Identification of MAGEA antigens as causal players in the development of tamoxifen-resistant breast cancer.

The antiestrogen tamoxifen is a well-tolerated, effective treatment for estrogen receptor-α-positive (ER+) breast cancer, but development of resistance eventually limits its use. Here we show that expression of MAGEA2, and related members of this cancer-testis antigen family, is upregulated in tamoxifen-resistant tumor cells. Expression of MAGEA2 in tumor lines grown in vitro or as xenografts led to continued proliferation in the presence of tamoxifen. At the molecular level, we demonstrate that MAGEA2 protein localizes to the nucleus and forms complexes with p53 and ERα, resulting in repression of the p53 pathway but increased ER-dependent signaling. In a series of ER+, tamoxifen-treated breast cancer patients, we show a highly significant (P=0.006) association between MAGEA (melanoma-associated antigen) expression and reduced overall survival, confirming the clinical significance of our observations.

Primary cell and micromass culture in assessing developmental toxicity.

Under the European Commission's New Chemical Policy both currently used and new chemicals should be tested for their toxicities in several areas, one of which was reproductive/developmental toxicity. Thousands of chemicals will need testing which will require a large number of laboratory animals. In vitro systems (as pre-screens or as validated alternatives) appear to be useful tools to reduce the number of whole animals used or refine procedures and hence decrease the cost for the chemical industry. Validated in vitro systems exist for developmental toxicity/embryotoxicity testing. Indeed, three assays have recently been validated: the whole embryo culture (WEC), the rat limb bud micromass (MM), and the embryonic stem cell test (EST). In this article, the use of primary embryonic cell culture, and in particular micromass culture, including a relatively novel chick heart micromass (MM) culture system has been described and compared to the validated D3 mouse embryonic stem cell (ESC) test.

Overexpression of TFAP2C in invasive breast cancer correlates with a poorer response to anti-hormone therapy and reduced patient survival.

The AP-2gamma transcription factor encoded by the TFAP2C gene is a member of a family of homologous DNA binding proteins that play essential roles during vertebrate embryogenesis but show a restricted pattern of expression in the adult. Elevated expression of the AP-2alpha and AP-2gamma family members has been associated with a number of neoplasms, particularly breast cancer. Here we present an exploratory immunohistochemical study of an archival primary breast tumour series (n = 75) with parallel clinicopathological data using a new, well-characterized antibody to AP-2gamma. Heterogeneous, exclusively nuclear expression of AP-2gamma was found in the epithelial and myoepithelial compartments of normal breast and within tumour epithelial cells. In the breast cancer series, the most notable association was a correlation between elevated levels of AP-2gamma and shortened patient survival (p = 0.0009*). This relationship was also conserved in ER-positive and ErbB2-negative patients; sub-groups generally considered to have a relatively good prognosis. When patient data for survival and duration of treatment response on anti-hormone therapy were examined by multivariate analysis, AP-2gamma was revealed in this study to be an independent predictor of outcome for both survival (p = 0.005) and response to anti-hormone therapy (p = 0.046). Studies using in vitro models confirmed that while tamoxifen response is associated with lower levels of AP-2gamma, acquisition of resistance to this and other anti-hormone measures (eg faslodex or oestrogen deprivation) is associated with high levels of nuclear AP-2gamma. Together these data suggest that elevated tumour AP-2gamma expression can contribute to the failure of cells to growth arrest following anti-hormone treatment and lead to sustained growth and poorer patient outcome.

Update on HER-2 as a target for cancer therapy: the ERBB2 promoter and its exploitation for cancer treatment.

Overexpression of the ERBB2 proto-oncogene is associated with amplification of the gene in breast cancer but increased activity of the promoter also plays a significant role. Members of two transcription factor families (AP-2 and Ets) show increased binding to the promoter in over-expressing cells. Consequently, strategies have been devised to target promoter activity, either through the DNA binding sites for these factors, or through another promoter sequence, a polypurine-polypyrimidine repeat structure. The promoter has also been exploited for its tumour-specific activity to direct the accumulation of cytotoxic compounds selectively within cancer cells. Our current understanding of the ERBB2 promoter is reviewed and the status of these therapeutic avenues is discussed.

Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator.

The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity and regulates gene transcription. Here we show that Cited2-/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2-/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.

Expression pattern of AP-2 transcription factors in cervical cancer cells and analysis of their influence on human papillomavirus oncogene transcription.

The AP-2 family of transcription factors consists of three known members, namely AP-2alpha, AP-2beta, and AP-2gamma. In experimental systems AP-2 factors possess tumor suppressor-like activities, and alterations in the AP-2 expression pattern have been described for some tumor entities. In addition, AP-2 has been implicated in the transcriptional control of human papillomaviruses (HPVs). We investigated here the expression pattern of AP-2alpha, AP-2beta, and AP-2gamma, as well as that of the cellular AP-2 target gene c-erbB-2, in a series of cervical cancer cell lines. In addition, we analyzed the influence of AP-2 factors on the activity of the HPV16 and HPV18 E6/E7 oncogene promoter. We found that, with the exception of HPV-negative C33A cells, all investigated cervical cancer cell lines expressed all three AP-2 family members, although at varying levels. No linear correlation between AP-2 and c-erbB-2 levels was observed. Although AP-2alpha, AP-2beta, and AP-2gamma can activate the c-erbB-2 promoter in reporter gene assays, they do not stimulate the HPV16 or HPV18 E6/E7 promoter. These results indicate that, although a rare event, loss of AP-2 expression occurs in cervical cancer cells. Moreover, AP-2alpha, AP-2beta, and AP-2gamma are neither sufficient nor required to activate the viral E6/E7 promoter.

Detoxification of endotoxin by endodontic irrigants and calcium hydroxide.

The effects of endodontic irrigants and calcium hydroxide on lipopolysaccharide (LPS; endotoxin) were analyzed using the highly selective technique of mass spectrometry/gas chromatography with selected ion monitoring. An aqueous solution of LPS was mixed with one of a variety of endodontic irrigants for 30 min. Because it is a commonly used interappointment dressing, calcium hydroxide was also applied to LPS for 1, 2, or 5 days. LPS inactivation was measured by quantitation of free fatty acid release. Water, EDTA, ethanol, 0.12% chlorhexidine, chlorhexidine + sodium hypochlorite, and sodium hypochlorite alone showed little breakdown of LPS. Long-term calcium hydroxide--as well as 30-min exposure to an alkaline mixture of chlorhexidine, ethanol, and sodium hypochlorite--did detoxify LPS molecules by hydrolysis of ester bonds in the fatty acid chains of the lipid A moiety.

Intracellular expression of the truncated extracellular domain of c-erbB-3/HER3.

The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length transmembrane protein and the latter a truncated extracellular fragment consisting of 140 amino acids of the c-erbB-3 protein followed by 43 unique residues. We have examined the expression of the two ERBB3 transcripts by Northern blotting in cancer cell lines and normal human fetal and adult tissues. We expressed the truncated receptor fragment and showed that it was glycosylated, probably with a single N-linked complex sugar chain, and that the protein was a 58-kDa disulphide-linked dimer. We were able to crosslink iodinated neuregulin (NRG)-1beta to the full-length solubilised receptor but not to the truncated dimeric protein. Using Western blot analysis, the truncated protein was shown to be present in cell lysates and, using immunoelectron microscopy, in vesicular structures within cells and associated with the plasma cell membrane.

Neuregulin 1-alpha expression in locally advanced breast cancer.

The human Neuregulin 1 (NRG1) gene encodes several alternatively spliced ligands that bind to both c-erbB-3 and c-erbB-4, members of the family of type 1 tyrosine kinase growth factor receptors. Antibodies raised to a synthetic peptide recognize selectively the alpha variant of NRG1. The NRG1-alpha isoforms' expression was studied in 115 locally advanced adenocarcinomas of the breast using immunohistochemistry. Absent or low levels of NRG1-alpha were found to be associated with poorer prognosis compared to tumours that had moderate to high levels of the protein.

Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of aldose reductase.

Lipid peroxidation represents a significant source of erythrocyte dysfunction and aging. Because the toxicity of lipid peroxidation appears to be in part due to aldehydic end products, we examined, in rat erythrocytes, the metabolism of 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and toxic lipid-derived aldehydes. Packed erythrocytes, 0.1 ml, completely metabolized 20 nmoles of HNE in 20 min. The glutathione conjugate of HNE and 4-hydroxynonanoic acid (HNA) represented 70 and 25% of the total metabolism, respectively. Approximately 70% of the metabolites were extruded to the medium. Upon electrospray ionization mass spectrometry, the glutathione conjugate resolved into two distinct species corresponding to glutathionyl HNE (GS-HNE) and glutathionyl 1,4-dihydroxynonene (GS-DHN). The concentration of GS-DHN formed was twice that of GS-HNE. Inhibition of aldose reductase by sorbinil and tolrestat led to a selective decrease in the formation of GS-DHN, although the extent of HNE glutathiolation was unaffected. Inhibitors of aldehyde or alcohol dehydrogenase, i.e., cyanamide and 4-methyl pyrazole, had no effect on the formation of HNA and GS-DHN, indicating that these enzymes are not significant participants in the erythrocyte HNE metabolism. Thus, oxidation to HNA, conjugation with glutathione, and further reduction of the conjugate by aldose reductase appear to be the major pathways of HNE metabolism in erythrocytes. These pathways may be critical determinants of erythrocyte toxicity due to lipid peroxidation-derived aldehydes.

Midazolam attenuates ketamine-induced abnormal perception and thought process but not mood changes.

PURPOSE: To determine the effects of midazolam, 30 ngxmL(-1), on altered perception, mood, and cognition induced by ketamine. METHODS: After ketamine was administered to achieve target concentrations of 50, 100, or 150 ngxmL in 11 volunteers, perception, mood, and thought process were assessed by a visual analog scale. Mini-Mental State examination (MMSE) assessed cognition. Boluses of midazolam, 30, 14.5, and 12 microgxkg(-1), were injected every 30 min to maintain the plasma concentration at 30 ngxmL(-1), which was reached 30 min after each injection. RESULTS: Ketamine produced changes in perception about the body (P < 0.01, 0.001, and 0.0001 at 30, 60, and 90 min), surroundings (P < 0.01 and 0.0001 at 60 and 90 min), time (P < 0.002 and 0.0001 at 60 and 90 min), reality (P < 0.001 and 0.0001 at 60 and 90 min), sounds (P < 0.002 at 90 min), and meaning (P < 0.05 at 90 min). Subjects felt less energetic and clearheaded (P < 0.02 and 0.05) during ketamine, midazolam, and their co-administration. Ketamine impaired thought process (P < 0.003 and 0.0001 at 60 and 90 min). Ketamine and midazolam decreased mean total MMSE and recall scores (P < 0.001 for both). Co-administration reduced the number of subjects with perceptual (body, P < 0.01 and 0.001 at 30 and 60 min) and thought process abnormalities. Within the range of observation, co-administration did not affect the changes in mood or recall. CONCLUSION: Midazolam attenuates ketamine-induced changes in perception and thought process.

Cofactor competition between the ligand-bound oestrogen receptor and an intron 1 enhancer leads to oestrogen repression of ERBB2 expression in breast cancer.

Overexpression of the ERBB2 proto-oncogene in breast tumours, which occurs in 25-30% of patients, correlates with poor prognosis. In oestrogen receptor (ER) positive breast epithelial cells oestrogens reduce ERBB2 mRNA and protein levels, an effect that is reversed in the presence of anti-oestrogens such as tamoxifen and ICI 182780. Our previous studies have shown that the major effect of oestrogen on ERBB2 expression is at the level of transcription and that this is mediated through a region within the ERBB2 first intron which can act as an oestrogen-suppressible enhancer in ER positive breast cells. In vitro footprinting of the smallest DNA fragment that retained full activity revealed four transcription factor binding sites. We report here that two of these sites are recognized by AP-2 proteins and the other two are bound by a variety of bZIP factors, including CREB and ATFI, with a major complex containing ATFa/ JunD. However, by using ER mutants it is clear that repression occurs essentially off the DNA. Indeed, the essential domain of the ER responsible for repression of the ERBB2 enhancer is a region termed AF2 which is required for the ligand-dependent association of non-DNA binding cofactors. We further demonstrate that one of these ER cofactors, SRC-1, can relieve oestrogen repression of the ERBB2 enhancer and conclude that these data fit with a model whereby the ER and the ERBB2 enhancer compete for this limiting, non-DNA binding cofactor. Thus, in oestrogenic conditions SRC-1 preferentially binds to the ER which effectively sequesters it thereby reducing enhancer activity, but in antioestrogenic media the cofactor is released from the ER and is therefore available to activate the ERBB2 enhancer.

Genetic prodrug activation therapy for breast cancer: A phase I clinical trial of erbB-2-directed suicide gene expression.

PURPOSE: This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS: Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS: The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. CONCLUSION: The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Insulation of a conditionally expressed transgene in an adenoviral vector.

Replication-defective recombinant adenoviruses provide an efficient system for in vivo gene transfer and numerous studies have demonstrated that this vector can accommodate tissue-specific promoters to restrict the expression of a transgene to a particular subset of cells. However, in some cases the selectivity of expression is lost when the tissue-specific promoter is placed in an adenoviral environment. In an attempt to restore the conditionality of expression of the transgene driven by the human ERBB2 promoter, we have flanked the expression cassette in 5' and 3' orientations with a 250 bp sequence containing the bovine growth hormone transcriptional stop signal for cloning into a recombinant adenovirus. The data presented here clearly demonstrate that these 'insulator' elements are able to restrict the expression of the transgene (herpes simplex thymidine kinase) to ERBB2-expressing cells and therefore to restore the selectivity mediated by the ERBB2 promoter. This approach could be generally useful to insulate expression cassettes in adenoviral vectors.

Identification and quantitation of N-(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry.

A sensitive, specific and reproducible method was developed for the quantitation of the hemoglobin (Hb) adduct N-(carboxymethyl)valine (CMV). This adduct is one of various products from the Maillard reaction, involving reducing sugars and amino acids, proteins or other molecules with a free amino group. Such adducts, including N epsilon-(carboxymethyl)lysine (CML), are called advanced glycation end products (AGE) and have been correlated with aging and severity of diabetes in human tissues. This method was developed to examine the CMV-Hb adduct as a possible AGE formed by reaction of Hb with glucose or other oxidation products. CMV was cleaved selectively from isolated globin using pentafluorophenyl isothiocyanate (PFPITC) in a modified Edman degradation at pH 9.5. The carboxyl group of the adduct was derivatized to its methyl ester with diazomethane. The resulting derivative, 5-isopropyl-1-(methyl acetate)-3-pentafluorophenyl-2-thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion (m/z 396) from synthesized CMV and N-(2-carboxyethyl)valine (molecular ion m/z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2-100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal-to-noise ratio greater than 5:1 using a 1:30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g-1 Hb in these species, respectively.

Immunohistochemical analysis reveals a tumour suppressor-like role for the transcription factor AP-2 in invasive breast cancer.

