Determination of urea content in urea cream by centrifugal partition chromatography.

The objective of this study is to establish a centrifugal partition chromatography (CPC) method for determination of the urea ingredient in urea cream. The mechanism of this method is that urea is determined by UV detector at 430 nm after being extracted from the cream and derivatized on line via Ehrlich reaction in rotor of CPC, where the reaction products dissolve in the mobile phase and the cream matrix retains in the stationary phase. The mixed solvent consisting of n-hexane, methanol, hydrochloric acid and p-dimethylaminobenzaldehyde with a ratio of 1000 mL:1000 mL:18 mL:2.0 g is used for solvent system of CPC. The CPC method proposed offers good precision and convenience without complex sample pretreatment processes.

JimMY on the stage: Linking DNA damage with cell adhesion and motility.

Cellular DNA undergoes constant assault from a wide range of genotoxic stress. In order to maintain genome integrity, cells develop a repertoire of sophisticated systems to detect DNA damage and mediate cellular responses to DNA damage. Defects in the DNA damage response have been implicated in a variety of disorders including aging and cancer. Tumor suppressor p53 is a key intermediate in DNA damage response by inducing cell cycle arrest to allow repair or promoting apoptosis to eliminate irreparably damaged cells. A recent study described a novel layer of p53-mediated cellular response to DNA damage, i.e., modulation of cell adhesion and motility. JMY, a p53 co-factor, was demonstrated to be a multifunctional protein that coordinates cell adhesion and motility with nuclear p53 response. These results suggest that abnormal JMY activity and/or localization could contribute to tumor invasion and reveal JMY as a potential therapeutic target.

Breast cancer metastasis driven by ErbB2 and 14-3-3zeta: A division of labor.

Metastasis remains the leading cause of cancer morbidity and mortality. ErbB2, a metastasis-promoting oncoprotein, is overexpressed in 50-60% of noninvasive ductal carcinoma in situ (DCIS). However, only 25% of invasive breast cancer (IBC) overexpress ErbB2, indicating that ErbB2 alone is not sufficient to drive metastasis and additional risk factors are necessary for the progression of ErbB2-overexpressing DCIS to IBC. A recent study published in Cancer Cell identified 14-3-3xi as a risk factor aiding the transition of ErbB2-overexpressing DCIS into IBC. Furthermore, the study elucidated molecular mechanisms by which ErbB2 and 14-3-3xi co-overexpression drives metastasis. Namely, ErbB2 promotes cell motility and migration via the activation of Src, while 14-3-3xi induces epithelial-mesenchymal transition by activating TGFbeta pathway to reduce cell adhesion. On the other hand, two studies recently published in British Journal of Cancer and Oncogene provide mechanistic insight into how ErbB2 signalling is transduced via Src, focal adhesion kinase and Ste20-like kinase to regulate focal adhesion turnover and modulate cell motility and migration. Taken together, these studies reveal that metastasis engages a variety of players that must show team spirit to win the game of spreading.

Wnt/Planar cell polarity signaling: a new paradigm for cancer therapy.

The evolutionarily conserved and developmentally important Wnt signaling pathway has traditionally been regarded as a critical player in tumorigenesis through the canonical Wnt/beta-catenin cascade. Nevertheless, accumulating evidence based on recent research has revealed the previously unacknowledged role of noncanonical Wnt/planar cell polarity (PCP) signaling in cancer progression, invasion and metastasis, and angiogenesis. This review describes the PCP signaling pathway and its ever-expanding components and modulators, highlights the most recent studies that provide insight into the link between PCP signaling and cancer, and, finally, proposes a model by which PCP signaling may promote cancer development. This review underscores the emerging theme that deregulated PCP signaling contributes to tumorigenesis, providing new potential targets for cancer therapy.

MERIT40 controls BRCA1-Rap80 complex integrity and recruitment to DNA double-strand breaks.

Rap80 targets the breast cancer suppressor protein BRCA1 along with Abraxas and the BRCC36 deubiquitinating enzyme (DUB) to polyubiquitin structures at DNA double-strand breaks (DSBs). These DSB targeting events are essential for BRCA1-dependent DNA damage response-induced checkpoint and repair functions. Here, we identify MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kD)/(C19orf62) as a Rap80-associated protein that is essential for BRCA1-Rap80 complex protein interactions, stability, and DSB targeting. Moreover, MERIT40 is required for Rap80-associated lysine(63)-ubiquitin DUB activity, a critical component of BRCA1-Rap80 G2 checkpoint and viability responses to ionizing radiation. Thus, MERIT40 represents a novel factor that links BRCA1-Rap80 complex integrity, DSB recognition, and ubiquitin chain hydrolytic activities to the DNA damage response. These findings provide new molecular insights into how BRCA1 associates with independently assembled core protein complexes to maintain genome integrity.

Inefficient proteasomal-degradation pathway stabilizes AP-2alpha and activates HER-2/neu gene in breast cancer.

HER-2/neu proto-oncogene is overexpressed in about one fourth of human breast cancers. AP-2 transcription factors bind to the HER-2/neu gene promoter and activate its expression. In a striking concurrence, anomalous abundance of AP-2alpha protein or its homolog AP-2gamma is also detected with HER-2/neu protein in mammary tumor-derived cell lines. This suggests that the deregulation of AP-2 is the preceding pathogenic event and probably the pivotal one in this type of mammary carcinogenesis. We examined the process of AP-2alpha gene expression in mammary carcinoma cell lines to identify where the aberration had occurred. We found no amplification of the AP-2alpha gene. Its promoter was marginally upregulated; however, it did not significantly increase the mRNA levels. When the AP-2alpha protein was examined, a remarkable stability was seen in breast cancer cell lines MDA-MB-453 and SK-BR-3, with a half-life of over 30 hr. This is sharply higher than the approximate 1 hr observed in mammary epithelial cell line MCF-10A and murine cell line NIH 3T3. Treatment of MCF-10A and NIH 3T3 cells with the proteasome inhibitor MG-132 showed that AP-2alpha was ubiquitinated and its level significantly increased. Moreover, this increase was accompanied by elevated levels HER-2/neu protein. In contrast, weaker ubiquitination of AP-2alpha was seen in MDA-MB-453 and SK-BR-3 cancer cells, and MG-132 treatment did not raise the AP-2alpha level any further. These results uncover that unusual stability is the main mechanism that raises the levels of AP-2 proteins, and in addition, provide the first clue that defective ubiquitin-dependent proteasomal-degradation pathway is possibly the prime cause that affects the HER-2/neu gene and culminates in breast cancer.

Functional characterization of the interacting domains of the positive coactivator PC4 with the transcription factor AP-2alpha.

The transcriptional positive cofactor 4 (PC4) physically interacts with the transcription factor, activator protein-2 (AP-2) alpha, and overexpression of PC4 results in a relief of the AP-2 transcriptional self-interference, which is induced by high levels of AP-2alpha expression. PC4 was initially described as a DNA-binding protein that enhances the activator-dependent transcription of class II genes in vitro, but it was later shown that PC4 could also act as a potent repressor of transcription on specific DNA structures such as single-stranded (ss) DNA, DNA ends and heteroduplex DNA. To further explore the functional domains of PC4 and its ssDNA-binding effect in the interaction with AP-2alpha and on AP-2 transcriptional activity, we investigated the C-terminal domain of PC4 (PC4-CTD) and several PC4 mutants in which the ssDNA binding function was interrupted. We found that the C-terminal domain of PC4 physically interacts with AP-2alpha and retains the function of full-length protein in relieving transcription self-interference of AP-2. A point-mutated form of PC4 within the C-terminal domain beta-ridge, PC4 W89A, or a triple mutant in the beta2-beta3 loop of PC4, F77A/K78G/K80G, inactivate the ability of PC4 to bind AP-2alpha and to relieve the transcription self-interference of AP-2alpha. In addition, point-mutated forms of AP-2alpha within the activation domain (AD) that inactivate AP-2 transcription activity also lose their self-interference function. Our data suggest that the C-terminal domain of the transcription cofactor PC4 is critical for AP-2alpha transcriptional interference that is mediated by the activation domain of AP-2alpha.

The human transcription factor activation protein-2 gamma (AP-2gamma): gene structure, promoter, and expression in mammary carcinoma cell lines.

Human activation protein-2 gamma (hAP-2gamma) is a key developmental transcription factor. It has been implicated in mammary carcinogenesis through its regulation of HER-2/neu proto-oncogene and estrogen receptor gene The hAP-2gamma gene is located on human chromosome 20q13.2. We cloned this gene, deduced its genomic structure, and mapped and analyzed its promoter. The hAP-2gamma gene contains seven exons. Primer extension analysis and 5'-rapid amplification of complementary DNA ends studies show that there is a single transcription start site 232 nt upstream of the translational start codon. The promoter lacks canonical binding sites for basal transcription factors such as TATA and CCAAT boxes, but contains a cluster of CpG islands and may rely on an initiator element for transcription. Deletion analyses of the promoter and chloramphenicol acetyl transferase reporter gene assays indicate that the sequence between -746 and -575 is important for its expression in mammary carcinoma cell lines. The hAP-2gamma gene is marginally activated in these cells suggesting that increased transcription partly contributes to its abundance. Architecture of the gene and promoter strikingly resembles that of hAP-2alpha, which is located on a different chromosome, suggesting a cognate origin. hAP-2alpha and hAP-2gamma have some common and some distinct roles in cells, and are likely the remarkable results of gene duplication, translocation and functional divergence through evolution.

Tumorigenic effect of transcription factor hAP-2alpha and the intricate link between hAP-2alpha activation and squelching.

Overexpression of human activator protein-2alpha (hAP-2alpha) is carcinogenic. Its aberrant regulation is the underlying tumorigenic event in the human teratocarcinoma cell line PA-1. In this cell line excess hAP-2alpha protein binds and sequesters coactivators, which interferes with the activity of other activators and with its own activity. The N-terminus of hAP-2alpha, which contains an activation domain, is critical in squelching and tumorigenicity. Mutation analyses of the N-terminus region showed that activation and squelching were intricately linked; nevertheless, squelching could occur in the absence of activity. Cells overexpressing squelching-proficient mutants grew efficiently on soft agar irrespective of their ability to activate transcription, which indicates that these cells are tumorigenic. Mutants that lacked both properties were nontumorigenic. These results suggest that squelching, but not activation, causes transformation and that the factors that are sequestered at this region are critical in tumorigenesis.