB DNA binding activity was blocked by chemical (curcumin, PDTC, or salicylic acid) or biological inhibitors (NIK-KM or IKK-beta DN), the cell viability was less in SiHa cells with cisplatin treatment, but these effects were not observed in HeLa cells. Similarly upon treatment with cisplatin SiHa cells had more activation of caspases compared to that seen in HeLa cells under conditions of NF-kappaB inhibition by biological or chemical inhibitors. These results suggest that NF-kappaB may contribute to the resistance of human cervical cancer cells to cisplatin and highlight the potential use of combination therapy involving cisplatin and NF-kappaB inhibitors.

Role of PI3K/Akt signaling in insulin‐like growth factor‐1 (IGF‐1) skin tumor promotion

Abstract: Overexpression of human IGF-1 with the bovine keratin 5 (BK5) promoter (BK5.IGF-1 transgenic mice) induces persistent epidermal hyperplasia and leads to spontaneous skin tumor formation. In previous work, PI3K and Akt activities were found to be elevated in the epidermis of BK5.IGF-1 transgenic mice compared to nontransgenic littermates. In the present study, we examined the importance of the PI3K/Akt signaling pathway in mediating the skin phenotype and the skin tumor promoting action of IGF-1 in these mice. Western blot analyses with epidermal lysates showed that signaling components downstream of PI3K/Akt were altered in epidermis of BK5.IGF-1 mice. Increased phosphorylation of GSK-3 (Ser(9/21)), TSC2(Thr(1462)), and mTOR(Ser(2448)) was observed. In addition, hypophosphorylation and increased protein levels of beta-catenin were observed in the epidermis of BK5.IGF-1 mice. These data suggested that components downstream of Akt might be affected, including cell cycle machinery in the epidermis of BK5.IGF-1 mice. Protein levels of cyclins (D1, E, A), E2F1, and E2F4 were all elevated in the epidermis of BK5.IGF-1 mice. Also, immunoprecipitation experiments demonstrated an increase in cdk4/cyclin D1 and cdk2/cyclin E complex formation, suggesting increased cdk activity in the epidermis of transgenic mice. In further studies, the PI3K inhibitor, LY294002, significantly blocked IGF-1-mediated epidermal proliferation and skin tumor promotion in DMBA-initiated BK5.IGF-1 mice. In addition, inhibition of PI3K/Akt with LY294002 reversed many of the cell cycle related changes observed in untreated transgenic animals. Collectively, the current results supported the hypothesis that elevated PI3K/Akt activity and subsequent activation of one or more downstream effector pathways contributed significantly to the tumor promoting action of IGF-1 in the epidermis of BK5.IGF-1 mice.

Targeting NAD(P)H:quinone oxidoreductase (NQO1) in pancreatic cancer

Abstract: NAD(P)H:quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species (ROS). The aim of our study was to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated increased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage-independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months.

Apigenin inhibits oxidative stress-induced macromolecular damage in N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinogenesis in Wistar albino rats

Abstract: Apigenin (4',5,7-trihydroxyflavone), a flavone subclass of flavonoid widely distributed in many herbs, fruits, and vegetables is a substantial component of the human diet and has been shown to possess a variety of biological activities including tumor growth inhibition and chemoprevention. Recent studies in several biological systems have shown that apigenin induces tumor growth inhibition, cell cycle arrest, and apoptosis. Free radical-induced degradation of polyunsaturated fatty acid results in electrophilic products and causes severe oxidative stress. Oxidative stress induced by free radicals, nonoxidizing species, electrophiles, and associated DNA damages have been frequently coupled with carcinogenesis. In the present study, the protective role of apigenin was examined against the oxidative stress caused by N-nitrosodiethylamine (NDEA) and phenobarbital (PB) in Wistar albino rats. Oxidative stress was measured in terms of lipid peroxidation (LPO) and protein carbonyl formation. Oxidative stress-induced DNA damage was measured by single cell gel electrophoresis (comet assay). Apigenin exhibited its antioxidant defense against NDEA-induced oxidative stress. We have observed minimal levels of LPO and DNA damage in apigenin-treated hepatoma bearing animals. Based on the results, we suggest that apigenin may be developed as a promising chemotherapeutic agent against the development of chemical carcinogenesis.