This paper describes the generation and characterization of a monoclonal antibody specific for two members of the AP-2 family of transcription factors, AP-2alpha and AP-2beta, and its subsequent application to archival primary breast tumour material. Nuclear localization of AP-2 was found in all expressing cases, but in general levels of immunostaining were low, with only 17 per cent of the 86 tumours examined showing very high expression levels. Nevertheless, data analysis of the whole patient series allowed the identification of significant relationships between levels of AP-2 and other important breast markers. Thus, expression of AP-2alpha/beta was found to correlate significantly with expression of both ER ( p=0.036*) and the universal cell-cycle inhibitor p21(cip) ( p=0.03*), but was inversely related to levels of the proto-oncogene ErbB2 ( p=0.008*). AP-2-positive tumours also showed a low rate of proliferation, with significantly reduced mitotic count and a lower tumour grade. There was no significant relationship with clinical parameters, but samples with adjacent normal tissue indicated that loss of the AP-2 marker was associated with disease progression from normal breast through to invasive disease. This was confirmed by examining separate series of pure normal and pure DCIS samples, both of which expressed significantly higher levels of AP-2 ( p=0.0001* in each case) than the invasive tumours. Overall, these findings implicate AP-2alpha/beta as having a role akin to that of a tumour suppressor in breast cancer.

Expression of AP-2 transcription factors in human breast cancer correlates with the regulation of multiple growth factor signalling pathways.

The AP-2 transcription factors are required for normal growth and morphogenesis during mammalian development. Previous in vitro studies have also indicated that the AP-2 family of proteins may be involved in the etiology of human breast cancer. The AP-2 genes are expressed in many human breast cancer cell lines, and critical AP-2-binding sites are present in both the ERBB-2 (HER2/neu) and estrogen receptor promoters. We have now characterized immunological reagents that enable specific AP-2 family members, including AP-2alpha and AP-2gamma, to be detected in human breast cancer epithelium. Data obtained with these reagents demonstrate that whereas AP-2alpha and AP-2gamma are both present in benign breast epithelia, there is a significant up-regulation of AP-2gamma expression in breast cancer specimens (P = 0.01). There was also a significant correlation between the presence of the AP-2alpha protein and estrogen receptor expression (P = 0.018) and between specimens containing both AP-2alpha/AP-2gamma proteins and ERBB-2 expression (P = 0.003). Furthermore, we detected an association (P = 0.04) between the expression of AP-2gamma and the presence of an additional signal transduction molecule implicated in breast cancer, the insulin-like growth factor I receptor. Analysis of the proximal promoter of the insulin-like growth factor I receptor revealed a novel AP-2-binding site. Thus, AP-2 proteins may directly regulate the transcription of this growth factor receptor. Taken together, these data strongly support a role for the AP-2 gene family in the control of cell growth and differentiation in breast cancer.

Expression of the c-erbB-4/HER4 protein and mRNA in normal human fetal and adult tissues and in a survey of nine solid tumour types.

The c-erbB-4/HER4 receptor belongs to the family of the type I growth factor receptors. Mouse monoclonal antibodies have been raised to the cytoplasmic domain of the c-erbB-4 receptor and characterized; the antibody HFR-1 has been used to determine the pattern of expression of the c-erbB-4 protein immunohistochemically in formalin-fixed, paraffin-embedded adult and fetal tissues. The expression of c-erbB-4 mRNA was determined by using 35S-labelled riboprobes and tissue in situ hybridization. c-erbB-4 is widely expressed in many adult and fetal tissues, including the lining epithelia of the gastrointestinal, urinary, reproductive, and respiratory tracts, as well as the skin, skeletal muscle, circulatory, endocrine, and nervous systems. The developing brain and heart notably express high levels of this receptor. The pattern of c-erbB-4 protein expression is also reported in a survey of common solid human cancers. Loss of expression was noted in 40-80 per cent of adenocarcinomas and up to 100 per cent of squamous cell carcinomas, whereas overexpression was observed in about 10-20 per cent of adenocarcinomas and astrocytomas. In general, the pattern of c-erbB-4 expression in normal tissues and cancers suggests that it tends to be associated with the differentiated compartment.

RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interaction with transcription factor AP-2.

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. We recently reported that inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers including E-cadherin. Here we show that both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 was demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibited its binding to both RB and AP-2. Taken together, our results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc.