Targeting to overexpressed glucose-regulated protein 78 in gastric cancer discovered by 2D DIGE improves the diagnostic and therapeutic efficacy of micelles-mediated system

The survivals of gastric cancer (GC) patients are associated with early diagnosis and effective treatments. Therefore, it is urgent for the discovery of early GC biomarkers and tumor-targeting therapeutics. The aim of this study was to uncover putative tissue biomarkers of GC using 2D DIGE and then apply one of these specific markers in GC treatment. We found three putative biomarkers of GC with significant differences in expression level compared to adjacent normal tissue, including glucose-regulated protein 78 (GRP78) and glutathione s-transferase pi (GSTpi) with increased expression level, and alpha-1 antitrypsin (A1AT) with reduced expression level. The overexpressed GRP78 was used as a targeted protein for guiding the drugs to tumor cells, leading to more effective treatment for GC xenografts. Our results demonstrated that the designated GRP78-binding peptide based on the sequence, WIFPWIQL, was selectively prone to recognize and bind to GC MKN45 cells in vitro, and also improve the delivery efficiency of polymeric micelles-encapsulated drugs into tumor cells and displayed better therapeutic outcome in experimental animals. This strategy of GRP78-mediated drug targeting system may bring chemotherapeutic drugs with more precise targeting to tumor cells, leading to minimize side effects on patients after chemotherapy.     

Overexpression of GRP78 and GRP94 is involved in colorectal carcinogenesis

Glucose-related proteins (GRPs) are ubiquitously expressed in the endoplasmic reticulum and assist in protein folding and assembly, consequently considered to be molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in samples taken from several different malignant tissues. To clarify the roles of both molecules in tumorigenesis and progression of colorectal carcinomas, immunohistochemistry (IHC) was performed on tissue microarrays containing colorectal carcinomas, adenomas and the non-neoplastic mucosa (NNM) using antibodies against GRP78 and GRP94. Their expression was correlated with the clinicopathological parameters of carcinomas. Both proteins were also studied in colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) by IHC and Western blot. There was a gradually increased GRP78 expression from colorectal NNMs, carcinomas, to low-grade and high-grade adenomas (P<0.05), while up-regulated GRP94 expression from NNM, low-grade adenoma, high-grade adenoma, to carcinoma (P<0.05). The expression was similar in all the carcinoma cell lines. GRP78 expression was negatively correlated with lymphatic invasion or low GRP94 expression of the carcinomas (P<0.05), while there was no correlation of GRP94 expression with other parameters of carcinomas (P>0.05). Multivariate analysis showed that venous invasion, lymph node metastasis and UICC staging (P<0.05), but not age, sex, tumor size, differentiation, depth of invasion, lymphatic invasion, GRP78 and GRP94 expression (P>0.05), were independent prognostic factors for carcinomas. It is suggested that up-regulated expression of GRP78 and GRP94 could possibly be involved in the pathogenesis of colorectal carcinomas.     

人胃癌组织中herg1基因和HERG1蛋白过度表达的临床病理意义的研究

目的探讨herg1基因及其表达的HERG1蛋白在胃癌中的表达情况,研究其与胃癌间的相关性。方法应用免疫组化方法（SP法）,RT—PCR技术及Westernblot方法对64例胃癌组织标本及对应正常胃粘膜组织中的herg1基因及HERG1蛋白的表达进行检测,并进行回顾性随访。结果（1）RT—PCR分析胃癌组织中herg1基因mRNA阳性率为100％（64／64）,正常组织中为0％（0／64）。（2）Western blot方法检测胃癌组织中HERG1蛋白表达阳性率为100％（64／64）,正常组织中为0％（0／64）。（3）免疫组化分析HERG1蛋白在胃癌组织的阳性率为81．25％（52／64）,与其病理类型无关,而在正常组织中未见明显表达,在伴有淋巴结转移或肝转移的胃癌herg1的阳性表达率为100％（10／10）,无转移者为78％（42／54）。Hegrl基因的表达与胃癌的淋巴转移、肝转移有相关性（P〈0．01）。结论Herg1基因和HERG1蛋白的过度表达参与了胃癌的发生发展过程,同时提示该基因和蛋白的检测可以预测胃癌组织的侵袭、转移倾向,为建立肿瘤转移的分子标志提供了理论和实验依据。     