Transactivating agonists of the EGF receptor require Tyr 845 phosphorylation for induction of DNA synthesis.

Abstract: Signaling networks play important roles in cancer progression. For example, overexpression of the epidermal growth factor receptor (EGFR) is a poor prognostic indicator in multiple tumor types. Recent studies have postulated that the EGFR functions as a central conduit for signaling by different classes of cell surface receptors. In this study, we demonstrated that c-Src-dependent phosphorylation of tyrosine 845 (Tyr 845) on EGFR was required for DNA synthesis induced by the G protein-coupled agonists, endothelin (ET) and lysophosphatidic acid (LPA), and the cytokine, growth hormone (GH), in murine fibroblast and breast cancer model systems. In addition, we showed that a dominant interfering form of signal transducer and activator of transcription (STAT)5b (a downstream effector of phospho-Tyr 845 [pY845] in fibroblasts) abrogates DNA synthesis induced by all agonists in the breast cancer model. To further characterize the role of Tyr 845, a pY845-containing peptide was microinjected into SKBr3 breast cancer cells and murine fibroblasts, and was found to ablate EGF-stimulated S-phase entry in both cell systems. Taken together, these findings suggested that pY845 is critical for DNA synthesis induced by a variety of mitogens and that its signaling effectors may include but are not limited to STAT5b.

Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes

Abstract: Apigenin is a nonmutagenic bioflavonoid that has been shown to be an inhibitor of mouse skin carcinogenesis induced by the two-stage regimen of initiation and promotion with dimethylbenzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). These DMBA/TPA-induced squamous cell carcinomas overexpress cyclooxygenase-2 (COX-2). Cyclooxygenases are key enzymes required for prostaglandin (PG) synthesis, converting the arachidonic acid (AA) released by phospholipase A2 into prostaglandins. A large body of evidence indicates that the inducible form of cyclooxygenase, COX-2, is involved in tumor promotion and carcinogenesis in a wide variety of tissue types, including colon, breast, lung, and skin. In the present study, we have determined that apigenin inhibited the TPA-induced increase in COX-2 protein and mRNA in the human keratinocyte cell line; HaCaT. The induction of COX-2 elicited by TPA correlated with increased activation of Akt kinase and cell treatment with the PI3 kinase inhibitor, LY294002, blocked TPA induction of COX-2. In cells treated with TPA and apigenin, the inhibition of COX-2 expression correlated with inhibition of Akt kinase activation. Apigenin-mediated inhibition of TPA-induced COX-2 expression was reversed by transient transfection with constitutively active Akt (CA-Akt). Chemical inhibitors of MEK (PD98059), p38 (SB202190), but not JNK (SP600125) blocked TPA induction of COX-2 although apigenin did not inhibit TPA-mediated COX-2 expression through these pathways. The TPA-induced release of AA from HaCaT cells was also inhibited by cell treatment with apigenin. These data show that apigenin inhibits TPA-mediated COX-2 expression by blocking signal transduction of Akt and that apigenin also blocks AA release, which may contribute to its chemopreventive activity.

Inhibition of the Akt, cyclooxygenase‐2, and matrix metalloproteinase‐9 pathways in combination with androgen deprivation therapy: Potential therapeutic approaches for prostate cancer

Abstract: Prostate cancer cells are generally dependent on androgen stimulation mediated by the androgen receptor (AR) for growth and survival, and, therefore, hormonal manipulation, such as castration and/or the use of AR antagonists, results in a regression of the cancer. However, this treatment very rarely leads to the “cure” of advanced disease, and cancers eventually become androgen-independent. A number of genes/pathways have been reported to be activated in prostate cancer, most of which are possibly associated with disease progression. In this article, among them, we focus on Akt (also known as protein kinase B), cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9, whose activities or expressions have been found to be regulated by androgens/AR. Previous studies by us and others, with androgen-sensitive prostate cancer cell lines, have demonstrated that androgen deprivation results in activation/overexpression of Akt, COX-2, and MMP-9 in cells. This suggests that androgen deprivation in clinical settings activates the Akt, COX-2, and MMP-9 pathways in prostate cancer, which may increase cell growth and in turn promote the transition to the androgen-independent state. We hypothesize that androgen deprivation, in combination with inhibition of the Akt, COX-2, and MMP-9 pathways, delays the androgen-independent transition and has more beneficial effects than hormonal therapy alone. © 2005 Wiley-Liss, Inc.

Epigenetic inactivation of CHFR in nasopharyngeal carcinoma through promoter methylation.

Abstract: Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers. Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. Epigenetic inactivation of CHFR through promoter CpG hypermethylation may lead to CIN and has been reported in several human cancers. In this study, we investigated the CHFR gene expression in a panel of nasopharyngeal carcinoma (NPC), prostate, ovarian, and breast cancer cell lines. We found that the expression of CHFR mRNA was significantly decreased or undetectable in all eight NPC cell lines as well as three human NPC xenografts, whereas non-malignant nasopharyngeal cell lines and other cancer cell lines tested expressed CHFR at relatively high levels. Hypermethylation of CHFR promoter region was also strongly correlated with decreased CHFR expression in NPC cell lines and xenografts. Treatment with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, led to restoration of CHFR expression in NPC cell lines. More importantly, hypermethylation of CHFR promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non-malignant tissues. These findings suggest that downregulation of CHFR is a common event in NPC cells which may be due to hypermethylation of the gene promoter region.

Estrogen Receptor α Increases Basal and Cigarette Smoke Extract-Induced Expression of CYP1A1 and CYP1B1, but not GSTP1, in Normal Human Bronchial Epithelial Cells

Abstract: Gender-specific estrogen receptor alpha (ERalpha) expression may plausibly influence lung carcinogenesis in females Initial genome-wide microarray studies confirmed that carcinogen metabolism genes (CYP1A1, CYP1B1) were those most responsive to cigarette smoke extract (CSE) in normal bronchial epithelial (NHBE) cells These two genes encoding phase I bioactivating enzymes and the GSTP1 gene encoding a phase II deactivating enzyme were then tested for induction by ERalpha NHBE cells (native ERalpha-) were transfected with wild-type ERalpha-adenoviral constructs, and then exposed to CSE, 17beta-estradiol (E2), and/or the ERalpha inhibitor, ICI 182,780 The expression levels of CYP1A1, CYP1B1, and GSTP1 were then determined by RNA-specific quantitative RT-PCR and immunoassay ERalpha increased the basal expression of CYP1B1 404-fold (P < 001) at the mRNA level and 65-fold at the protein level ERalpha also increased the CSE-induced mRNA expression of CYP1B1 226-fold (P < 001), but not the protein expression ERalpha did not alter the CYP1A1 mRNA levels, but did increase protein expression 20-fold (P < 001) on CSE exposure, and 62-fold (P < 001) upon E2 exposure These effects could be inhibited by ICI 182,780 ERalpha did not alter the expression of GSTP1 Chromatin immunoprecipitation assay (ChIP) assay confirmed ERalpha binding to CYP1B1 promoter near the transcription start site These results suggest that ERalpha regulates the CYP1B1 expression at a transcriptional level, and CYP1A1 expression at a translational level These data raise the possibility that inter-gender differences in expression of ERalpha that are known to exist in human lung may contribute to inter-individual expression differences in CYP1A1 and CYP1B1, and to differences in carcinogen metabolism and mutation

Artepillin C in Brazilian propolis induces G0/G1 arrest via stimulation of Cip1/p21 expression in human colon cancer cells

Abstract: Potential chemopreventive agents exist in foods. Artepillin C in Brazilian propolis was investigated for its effects on colon carcinogenesis. We had found that artepillin C was a bioavailable antioxidant, which could be incorporated into intestinal Caco-2 and hepatic HepG2 cells without any conjugation and inhibited the oxidation of intracellular DNA. Artepillin C was then added to human colon cancer WiDr cells. It dose-dependently inhibited cell growth, inducing G(0)/G(1) arrest. The events involved a decrease in the kinase activity of a complex of cyclin D/cyclin-dependent kinase 4 and in the levels of retinoblastoma protein phosphorylated at Ser 780 and 807/811. The inhibitors of the complex, Cip1/p21 and Kip1/p27, increased at the protein level. On the other hand, Northern blotting showed that artepillin C did not affect the expression of Kip1/p27 mRNA. According to the experiments using isogenic human colorectal carcinoma cell lines, artepillin C failed to induce G(0)/G(1) arrest in the Cip1/p21-deleted HCT116 cells, but not in the wild-type HCT116 cells. Artepillin C appears to prevent colon cancer through the induction of cell-cycle arrest by stimulating the expression of Cip1/p21 and to be a useful chemopreventing factor in colon carcinogenesis.

Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair.

Abstract: The ubiquitin-proteasome pathway is fundamental to synchronized continuation of many cellular processes, for example, cell-cycle progression, stress response, and cell differentiation. Recent studies have shown that the ubiquitin-proteasome pathway functions in the regulation of nucleotide excision repair (NER) in yeast. In order to investigate the role of the ubiquitin-proteasome pathway in the NER of mammalian cells, global genomic repair (GGR), and transcription-coupled repair (TCR) were examined in a mouse ts20 cell line that harbors a temperature-sensitive ubiquitin-activating enzyme (E1). We found that E1 inactivation-induced ubiquitination deficiency decreased both GGR and TCR, indicating that the ubiquitination system is involved in the optimization of entire NER machinery in mammalian cells. We specifically inhibited the function of 19S proteasome subunit by overexpressing 19S regulatory complex hSug1 or its mutant protein hSug1mk in repair competent human fibroblast, OSU-2, cells and compared their capacity for NER. The results showed that 19S regulatory complex positively modulates NER in cells. In addition, we treated OSU-2 cells with the inhibitors of 20S subunit function, MG132 and lactacystin, and demonstrated that the catalytic activity of 20S subunit is also required for efficient NER. Moreover, the UV-induced recruitment of repair factor xeroderma pigmentosum protein C (XPC) to damage sites was negatively affected by treatment of repair competent cells with MG132. Taken together, we conclude that the ubiquitin-proteasome pathway has a positive regulatory role for optimal NER capacity in mammalian cells and appears to act through facilitating the recruitment of repair factors to DNA damage sites.

Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to apoptotic stress.

Abstract: Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

In vivo mutational analysis of liver DNA in gpt delta transgenic rats treated with the hepatocarcinogens N-nitrosopyrrolidine, 2-amino-3-methylimidazo[4,5-f]quinoline, and di(2-ethylhexyl)phthalate.

Abstract: In order to cast light on carcinogen-specific molecular mechanisms underlying experimental hepatocarcinogenesis in rats, in vivo mutagenicity and mutation spectra of known genotoxic rat hepatocarcinogens N-nitrosopyrrolidine (NPYR), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as well as the nongenotoxic hepatocarcinogen di(2-ethylhexyl)phthalate (DEHP) and the noncarcinogen acetaminophen (AAP), were investigated in guanine phosphoribosyltransferase (gpt) delta transgenic rats, a recently developed animal model for genotoxicity analysis. After 13-wk treatment, glutathione S-transferase placental form (GST-P)-positive liver cell foci were significantly increased in NPYR-treated and IQ-treated rats. In the DEHP-treated rats, marked hepatomegaly with centrilobular hypertrophy of hepatocytes occurred, although GST-P staining was consistently negative. Positive mutagenicity was detected in IQ- and NPYR-treated rats. Mutant frequencies (MFs) in the liver DNA were 188.0 x 10(-6) and 56.5 x 10(-6), approximately 35-fold and 10-fold higher, respectively, than that of nontreatment control rats (5.5 x 10(-6)). There were no increases in MFs in the DEHP- or AAP-treated rats as compared to the nontreatment control value. IQ induced mainly base substitutions leading to G:C to T:A transversions (56.9%) and deletions of G:C base pairs. In contrast, NPYR primarily caused specific A:T to G:C transitions (49.3%), which are very rare in the other groups. These data provided support for the conclusion that IQ and NPYR hepatocarcinogenesis depends on genotoxic processes and specific DNA adduct formation while DEHP exerts its influence via a nongenotoxic promotional pathway. Our data also indicate that analysis of specific in vivo mutational responses with transgenic animal models can provide crucial information for understanding the molecular mechanisms underlying chemical carcinogenesis.

Loss of heterozygosity and transcriptome analyses of a 1.2 Mb candidate ovarian cancer tumor suppressor locus region at 17q25.1-q25.2.

Abstract: Loss of heterozygosity (LOH) analysis was performed in epithelial ovarian cancers (EOC) to further characterize a previously identified candidate tumor suppressor gene (TSG) region encompassing D17S801 at chromosomal region 17q25.1. LOH of at least one informative marker was observed for 100 (71%) of 140 malignant EOC samples in an analysis of 6 polymorphic markers (cen-D17S1839-D17S785-D17S1817-D17S801-D17S751-D17S722-tel). The combined LOH analysis revealed a 453 kilobase (Kb) minimal region of deletion (MRD) bounded by D17S1817 and D17S751. Human and mouse genome assemblies were used to resolve marker inconsistencies in the D17S1839-D17S722 interval and identify candidates. The region contains 32 known and strongly predicted genes, 9 of which overlap the MRD. The reference genomic sequences share nearly identical gene structures and the organization of the region is highly collinear. Although, the region does not show any large internal duplications, a 1.5 Kb inverted duplicated sequence of 87% nucleotide identity was observed in a 13 Kb region surrounding D17S801. Transcriptome analysis by Affymetrix GeneChip and reverse transcription (RT)-polymerase chain reaction (PCR) methods of 3 well characterized EOC cell lines and primary cultures of normal ovarian surface epithelial (NOSE) cells was performed with 32 candidates spanning D17S1839-D17S722 interval. RT-PCR analysis of 8 known or strongly predicted genes residing in the MRD in 10 EOC samples, that exhibited LOH of the MRD, identified FLJ22341 as a strong candidate TSG. The proximal repeat sequence of D17S801 occurs 8 Kb upstream of the putative promoter region of FLJ22341. RT-PCR analysis of the EOC samples and cell lines identified DKFZP434P0316 that maps proximal to the MRD, as a candidate. While Affymetrix technology was useful for initially eliminating less promising candidates, subsequent RT-PCR analysis of well-characterized EOC samples was essential to prioritize TSG candidates for further study.

Amplification and overexpression of Aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells

Abstract: Immortalization is an early and essential step of human carcinogenesis Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells The chromosome 20q harbors genes including TGIF2 (20q112-q12), AIB1 (20q12), PTPN1 (20q131), ZNF217 (20q132), and AURKA (20q132-q133), which were previously reported to be amplified and overexpressed in ovarian cancers Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q132-133) where AURKA resides DNA amplification of AURKA was also confirmed using semi-quantitative PCR Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis

Use of mononucleotide repeat markers for detection of microsatellite instability in mouse tumors

Abstract: Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR-deficient mice with four mononucleotide repeats with polyA(24-37) tracts and three new markers with extended polyA(59-67) tracts. All seven markers were sensitive to MSI in MMR-deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR-deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono- and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.

Effects of dietary energy repletion and IGF-1 infusion on the inhibition of mammary carcinogenesis by dietary energy restriction

Abstract: Dietary energy restriction (DER) is a potent inhibitor of mammary carcinogenesis, but the responsible mechanisms are not fully understood. In a number of model systems, DER is associated with a decrease in circulating levels of IGF-1. Moreover, we have recently reported that protection against cancer is lost, and plasma IGF-1 levels are restored to control values when animals are re-fed, i.e., energy repleted (DER-REP). Accordingly, an experiment was designed to determine if infusion of IGF-1 could mimic the effect of DER-REP on the carcinogenic response in animals that were DER. Following 1-methyl-1-nitrosourea injection (50 mg/kg), rats were fed either ad libitum (AL) or 40% DER. After 6 wk, the DER group was divided into three groups: (1) continued DER, (2) DER-REP, or (3) continued DER and infused with 120 μg rh-IGF-1/d (INF) for a duration of 8 d. DER reduced mammary cancer incidence and multiplicity (P < 0.01) versus AL rats. In rats that were DER-REP, cancer incidence increased 1.4-fold and multiplicity increased by 3.6-fold versus DER rats. Plasma IGF-1 were reduced by DER (P < 0.01), an effect that was reversed by DER-REP (P < 0.05). INF increased plasma IGF-1 versus DER rats (P < 0.01) but did not reverse the carcinogenic response. Plasma IGFBP-3 levels were reduced by DER (P < 0.01), but elevated by either REP or INF. Thus, an 8-d period of refeeding following chronic DER (DER-REP) reversed the anticancer effects of DER, and 8 d of IGF-1 infusion without refeeding (INF) did not mimic the effects of the DER-REP on the carcinogenic response. © 2004 Wiley-Liss, Inc.

ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.

Abstract: Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

Upstream stimulatory factor (USF) as a transcriptional suppressor of human telomerase reverse transcriptase (hTERT) in oral cancer cells.

Abstract: Telomerase activity is suppressed in normal human somatic tissues but is activated in cancer cells and immortal cell lines. The reverse transcriptase (RT) subunit human telomerase reverse transcriptase (hTERT) is the key regulator of telomerase activity. The hTERT promoter contains E-box elements and may allow upstream stimulatory factor (USF), a basic helix-loop-helix (bHLH) leucine zipper family proteins, to bind and regulate the expression. In this study, we investigated whether and how USF effect on hTERT. Through luciferase reporter assays, we found that both USF1 and USF2 possess a comparable effect on the inhibition of hTERT expression. Immunoprecipitation (IP) and immunoblotting (IB) analysis reveal that the suppression of hTERT by USF was not through the interaction of USF with c-myc or mad, nor disturbed the cellular protein levels of those. In gel mobility shift and chromatin immunoprecipitation (CHIP) assays, we found that the USF suppression is through direct binding at the E-box site of hTERT promoter and rendering the effect actively. Analysis on clinical normal and tumor tissues reveal that the expression of USF1 and USF2 was lower in the tumor tissues, correlated with hTERT expression and telomerase activity. Taking together, our results demonstrate that USF is a negative transcriptional repressor for hTERT in oral cancer cells. It is possible that USF lose the inhibitory effect on hTERT expression leading to telomerase reactivation and oral carcinogenesis.

Mammary glands of sexually immature rats are more susceptible than those of mature rats to the carcinogenic, lethal, and mutagenic effects of N-nitroso-N-methylurea

Abstract: Knowing that the prepubertal period is a time of enhanced susceptibility for radiation-induced human breast cancer, we used the Fischer 344 rat model to explore the age-differential susceptibility of the mammary gland to the carcinogenic, lethal, and mutagenic effects of two structurally diverse chemical carcinogens, N-nitroso-N-methylurea (NMU), and 7,12-dimethylbenz(a)anthracene (DMBA). Mammary carcinoma incidences and multiplicities were significantly greater in immature than mature NMU-treated rats while mammary carcinoma incidences and multiplicities were significantly lower in immature than mature DMBA-treated rats. The survival of mammary clonogens of mature NMU-treated rats in limiting dilution transplantation assays was greater than that of the survival of mammary clonogens of immature NMU-treated rats. No differences were found in the survival of mammary cells from immature and mature rats exposed to DMBA. Although there were no mutation spectra differences, mammary epithelial cells of immature NMU-treated rats had greater mutation frequencies than those of mature NMU-treated rats. Together these results support the hypothesis that the mammary gland of immature rats is more susceptible to the carcinogenic, lethal, and mutagenic effects of alkylating agents represented by NMU in a carcinogen-class-specific manner. Further, the results suggest the importance of mechanistic and epidemiological studies of the susceptibility of the prepubertal breast to specific carcinogens such as alkylating agents.

Activation of Wnt/β-catenin/Tcf signaling in mouse skin carcinogenesis

Abstract: Although Wnt/β-catenin/Tcf signaling pathway has been shown to be an important factor in the development of many malignancies including colorectal, ovarian, prostate, and many other cancers, little is known about its role in non-melanoma skin cancers. Here, we report the first evidence that β-catenin/Tcf signaling pathway is constitutively activated in non-melanocytic skin tumors induced by two stage chemical carcinogenesis protocol. Mouse skin tumors showed cytoplasmic and nuclear accumulation of β-catenin, and upregulation of β-catenin/Tcf target genes (c-myc and c-jun). We found high levels of skin-expressed Wnt proteins (Wnt 3, 4, and 10b) in different parts of the tumors, likely representing key upstream events in β-catenin/Tcf activation during mouse skin carcinogenesis. Inhibition of β-catenin/Tcf signaling by ectopic expression of dominant negative Tcf4 resulted in significant inhibition of growth in squamous cell carcinoma cells. A role of the constitutive activation of β-catenin/Tcf signaling in skin carcinogenesis is discussed. © 2005 Wiley-Liss, Inc.

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects†

Abstract: Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.