ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex with p-p53 and p-ERK in response to DNA damage.

DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK).

Effect of combined treatment with progesterone and tamoxifen on the growth and apoptosis of human ovarian cancer cells.

Progesterone has a potential protective effect against ovarian carcinoma induced by estrogen. Progesterone is also known to cause apoptosis while tamoxifen induces growth arrest. Therefore, we attempted to determine whether combined treatment with progesterone and tamoxifen has a synergistic effect on anti-cancer activity.

Higher DNA repair activity is related with longer replicative life span in mammalian embryonic fibroblast cells.

Since the detailed comparison of DNA repair activities among mammalian embryonic fibroblast cells with different replicative life spans has not been investigated, we tested DNA repair activities in embryonic fibroblast cells derived from mammals including human, dog, rat, and mouse. The cell viability after treatment of four DNA damage agents appeared to be decreased in the order of human embryonic fibroblasts (HEFs) > dog embryonic fibroblasts (DEFs) > rat embryonic fibroblasts (REFs) > mouse embryonic fibroblasts (MEFs) although statistical significance was lacking. The amounts of strand breaks and AP (apurinic/apyrimidinic) sites also appear to be decreased in the order of HEFs > DEFs > REFs ≥ MEFs after treatment of DNA damage agents.

Calorie restriction (CR) reduces age-dependent decline of non-homologous end joining (NHEJ) activity in rat tissues.

Even though CR has shown to enhance base excision repair (BER) and nucleotide excision repair (NER) capacities, it has not been reported whether CR can enhance non-homologous end joining (NHEJ) activity. To examine the effect of CR on NHEJ activity, ad libitum (AL)- and calorie restricted (CR)-dieted rats were used. Age-dependent decline of NHEJ activity was apparent in the lung, liver, and kidney and appeared to be slightly decreased in spleen.

ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to DNA damage in normal human embryonic fibroblast (HEF) cells.

Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level.

Nitric oxide is an essential mediator for neuronal differentiation of rat primary cortical neuron cells.

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells.

WDNM1 is associated with differentiation and apoptosis of mammary epithelial cells.

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells.

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells.

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha.

Broad spectrum identification of SUMO substrates in melanoma cells.

Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles.

Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.

Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria.

A tagging-via-substrate technology for genome-wide detection and identification of farnesylated proteins.

Protein farnesylation is one of the most common lipid modifications and has an important role in the regulation of various cellular functions. We have recently developed a novel proteomics strategy, designated the tagging-via-substrate (TAS) approach, for the detection and proteomic analysis of farnesylated proteins. This chapter describes the principle of TAS technology and details the method for detection and enrichment of farnesylated proteins.

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively.

Proteomic analysis of integral plasma membrane proteins.

Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins.