Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases.

ECM1 overexpression is an independent predictor of poor prognosis in primary breast carcinomas, however the mechanisms by which ECM1 affects tumor progression have not been completely elucidated. ECM1 was silenced in the triple-negative breast cancer cell lines Hs578T and MDAMB231 using siRNA and the cells were evaluated for changes in morphology, migration, invasion and adhesion. Actin cytoskeleton alterations were evaluated by fluorescent staining and levels of activated Rho GTPases by pull down assays. ECM1 downregulation led to significantly diminished cell migration (p = 0.0005 for Hs578T and p = 0.02 for MDAMB231) and cell adhesion (p < 0.001 for Hs578T and p = 0.01 for MDAMB231). Cell invasion (matrigel) was reduced only in the Hs578T cells (p < 0.01). Silencing decreased the expression of the prometastatic molecules S100A4 and TGFßR2 in both cell lines and CD44 in Hs578T cells. ECM1-silenced cells also exhibited alterations in cell shape and showed bundles of F-actin across the cell (stress fibers) whereas NT-siRNA treated cells showed peripheral membrane ruffling. Downregulation of ECM1 was also associated with an increased F/G actin ratio, when compared to the cells transfected with NT siRNA (p < 0.001 for Hs578T and p < 0.00035 for MDAMB231) and a concomitant decline of activated Rho A in the Hs578T cells. Re-expression of S100A4 in ECM1-silenced cells rescued the phenotype in the Hs578T cells but not the MDAMB231 cells. We conclude that ECM1 is a key player in the metastatic process and regulates the actin cytoskeletal architecture of aggressive breast cancer cells at least in part via alterations in S100A4 and Rho A.

The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis.

TFAP2C/AP-2γ influences development of the mammary gland and regulates patterns of gene expression in luminal and HER2-amplified breast cancer. The roles of TFAP2C in mammary gland tumorigenesis and in pathways critical to cancer progression remain poorly understood. To gain greater insight into oncogenic mechanisms regulated by TFAP2C, we examined mammary tumorigenesis in MMTV-Neu transgenic female mice with or without conditional knockout (KO) of Tcfap2c, the mouse homolog of TFAP2C. Loss of Tcfap2c increased the latency of tumorigenesis and tumors that formed demonstrated reduced proliferative index and increased apoptosis. In addition, tumors formed in Tcfap2c KO animals had a significant reduction in Egfr levels without a change in the expression of the Neu oncogene. The MMneu-flAP2C cell line was established from tumor tissue derived from MMTV-Neu/Tcfap2c(L/L) control animals and parallel cell lines with and without expression of Tcfap2c were created by transduction with adenovirus-empty and adenovirus-Cre, respectively. KO of Tcfap2c in vitro reduced activated phosphorylated-Erk, decreased cell viability, repressed tumor growth and was associated with attenuation of Egfr expression. Chromatin immunoprecipitation and direct sequencing and expression analysis confirmed that Egfr was a Tcfap2c target gene in murine, as well as human, mammary carcinoma cells. Furthermore, decreased viability of mammary cancer cells was directly related to Egfr functional blockade. We conclude that TFAP2C regulates tumorigenesis, cell growth and survival in HER2-amplified breast cancer through transcriptional regulation of EGFR. The findings have important implications for targeting the EGFR pathway in breast cancer.

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis.

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.

Transcriptional regulation of the GPX1 gene by TFAP2C and aberrant CpG methylation in human breast cancer.

The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. TFAP2C is a transcription factor that has a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines. Comparing the genomic binding sites for TFAP2C, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knockdown of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5'-aza-2-deoxycytidine (5'-aza-dC) (an inhibitor of DNA methylation) allowed TFAP2C to bind to the GPX1 promoter resulting in the activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in ∼20% of primary breast cancers and a highly significant correlation between the TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells.

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Methylation of the p16(INK4a) promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells.

Human keratinocytes grown in co-culture with fibroblast feeder cells have an extended in vitro lifespan and delayed accumulation of the tumor suppressor protein p16(INK4a) when compared to the same cells grown on tissue culture plastic alone. Previous studies have indicated that human keratinocytes can be immortalized by telomerase activity alone when grown in co-culture with feeder cells, suggesting that loss of the p16(INK4a)/Rb pathway is not required for immortalization. Using two independent human keratinocyte cell strains, we found that exogenous telomerase expression and co-culture with feeder cells results in efficient extension of lifespan without an apparent crisis. However, when these cells were transferred from the co-culture environment to plastic alone they experienced only a brief period of slowed growth before continuing to proliferate indefinitely. Examination of immortal cell lines demonstrated p16(INK4a) promoter methylation had occurred in both the absence and presence of feeder cells. Reintroduction of p16(INK4a) into immortal cell lines resulted in rapid growth arrest. Our results suggest that p16(INK4a)/Rb-induced telomere-independent senescence, although delayed in the presence of feeders, still provides a proliferation barrier to human keratinocytes in this culture system and that extended culture of telomerase-transduced keratinocytes on feeders can lead to the methylation of p16(INK4a).

