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.

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.

AP2alpha alters the transcriptional activity and stability of p53.

AP2alpha and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2alpha are mediated through p21WAF1/CIP1 and the ability for AP2alpha to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305-375. Analysis of 26 distinct p53 alleles established a correlation between AP2alpha binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2alpha to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2alpha overexpression. We conclude that AP2alpha has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester.

DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA-3'-phosphotyrosyl-enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'-pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein-DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.

Prevalence and phylogenetic characterisation of TT-virus in the blood donor population of Auckland, New Zealand.

TT-virus (TTV, patient initials: T.T.), a novel DNA virus, was first isolated in Japan in 1997 from serum of a patient with post-transfusion hepatitis of unknown aetiology. To date, the contribution of TTV to liver disease remains doubtful. The potential for transmission via blood and blood products makes it essential to establish the prevalence of TTV viraemia in the blood donor population. 413 blood donor serum samples were chosen randomly, the DNA was extracted and TTV-specific DNA amplified by nested polymerase chain reaction (PCR). TTV infection was present in 13 out of 413 (3.15%) blood donors in the Auckland region of New Zealand using a set of primers targeting open reading frame (ORF) 1. These 13 amplification products (264 bp) were sequenced and TTV genotypes determined. Alignment with published TTV sequences showed that seven (53.8%) of the thirteen positive serum samples belonged to genotype 1, five (38.5%) belonged to genotype 2 and one (7.7%) could not be classified as either genotype 1 or 2. One hundred twenty-seven blood donor serum samples were retested with a second set of primers targeting the 5' region of the TTV genome in a single round PCR. Forty-three samples were positive for TTV DNA with these primers resulting in a prevalence of 37%. The data demonstrate that TTV is present among New Zealand blood donors and support the need for further investigation into the natural history of TTV infection.

Malarial antibodies in Auckland blood donors.

AIM: To determine the malarial exposure characteristics of "malarial risk" blood donors and measure the potential infectivity of their donations using a commercially available malarial antibody screening kit. METHOD: Malarial risk donors were identified according to standard protocols, questioned as to their degree of exposure to malaria and blood samples were tested for malarial antibodies using an enzyme immunoassay kit. The kit used detects IgG antibodies to P. falciparum, shows 50% crossreactivity with P. vivax and some crossreactivity with P. ovale. Antibody positive samples were further checked by a direct immunochromatographic test for P. falciparum. RESULTS: We found 1.7% of the donors who were classified as a "malarial risk" to be positive for IgG malarial antibodies. None of these antibody positive samples was positive by the direct immunochromatographic test for P. falciparum. CONCLUSION: These results indicate that none of these donors tested were a risk of transmitting P. falciparum, the major and most serious cause of transfusion transmitted malaria. The introduction of malarial testing of malarial risk blood donors in Auckland, currently deferred for plasma donation only, could potentially recover 2300 units of red cells per year.

Non-insulin-dependent diabetes mellitus in New Zealand Maori: a relationship with Class I but not Class II histocompatibility locus antigens.

AIMS: To investigate the possibility of a relationship between the major histocompatibility complex (MHC) and non-insulin-dependent diabetes mellitus (NIDDM) in Maori. Such relationships have previously been shown in non-European races with a high incidence of NIDDM. METHODS: We performed serological Class I and PCR-SSP Class II HLA typing on 44 Maori with NIDDM and renal failure and compared the results with normal Maori. RESULTS: A strong relationship with the HLA-B40 groups of antigens (relative risk 5.1 chi 2 = 16.8, p < 0.001) was found; this was mainly attributable to HLA-B48 and HLA-B60. There was no HLA Class II relationship. CONCLUSION: The relationship with HLA-B40 antigens suggests that the MHC or other genes on chromosome 6 play a role in NIDDM in Maori.

A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene.

A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmond's classification.

Mutations causing coagulation factor XIII subunit A deficiency: characterization of the mutant proteins after expression in yeast.

We identified the mutations causing factor XIII A subunit deficiency in two families. Two distinct mutations were identified in the S family: the nonsense mutation Tyr 441-->stop in exon 11, inherited through the paternal line, and the missense mutation Asn 60-->Lys in exon 3, inherited through the maternal line. Two members of the J family were heterozygous for the previously described type 3 A subunit. The substitution giving rise to the type 3 variant was found to be Gly 501-->Arg in exon 12. The Asn 60-->Lys and Gly 501-->Arg mutations were constructed in cDNA clones and expressed in yeast (Saccharomyces cerevisiae AH22). Although mRNA could be detected, protein containing the Asn 60-->Lys substitution could not be detected, suggesting extreme instability or susceptibility to proteolysis. A subunits containing the Gly 501-->Arg substitution were expressed and found to be enzymatically active in fresh yeast lysates. This variant has thermal instability and lost activity during storage or purification. Gel filtration studies suggested that the type 3 variant assembled as a dimer, as do normal A subunits. The data suggest that the Gly 501-->Arg (Type 3 variant) would cause severe factor XIII deficiency if inherited in the homozygous form or as a compound heterozygote with another deleterious mutation.

HLA class I gene, antigen and haplotype frequencies in New Zealand Maori and Europeans.

AIM: To determine class I HLA gene, antigen and haplotype frequencies for New Zealand Maori and Europeans. METHODS: Statistical analysis was performed using accumulated data from Maori (n = 576) and European (n = 1747) parentage studies. RESULTS: HLA class I gene, antigen and haplotype frequencies are as tabulated. Significant statistical differences were shown to exist between New Zealand Maori, New Zealand Europeans and Europeans. Gene frequencies of HLA A1, 3, 9, 11, 19 and HLA B7, 8, 12, 16, 22, and 40 differed significantly between New Zealand Maori and New Zealand European data. Gene frequencies of HLA A1 and B12 differed significantly between New Zealand European and European data. CONCLUSIONS: This HLA frequency data can be used for calculating more reliable indices of paternity or for determining the potential availability of matched organs for transplantation, as well as for anthropological studies. The data may also be useful in forensic investigations.

The hospital transfusion committee: a step towards improved quality assurance.

Quality assurance has an important contribution to make in the judicious use of scarce resources. Auckland Hospital has established a transfusion committee because there was an escalating usage of blood and blood products which are expensive prescription medicines. A pilot audit of red cell transfusions indicated that 29% of red cell transfusions may have been unnecessary. A wide range of initiatives at Auckland Hospital has reduced blood product usage. Inappropriate use of blood carries an opportunity cost and may subject patients to unnecessary risk of reactions, including potential disease transmission. Strategies which need to be employed by transfusion committees include the introduction of clinical audit, physician education, restrictions on availability, and clinical budgeting. It is recommended that transfusion committees be set up in all major hospitals.