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Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the

Additional file 5: Figure S4. Protein interaction network for specific DEGs in L. brandtii (A) and L. mandarinus(B) brain under hypoxia. Pink squares represent upregulated proteins, and green squares represent downregulated proteins; size of the squares represents the importance of the protein in the network, with bigger squares indicating a greater importance; the thickness of line between the squares represents the strength of the association between two proteins, with a wider line indicating a stronger correlation.

Additional file 1: Table S1. Illumina sequencing data of the analyzed samples. Table S2. Length distribution and quality metrics of unigenes in L. mandarinus and L. brandtii. Table S3. Functional annotation results for L. mandarinus and L. brandtii transcriptomes. Table S4. Annotated DEGs for L. mandarinus and L. brandtii. Table S5. Significantly enriched GO terms for up- and downregulated DEGs in L. mandarinus and L. brandtii under severe hypoxia. Table S6. Enriched KEGG pathways for up- and downregulated DEGs in L. mandarinus and L. brandtii under severe hypoxia. Table S7. Results of RNA extraction from L. mandarinus and L. brandtii brain tissue. Table S8. RT-qPCR primers for the validation of RNA-Seq data. Table S9. Characteristics of L. mandarinus and L.brandtii samples.

Additional file 3: Figure S2. DEGs in the brains of L. mandarinus and L. brandtii under severe hypoxia vs. normoxia. FC, fold change; FDR, false discovery rate. Red, blue, and green dots represent up- and downregulated and unchanged genes, respectively.

Additional file 4: Figure S3. Gene-pathway networks for DEGs in L. mandarinus (A) and L. brandtii (B) under severe hypoxia determined using the Clue GO tool in Cytoscape. Small and large dots represent DEGs and enriched pathways, respectively. Genes indicated in blue are upregulated. Lines between dots represent connections between the genes and the pathways in the network; genes with more connections are more important in the network.

Additional file 2: Figure S1. Classification of annotated transcriptome in the Eukaryotic Orthologous Group (KOG). A to Z represent the specific entry information of the KOG annotated by unigenes.

Background Severe hypoxia induces a series of stress responses in mammals; however, subterranean rodents have evolved several adaptation mechanisms of energy metabolisms and O2 utilization for hypoxia. Mammalian brains show extreme aerobic metabolism. Following hypoxia exposure, mammals usually experience irreversible brain damage and can even develop serious diseases, such as hypoxic ischemic encephalopathy and brain edema. To investigate mechanisms underlying the responses of subterranean rodents to severe hypoxia, we performed a cross-species brain transcriptomic analysis using RNA sequencing and identified differentially expressed genes (DEGs) between the subterranean rodent Lasiopodomys mandarinus and its closely related aboveground species L. brandtii under severe hypoxia (5.0% O2, 6 h) and normoxia (20.9% O2, 6 h). Results We obtained 361 million clean reads, including 69,611 unigenes in L. mandarinus and 69,360 in L. brandtii. We identified 359 and 515 DEGs by comparing the hypoxic and normoxia groups of L. mandarinus and L. brandtii, respectively. Gene Ontology (GO) analysis showed that upregulated DEGs in both species displayed similar terms in response to severe hypoxia; the main difference is that GO terms of L. brandtii were enriched in the immune system. However, in the downregulated DEGs, GO terms of L. mandarinus were enriched in cell proliferation and protein transport and those of L. brandtii were enriched in nuclease and hydrolase activities, particularly in terms of developmental functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that upregulated DEGs in L. mandarinus were associated with DNA repair and damage prevention as well as angiogenesis and metastasis inhibition, whereas downregulated DEGs were associated with neuronal synaptic transmission and tumor-associated metabolic pathways. In L. brandtii, upregulated KEGG pathways were enriched in the immune, endocrine, and cardiovascular systems and particularly in cancer-related pathways, whereas downregulated DEGs were associated with environmental information processing and misregulation in cancers. Conclusions L. mandarinus has evolved hypoxia adaptation by enhancing DNA repair, damage prevention, and augmenting sensing, whereas L. brandtii showed a higher risk of tumorigenesis and promoted innate immunity toward severe hypoxia. These results reveal the hypoxic mechanisms of L. mandarinus to severe hypoxia, which may provide research clues for hypoxic diseases.

The human homologue of the mouse double minute 2 (MDM2) is known to be overexpressed in a variety of human malignancies. As one of E3 ubiquitin–protein ligases, MDM2 interacts with the tumour suppressor p53 by mediating ubiquitination and degradation of p53. Since abnormally expressed proteins can induce autoimmune response, to further examine whether sera from patients with esophageal squamous cell carcinoma (ESCC) exhibited immunoreactivity against MDM2 and p53, autoantibody responses to MDM2 and p53 were evaluated by enzyme-linked immunosorbent assay (ELISA) in sera from patients with ESCC and normal individuals. Positive results were also confirmed by Western blotting and indirect immunofluorescence assay. The results demonstrated that the positive rate of autoantibody against p53 and MDM2 in ESCC sera was 22.9% (36/157) and 14.0% (22/157), whereas this rate was 0% (0/85) and 1.2% (1/85), respectively, in normal individuals. Some of the sera with antibodies specific for MDM2 also contained antibodies against p53. And there was an increase of positive antibody reactions reaching a frequency of 35% (55/157) combination with MDM2 and p53. This was significantly higher than the frequency of antibodies in normal individuals (P

HCC1/CAPERα is considered to be a novel human tumor-associated antigen, and the tumor-specific immunity of HCC1/CAPERα has been reported in several types of cancer. However, there was very limited evidence indicating its function in tumorigenesis. In the present study, to elucidate the roles and underlying molecular mechanism of HCC1/CAPERα in lung cancer, we examined the expression of HCC1/CAPERα in human non-small cell lung cancer (NSCLC) cell line and NSCLC tissue microarray (TMA). Immunohistochemistry with TMA was performed to detect HCC1/CAPERα expression in NSCLC and adjacent lung tissues. NSCLC cell line constitutively transfected by pcDNA3.1-HCC1/CAPERα, and empty pcDNA3.1 vector were used. These cells were analyzed by Western blot, MTT, immunofluorescence, wound healing assay, and transwell assays. It was found that HCC1/CAPERα was mainly localized in the nucleus of the lung cancer cells and overexpression of HCC1/CAPERα may promote lung cancer cells proliferation and increase cells migration. The frequency of HCC1/CAPERα expression in NSCLC tissues was significantly higher than that in adjacent and normal tissues (P < 0.01). Our data suggest that overexpression of HCC1/CAPERα may increase the proliferation and migration of NSCLC cells, and HCC1/CAPERα could be a promising biomarker for lung cancer.

There is an urgent need to identify relevant tumor markers showing high sensitivity and specificity for early immunodiagnosis of breast cancer. Autoantibodies directed against tumor-associated antigens (TAAs) have been shown to be relevant tumor markers. The purpose of this study was to evaluate whether autoantibodies to a tumor-associated antigen p90/CIP2A can be used as diagnostic markers in breast cancer. In this study, autoantibody responses to p90/CIP2A were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay in sera from patients with breast cancer and normal human individuals. The results have demonstrated that p90/CIP2A can induce a relatively higher frequency of autoantibody response in breast cancer (19.1 %) compared to the sera of normal individuals (2.3 %). The frequency of p90/CIP2A expression in breast cancer tissues was significantly higher than that in adjacent normal tissues (P

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. Serum alpha-fetoprotein (AFP) is the conventional biomarker currently used in clinical diagnosis of this malignancy. However, AFP is not reliable for early diagnosis, and especially the sensitivity and specificity of AFP in HCC diagnosis are not optimal. Early detection of HCC is an important issue because of the very poor prognosis and usually no more than 6 months survival after diagnosis. Therefore, there is a need for the development of more sensitive and specific methods that can supplement AFP in the early detection of this cancer. In this study, autoantibody responses to 14-3-3ζ in HCC were evaluated by enzyme-linked immunosorbent assay (ELISA), western blot, and indirect immunofluorescence assay. Immunohistochemistry (IHC) with tissue array slides was also performed to analyze protein expression of 14-3-3ζ in HCC and control tissues. The prevalence of autoantibodies against 14-3-3ζ was 16.7% (28/168) in HCC, which was significantly higher than that in liver cirrhosis (LC), chronic hepatitis (CH), and normal human sera (NHS) (P 0.01). The average titer of autoantibodies against 14-3-3ζ in HCC sera was higher compared to that in LC, CH, and NHS (P 0.01). In the further study, anti-14-3-3ζ antibodies have been detected in the sera from several HCC patients with serial bleeding samples. A stronger reactive band with 14-3-3ζ in western blot can be seen in sera at 9 months before the clinical diagnosis of HCC. Our preliminary data indicate that anti-14-3-3ζ autoantibodies may be potential biomarkers for early-stage HCC screening and diagnosis.

Peroxiredoxin 1 (Prdx1) is an antioxidant and plays an important role in H2O2-mediated cell signaling. We previously found that the expression level of Prdx1 was elevated in esophagus squamous cell carcinoma (ESCC) tissue using a proteomics approach. Since overexpressed protein can induce an autoimmune response, to further examine whether serum from ESCC patients exhibits immunoreactivity against Prdx1, autoantibody responses to Prdx1 were evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with ESCC and normal individuals. Immunohistochemical study with tissue array slides and western blot analysis with cancer cell lines were also performed to analyze the protein expression profiles of Prdx1 in ESCC tissues and cancer cell lines. The results demonstrated that the positive rate of autoantibody against Prdx1 in ESCC sera was 13.2% (9/68), whereas this rate was 0% (0/89) in normal individuals. Data also showed that expression of Prdx1 was significantly increased in ESCC tissues when compared to expression in paired adjacent normal tissues (P

Somatostatin (SST) exhibits a wide range of physiological functions, including the regulation of tumor cell growth. Octreotide (OCT) is a synthetic analogue of SST that can be used to slow gastrointestinal bleeding, inhibit the release of growth hormone and impede gastrointestinal tumor growth. The aim of the present study was to investigate the molecular mechanism of OCT underlying the inhibition of gastric cancer cell proliferation. Proteins of interest were detected using western blotting, and the zinc finger protein (ZAC)-P300 complex was quantified using co-immunoprecipitation. P300-histone acetyltransferase (P300-HAT) activity was determined spectrophotometrically. The results showed that OCT decreased the phosphorylation of Akt which caused the level of ZAC to increase. In turn, the interaction between ZAC and P300 increased the activity of P300-HAT; ultimately, the phosphorylation of serine 10 in histone H3 (pS10-H3) was decreased and the acetylation of lysine 14 in histone H3 (acK14-H3) was increased. These results suggest that OCT attenuates SGC-7901 cell proliferation by enhancing P300-HAT activity through the interaction of ZAC and P300, causing a reduction in pS10-H3 and an increase in acK14-H3. These findings provide insight for future research on OCT and further demonstrate the potential of OCT to be used as a therapeutic agent for gastric cancer.

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.

Background Cancerous inhibitor of PP2A (CIP2A) is a recently characterized oncoprotein, which promotes cancer cell proliferation. But the role of CIP2A in lung cancer progression is still not well understood. Methods The expression level of CIP2A in lung cancer tissues was examined by immunohistochemistry. CIP2A-associated cell proliferation was performed by knock down or overexpression of CIP2A in lung cancer cells. Phospho-array was used to screen kinase candidates related to expression change of CIP2A. Western-blot and luciferase reporter assay were used to validate phospho-array results. Results Overexpression of CIP2A in lung cancer not only triggers immune response in lung cancer patients but also promotes lung cancer cell proliferation. By phospho-array, several kinase candidates were identified, one of which is c-Jun activated kinases (JNK). The knock down of CIP2A decreased JNK phosphorylation, and the phosphorylation of downstream transcriptional factors, ATF2 and c-Jun, whose transcriptional activity were decreased as well. Furthermore, the expression level of CIP2A also affected the phosphorylation of the upstream kinase of JNK, MKK4/MKK7. At last, treatment with JNK inhibitor partially abolished CIP2A-induced cell proliferation. Conclusion CIP2A is a tumor-associated autoantigen in lung cancer, which promote lung cancer proliferation partially through MKK4/7-JNK signaling pathway.

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized endogenous inhibitor of the phosphatase activity of protein phosphatase 2A (PP2A), which extends the half-life of oncogenic protein c-myc and promotes in vivo tumor growth. The function of CIP2A in cancer progression is still poorly understood. To uncover the underlying mechanism of CIP2A-mediated cell proliferation, we implemented a two-dimensional electrophoresis (2DE)-based proteomic approach to examine lung cancer cell H1299 with and without CIP2A. We found 47 proteins differentially expressed where 19 proteins were upregulated and 28 proteins were downregulated. These were categorized into functional groups such as metabolism (25%), transcriptional and translational control (23%), and the signaling pathway and protein degradation (20%). On one hand, we validate our proteomic work by measuring the metabolic change. The knockdown of CIP2A decreased the expression of LDH-A as well as the enzymatic activity, accompanying with a decreased lactate production, an increased NADH/NAD+ ratio and ROS production. On the other hand, we found that CIP2A may regulate CREB activity through bioinformatics analysis. Our following experiments showed that, CIP2A positively regulated the phosphorylation of CREB in response to the serum treatment. Therefore, our proteomic study suggested that CIP2A mediates cancer progression through the metabolic pathway and intracellular signaling cascade.

Matrix attachment region (MAR) is the DNA fragment that could bind to the nuclear matrix. Recently, many experiments have demonstrated that MAR can increase overall levels of foreign gene expression and decrease variability of expression. MARs have been isolated from yeast, plants, animals and human, but not from algae. In order to isolation the MAR from unicellular alga: Dunaliella salina, we first created a random MAR library as follows: The nuclear matrix were prepared from isolated nuclei by extracting with 25mM lithium diiodosalicylate (LIS) and then digested with restriction enzyme, the residual DNA fragments in the nuclear matrices were cloned into the pUC18 to construct the MAR library. Secondly, the interactions of several of the cloned DNA fragments with nuclear matrices were tested by an in vitro binding assay in which E.coli DNA was used as competitor. Some of DNA fragments could bind specifically to the nuclear matrices and four of them contained sequences related to known consensus motifs and a hairpin loop structure of MAR.

Many studies demonstrated that the immune system can recognize the antigenic changes in cancer cells, and further develop autoantibodies against these antigens which have been called tumor-associated antigens (TAAs). During transition from chronic liver disease to hepatocellular carcinoma (HCC), autoantibodies can appear which are not detected in the pre-malignant conditions. In order to explore the possibility and usefulness of using a mini-array of multiple TAAs as an approach in the diagnosis of HCC, a total of 14 TAAs, including IMP1, IMP2/p62, IMP3/Koc, p53, Survivin, p16, CAPERα, CIP2A/p90, RalA, NPM1, MDM2 and 14-3-3 ζ, c-Myc and CyclinB1, were selected for expression of full-length recombinant proteins. Antibodies against 14 TAAs in 30 sera from patients with chronic hepatitis, 30 sera from patients with liver cirrhosis and 76 sera from patients with HCC as well as sera from 16 HCC patients with serial bleeding samples were detected by enzyme-linked immunosorbent assay (ELISA). Positive results were also confirmed by Western blotting. Antibody frequency to any individual TAA in HCC varied from 6.6% to 21.1%. With the successive addition of TAAs to the panel of TAAs, there was a stepwise increase of positive antibody reactions. According to the evaluation indexes for accuracy of screening test, the sensitivity and specificity in using a panel of 14 TAAs for immunodiagnosis of HCC was 69.7% and 83.0%, respectively. The interesting finding was that these anti-TAA autoantibodies could be detected at least one year before the diagnosis of HCC, suggesting that using a penal of 14 TAAs for enhancing autoantibody detection would be a valuable approach in the early diagnosis of HCC. The mini-array of 14 TAAs can identify 43.8% HCC patients who shows serum alpha-fetoprotein (AFP) negative. This study deals with the concept of “cancer immunomics” which allows a global analysis of the auto-antibodies against antigens in a neoplasm. It also determinates that malignant transition to HCC is associated with increased autoantibody responses to certain cellular proteins. In summary, the data from this study further support the hypothesis that a customized TAA array can constitute promising and powerful tool for enhancing the serological anti-TAA antibody detection of HCC, especially in patients with serum AFP negative. Citation Format: Liping Dai, Mei Liu, Qing Zhu, Xinxin Liu, Yurong Chai, Pengfei Ren, Kaijuan Wang, Chunhua Song, Peng Wang, Mingan Wang, Eng M. Tan, Jianying Zhang. Using immunomic approach to enhance tumor-associated autoantibody detection in diagnosis of hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 885. doi:10.1158/1538-7445.AM2014-885

Autoantibodies to autologous cellular antigens in cancer patients have been considered as reporters identifying antigenic changes in cellular factors involved in the transformation process. We have made use of such autoantibodies from hepatocellular carcinoma patients to immunoscreen a cDNA expression library and have identified a cytoplasmic RNA-binding protein p62. Autoantibodies to p62 have been detected in up to 21% of a cohort of hepatocellular carcinoma (HCC) patients. Further study has demonstrated that p62 is a member of insulin-like growth factor 2 mRNA binding protein family (IMPs), which is an isoform of IMP2. The three members of the IMP family (IMP1, 2 and 3) have been found to regulate translation of insulin-like growth factor 2 (IGF2) mRNA in a post-transcriptional manner. Since several studies have suggested that IMP1 and IMP3 play important roles in cancer progression, whether p62/IMP2 can also play a role in cancer formation remains to be investigated. In this study, we intend to interrogate the role of p62/IMP2 in HCC formation. We found that the expression of p62/IMP2 (57.5%) is significantly higher in HCC specimen than that in normal liver tissue (15.4%) by immunohistochemistry study. Furthermore, p62/IMP2 is detected to be overexpressed in several liver cancer cell lines. Depletion of p62/IMP2 via lentivirus-mediated knockdown in SNU449 liver cancer cell line decreased the cell proliferation. On the contrary, the ectopic overexpression of p62/IMP2 increased cell proliferation. Our data suggested that p62/IMP2 is overexpressed in liver cancer and is functionally correlated with cancer cell growth. Citation Format: Ningjing Lei, Bo Peng, Yang Li, Yurong Chai, Jianying Zhang. p62/IMP2 promotes hepatocellular carcinoma (HCC) progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5381. doi:10.1158/1538-7445.AM2013-5381