AP-2alpha and AP-2gamma are transcriptional targets of p53 in human breast carcinoma cells.

Activating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.

V-Ha-Ras overexpression induces superoxide production and alters levels of primary antioxidant enzymes.

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

v-Ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.

Matrix metalloproteinase 9 (MMP-9) degrades basement membrane type IV collagen and is expressed during cellular migration and invasion. Here we show that v-Ha-Ras overexpression in rat kidney epithelial cells (REC) caused upregulation of MMP-9 gene expression in part by increasing cellular oxidant levels. v-Ha-Ras mediated the production of superoxide in Ras-transfected cells, which was associated with upregulated MMP-9 gene expression. Conversely, v-Ha-Ras expression decreased steady-state levels of mRNAs from tissue inhibitor of metalloproteinase 1 (TIMP-1), an inhibitor of MMP-9; plasminogen activator inhibitor 1 (PAI-1), which indirectly activates MMP-9 by increasing plasmin levels; and collagen IV, a substrate of MMP-9 and a major component of basement membrane. Gel mobility shift assays demonstrated that Ras overexpression enhanced NF-kappaB, but not AP-1 DNA binding to motifs in the MMP-9 gene promoter. The Ras-induced increase in NF-kappaB DNA binding could be inhibited by treatment with the antioxidants N-acetyl-L-cysteine and glutathione monoester, suggesting that intracellular oxidant levels can mediate MMP-9 transcription. Our findings identify an important role for Ras in the regulation of MMP-9 expression, and suggest that increased superoxide production can upregulate MMP-9 expression and thus contribute to malignant conversion.

Constitutive activation of transcription factor AP-2 is associated with decreased MnSOD expression in transformed human lung fibroblasts.

Activator protein-2 (AP-2) is a transcription factor with transactivating and transrepressing potential in different promoter contexts. AP-2 contains seven cysteines, and its in vitro DNA binding activity is redox-sensitive. Superoxide dismutase-2 (SOD2), which encodes the antioxidant enzyme manganese superoxide dismutase (MnSOD), is a putative tumor suppressor gene whose loss of expression is associated with the malignant phenotype. SOD2 promoter mutations that generate new AP-2 sites are associated with loss of MnSOD expression in cancer cells. In the current study, we have identified an inverse expression pattern between AP-2 and MnSOD in normal versus transformed human cells. MRC5 cells are a normal human lung fibroblast cell strain that is mortal and senesces after a certain number of passages in vitro. MRC5-VA is a simian virus transformed variant of MRC5. We determined the levels of expression of MnSOD and AP-2 in these two cell types at the levels of mRNA, protein, and activity. Our results indicated that MnSOD expression was significantly decreased in MRC5-VA cells compared with MRC5 cells at each level of investigation, whereas AP-2 showed an opposing pattern of expression and DNA binding activity. These results suggest that AP-2 may participate in the mechanism(s) underlying decreased expression of SOD2 in transformed cells.

Expression of AP-2 alpha in SV40 immortalized human lung fibroblasts is associated with a distinct pattern of cytosine methylation in the AP-2 alpha promoter.

Activator protein-2 alpha (AP-2 alpha) is a cell type-specific, developmentally regulated, transcription factor that has been implicated as a critical regulator of gene expression during vertebrate development and carcinogenesis. We found that AP-2 alpha was differentially expressed in the normal human lung fibroblast cell strains WI38, MRC-5 and their respective SV40-transformed cell counterparts WI38-VA, MRC-5VA. Since CpG methylation within genetic regulatory regions has been implicated as a mechanism of gene regulation, we investigated the CpG methylation status of the AP-2 alpha gene promoter in these cells. High resolution mapping of methylated cytosines revealed that differential expression of the AP-2 alpha gene in normal human lung fibroblasts and their SV40-transformed counterparts was associated with distinct patterns of cytosine methylation in the AP-2 alpha promoter just 5' to the transcription initiation site. Site-specific methylation was positively correlated with increased AP-2 alpha gene expression in both transformed cell lines investigated. Interestingly, one of the two major centers of hypermethylation in the transformed cells encompassed the cis-element for the AP-2 repressing transcription factor AP-2rep (KLF12). Finally, a sequence variation in human lung fibroblasts relative to the published sequence revealed a previously unidentified AP-2 binding site at position -528 with respect to the transcription initiation site that overlapped the AP-2rep site. Our results suggest that transcriptional activation of AP-2 alpha in the SV40-transformed cells is mediated, at least in part, by site-specific methylation of a negative regulatory cis-element in the AP-2 alpha promoter.

Phospholipid hydroperoxide glutathione peroxidase protects against singlet oxygen-induced cell damage of photodynamic therapy.

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an important enzyme in the removal of lipid hydroperoxides (LOOHs) from cell membranes. Cancer treatments such as photodynamic therapy (PDT) induce lipid peroxidation in cells as a detrimental action. The photosensitizers used produce reactive oxygen species such as singlet oxygen ((1)O(2)). Because singlet oxygen introduces lipid hydroperoxides into cell membranes, we hypothesized that PhGPx would provide protection against the oxidative stress of singlet oxygen and therefore could interfere with cancer treatment. To test this hypothesis, human breast cancer cells (MCF-7) were stably transfected with PhGPx cDNA. Four clones with varying levels of PhGPx activity were isolated. The activities of other cellular antioxidant enzymes were not influenced by the overexpression of PhGPx. Cellular PhGPx activity had a remarkable inverse linear correlation to the removal of lipid hydroperoxides in living cells (r = -0.85), and correlated positively with cell survival after singlet oxygen exposure (r = 0.94). These data demonstrate that PhGPx provides significant protection against singlet oxygen-generated lipid peroxidation via removal of LOOH and suggest that LOOHs are major mediators in this cell injury process. Thus, PhGPx activity could contribute to the resistance of tumor cells to PDT.

A family of AP-2 proteins down-regulate manganese superoxide dismutase expression.

Manganese superoxide dismutase (Mn-SOD) is a primary antioxidant enzyme whose expression is essential for life in oxygen. Mn-SOD has tumor suppressor activity in a wide variety of tumors and transformed cell systems. Our initial observations revealed that Mn-SOD expression was inversely correlated with expression of AP-2 transcription factors in normal human fibroblasts and their SV-40 transformed counterparts. Thus we hypothesized that AP-2 may down-regulate Mn-SOD expression. To examine the functional role of AP-2 on Mn-SOD promoter transactivation we cotransfected AP-2-deficient HepG2 cells with a human Mn-SOD promoter-reporter construct and expression vectors encoding each of the three known AP-2 family members. Our results indicated that AP-2 could significantly repress Mn-SOD promoter activity, and that this repression was both Mn-SOD promoter and AP-2-specific. The three AP-2 proteins appeared to play distinct roles in Mn-SOD gene regulation. Moreover, although all three AP-2 proteins could repress the Mn-SOD promoter, AP-2alpha and AP-2gamma were more active in this regard than AP-2beta. Transcriptional repression by AP-2 was not a general effect in this system, because another AP-2-responsive gene, c-erbB-3, was transactivated by AP-2. Repression of Mn-SOD by AP-2 was dependent on DNA binding, and expression of AP-2B, a dominant negative incapable of DNA binding, relieved the repression on Mn-SOD promoter and reactivated Mn-SOD expression in the AP-2 abundant SV40-transformed fibroblast cell line MRC-5VA. These results indicate that AP-2-mediated transcriptional repression contributes to the constitutively low expression of Mn-SOD in SV40-transformed fibroblasts and suggest a mechanism for Mn-SOD down-regulation in cancer.

Inhibition of the 26S proteasome induces expression of GLCLC, the catalytic subunit for gamma-glutamylcysteine synthetase.

The majority of short- and long-lived cellular proteins are degraded by the activities of the 26S proteasome, a large multi-catalytic protease. Its unique function places it as a central regulatory activity for many important physiological processes. Lactacystin is a very specific 26S proteasome inhibitor and represents an excellent tool for demonstrating that a pathway exhibits proteasome-dependent biochemical regulation. Exposure of HepG2 cells to lactacystin resulted in robust elevation of GLCLC mRNA levels, followed by an increase in GSH concentrations. GLCLC is the gene that encodes the catalytic subunit for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the synthesis of glutathione (GSH). Inhibition of non-proteasome, protease activities did not induce GLCLC. Gel mobility shift assays and expression of CAT activity from heterologous reporter vectors identified Nrf2 mediation of the GLCLC antioxidant response element, ARE4, as the mechanism by which lactacystin induced GLCLC. These studies have identified 26S proteasome activity as a central regulatory pathway for glutathione synthesis.

Epigenetic silencing of maspin gene expression in human breast cancers.

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.

Superoxide generation in v-Ha-ras-transduced human keratinocyte HaCaT cells.

The oncogenic ras protein controls signal-transduction pathways that are critical for cell proliferation and tumorigenesis. Here, we demonstrate that v-Ha-ras-transduced human keratinocyte HaCaT cells produced significantly larger amounts of superoxide than did control cell lines. The superoxide generation was mediated by the transduced ras protein, because superoxide generation was modified by an inhibitor, lovastatin, that inhibits ras farnesylation during ras protein maturation. Superoxide generation was also inhibited by diphenylene iodonium, an inhibitor of flavoproteins, including NADPH oxidase, but not by rotenone, an inhibitor of the respiratory chain of the mitochondria. Those observations suggested that a phagocytic-like NADPH oxidase exists in keratinocytes that could be activated by the dominant activated v-Ha-ras and produce superoxide. Overexpression of manganese-containing superoxide dismutase and copper and zinc-containing superoxide dismutase cDNA via adenovirus infection also attenuated superoxide generation. Previous work has demonstrated that extracellular superoxide dismutase (SOD) can lower superoxide generation; this is the first report that intracellular SOD could also modify the amount of superoxide production from the cells. This report implies that superoxide radical may act as a second messenger molecule of oncogenic ras.

Decreased expression of manganese superoxide dismutase in transformed cells is associated with increased cytosine methylation of the SOD2 gene.

Tumor cells express lower levels of manganese superoxide dismutase (MnSOD) than their normal counterparts. Enforced expression of MnSOD reverses the malignant phenotype of many transformed cells, suggesting that SOD2 is a tumor suppressor. The SOD2 gene contains a large CpG island spanning > 3.5 kb that starts near the 5' edge of the promoter and extends into intron 2. We hypothesized that the difference in SOD2 expression between tumor cells and their normal cell counterparts might be secondary to differences in their cytosine methylation patterns in this CpG island. To test this hypothesis, we analyzed the methylation status of the SOD2 gene in two cell line models that show differential MnSOD expression between normal and SV40-transformed cells: WI38 and MRC5 and their SV40-transformed variants, WI38-VA and MRC5-VA. We subdivided the SOD2 gene CpG island into 10 individual regions for analysis by bisulfite genomic sequencing. A region located in intron 2 displayed a significant increase in cytosine methylation in both transformed cell lines that expressed low levels of MnSOD mRNA compared with their normal cell counterparts. Recent studies by others have shown that SOD2 intron 2 is a potent transcriptional enhancer. The association between increased cytosine methylation of the SOD2 intron 2 region and decreased MnSOD expression in transformed cells compared with their normal counterparts suggests that an epigenetic mechanism contributes to the differential SOD2 gene expression between these normal and SV40-transformed cells.

Transcription factor AP-2 mRNA and DNA binding activity are constitutively expressed in SV40-immortalized but not normal human lung fibroblasts.

Large T antigen (LT) expressed by the oncogenic DNA virus SV40 transforms cells by interacting with and perturbing the normal function of several important cellular proteins including P53, RB, c-MYC, and AP-2. AP-2 binds to regulatory elements within the SV40 enhancer and is therefore of particular interest for mechanisms relating to viral transcription, replication, and packaging. LT antigen has been previously shown to inhibit transcription factor AP-2 from binding to its cognate cis-element in DNA in vitro, and this is believed to occur through a direct physical interaction between the LT and AP-2 proteins. Recently LT and AP-2 were shown to interact at the protein level in vivo and this interaction appeared to mediated by the RB protein. Although LT inhibited AP-2 DNA binding in vitro, the effects of LT on AP-2 expression and DNA binding activity in vivo have not been previously reported. We report here that transcription factor AP-2alpha is constitutively expressed in SV40-transformed cells compared to their normal cell counterparts. The overexpression of AP-2alpha in SV40 transformed cells occurred at the levels of mRNA, protein, and DNA binding activity. The increase in AP-2 DNA binding in vivo was particularly interesting since previous studies in vitro would have predicted that AP-2 DNA binding should be decreased in the presence of LT. AP-2 is a plieotropic regulator of gene expression, activating some and repressing others. Thus, increased cellular AP-2 activity may be an important downstream effector for the transforming ability of SV40.

Differential DNA methylation of the p16 INK4A/CDKN2A promoter in human oral cancer cells and normal human oral keratinocytes.

The p16 INK4A tumor suppressor gene participates in establishing and maintaining the malignant phenotype of a variety of cancer cell lines and primary tumors. Recently it has been observed that p16 expression is lost in oral cavity cancer cell lines in the presence of a normal intact gene. To examine the role of DNA methylation as an explanation for these findings, we analyzed the DNA methylation patterns of the p16 INK4A promoter in DNA isolated from primary cultures of normal human oral keratinocytes and squamous cell carcinoma (SCC-15) oral cancer cells using bisulfite genomic sequencing. Our results demonstrated striking differences in the methylation status of the 5' CpG island of the p16 gene between normal and cancer cells. Normal human oral keratinocytes showed practically no methylation of the p16 INK4A promoter, while SCC-15 oral cancer cells showed almost complete methylation in this region. These data implicate DNA methylation as a mechanism for transcriptional silencing of the p16 INK4A gene in oral cancer cells.