Comparative analysis of the protein profiles from primary gastric tumors and their adjacent regions: MAWBP could be a new protein candidate involved in gastric cancer

The study of tumor biomarkers is generally facilitated by the adoption of proteomic strategies. With limitations of techniques and individual varieties of biological samples, the biomarkers for gastric cancer (GC) have not reached a common agreement derived from the proteomic investigations. Herein, we reported a new set of data for screening the biomarkers from the gastric tissues, on the basis of the proteomic strategy developed in our laboratory. Ten pairs of the clinic samples were collected and treated with protein extraction. The gastric proteins were well-resolved by 2-DE, and the GC-associated proteins were identified by MALDI-TOF/TOF MS following image analysis, including 12 up-regulated and 13 down-regulated unique ones. MAWBP was found to be one of the new GC proteins which appeared with lower expression in the GC tissues. We expanded a systematical examination to deeply pursue the relevance between MAWBP and GC. Quantitatively, we measured the expression of MAWBP with Western blot and Real-Time PCR. Extendedly, we estimated the existence of MAWBP with immunohistochemical staining in a large number of the GC cases. Specifically, we inquired whether MAWD, a protein with high affinity to MAWBP, could coexpress and interact with MAWBP in vivo. On the basis of all the results, we concluded that MAWBP could be a new GC-related protein even though its physiological roles remain unexplored.     

Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.     

Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer

The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human samples by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Mutation at Intronic Repeats of the Ataxia-Telangiectasia Mutated (ATM) Gene and ATM Protein Loss in Primary Gastric Cancer with Microsatellite Instability

Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC.     

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27^(Ser15), p-HSP27^(Ser78), p-HSP27^(Ser82), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27^{(Ser15, Ser78, Ser82)} and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.     

GRP78 的表达与非小细胞肺癌生物学特性及预后的关系

目的:探讨GRP78在非小细胞肺癌和癌旁组织中的表达情况,并研究其与生物学特征及临床预后的关系 方法:收集非小细胞肺癌术后切除标本88例,及其癌旁组织20例作为对照.采用免疫组织化学方法检测GRP78的表达.结果:GRP78在非小细胞肺癌组织和癌旁组织中的表达有统计学差异.GRP78的表达与非小细胞肺癌的临床分期、分化程度有关,而与患者性别、年龄和病理类型无关.非小细胞肺癌中GRP78高表达的患者生存时间短于GRP78低表达的患者.GRP78的表达情况是影响非小细胞肺癌患者手术预后的独立危险因素.结论:非小细胞肺癌患者的GRP78的表达可能与肿瘤细胞的发生及发展有关,GRP78可以作为一个预测非小细胞肺癌患者预后的分子标志物.     

Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma

To identify proteins linked to the pathogenesis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), we profiled protein expression levels in samples of HCC. To identify essential proteins, ten samples of HCV-related HCC were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These experiments revealed increased levels of nine proteins in cancerous tissues compared to levels in corresponding noncancerous liver tissues. We focused on four members of the heat shock protein 70 family: 78 kDa glucose-regulated protein (GRP78), heat shock cognate 71 kDa protein (HSC70), 75 kDa glucose-regulated protein (GRP75), and heat shock 70 kDa protein 1 (HSP70.1). These results were confirmed by immunoblot analysis. In an additional 11 samples, the same expression patterns of these four proteins were observed. In total, 21 samples showed statistically significant up-regulation of GRP78, GRP75 and HSP70.1 in cancerous tissues. HSC70 showed a tendency toward overexpression. There has been no report describing overexpression of these four proteins simultaneously in HBV-related HCC as well as nonviral HCC. Our results suggest that these four proteins play important roles in the pathogenesis of HCV-related HCC and could be molecular targets for diagnosis and treatment of this disease.     

Differential expression of glucose-regulated protein 78 during spermatogenesis

The aim of the present study was to isolate and identify proteins involved in the process of spermatogenesis. To achieve this goal we used the technique of proteomic analysis. Comparison of testis protein patterns obtained by high-resolution two-dimensional gel electrophoresis from 1-week- and 7-week-old mice showed significant differences in protein spot intensities. Subsequently several of these variant protein spots were identified by mass spectrometry. Glucose-regulated protein (GRP) 78 (Bip) was one of them. GRP78, expressed at a lower level in 1-week-old mouse testes compared to 7-week-old mouse testes, is a member of the heat shock 70 protein family. It has recently been shown to be important for protecting cells from apoptosis in somatic cells, especially in progressively growing tumor cells. Further, immunohistochemical (IHC) analyses of mouse and human testes sections were performed to determine the cellular distribution of this protein. A strong GRP78 staining was seen beginning with pachytene spermatocytes. These findings suggested that GRP78 might perform an important function in the process of spermatogenesis.The first two authors contributed equally to the work. The research was supported by grants from China National 973 (G1999055901) and the Chinese Natural Science Foundation (30170485)     

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data‐independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down‐regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.     

m6A methylation mediates LHPP acetylation as a tumour aerobic glycolysis suppressor to improve the prognosis of gastric cancer

LHPP, a histidine phosphatase, has been implicated in tumour progression. However, its role, underlying mechanisms, and prognostic significance in human gastric cancer (GC) are elusive. Here, we obtained GC tissues and corresponding normal tissues from 48 patients and identified LHPP as a downregulated gene via RNA-seq. qRT-PCR and western blotting were applied to examine LHPP levels in normal and GC tissues. The prognostic value of LHPP was elucidated using tissue microarray and IHC analyses in two independent GC cohorts. The functional roles and mechanistic insights of LHPP in GC growth and metastasis were evaluated in vitro and in vivo. The results showed that LHPP expression was significantly decreased in GC tissues at both the mRNA and protein levels. Multivariate Cox regression analysis revealed that LHPP was an independent prognostic factor and effective predictor in patients with GC. The low expression of LHPP was significantly related to the poor prognosis and chemotherapy sensitivity of gastric cancer patients. Moreover, elevated LHPP expression effectively suppressed GC growth and metastasis in vitro and in vivo. Mechanistically, the m6A modification of LHPP mRNA by METTL14 represses its expression; LHPP inhibits the phosphorylation of GSK3b through acetylation and mediates HIF1A to inhibit glycolysis, proliferation, invasion and metastasis of gastric cancer cells. Together, our findings suggest that LHPP is regulated by m6A methylation and regulates the metabolism of GC by changing the acetylation level. Thus, LHPP is a potential predictive biomarker and therapeutic target for GC.Subject terms: Gastric cancer, Predictive markers     

Expression of programmed cell death 5 gene involves in regulation of apoptosis in gastric tumor cells

The protein of programmed cell death 5 (PDCD5) is believed to participate in regulation of apoptosis. Although PDCD5 is reducibly expressed in various human tumors, it is not clear which expression level of PDCD5 is in gastric cancer (GC). In this study, we have systematically employed the approaches of RT-PCR, Real- time PCR, Immunohistochemistry (IHC), Immunofluorescence staining (IFS) and Western blot to determine the PDCD5 expression in GC cells and primary tumors, at mRNA and protein level, respectively. Our data revealed that the positive rate of PDCD5 expression in the gastric tumor tissues was significantly less than that of the normal tissues (14 out of 102 vs 36 out of 51), whereas, the decreased expression of PDCD5 protein was well correlated with the up-regulated expression of Bcl-2 in these tissues, and the up-regulated expression and nuclear translocation of PDCD5 protein were verified in the apoptotic GC cells induced by Diallyl trisulfide (DATS). Furthermore, the survival curve has suggested that the more PDCD5 expressions were found in the patients, the longer the survival periods were. Therefore, our observations lay down a reasonable postulation that PDCD5 may play a key role to regulate the apoptotic processes in the GC cells and gastric tumors.     

Comparative proteomic analysis of cancerous and adjacent normal lung tissues

Lung cancer is the leading cause of cancer-related mortality in industrialized countries. Unfortunately, most lung cancers are found too late for a cure, therefore early detection and treatment is very important. We have applied proteomic analysis by using two-dimensional gel electrophoresis and peptide mass fingerprinting techniques for examination of cancerous and adjacent non-cancerous lung tissues from the same patient. The aim of the study was to find proteins, which could be used as biomarkers for diagnosis and monitoring of this disease. Indeed, we found differences in expression of several proteins, related to various cellular activities, such as, chaperoning (e,g. GRP96, GRP78, HSP27), metabolism and oxidation stress (e.g. L-fucose, GST), cytoskeleton (e.g., tubulin beta 2/3, beta actin), cell adhesion (e.g. annexin A5/3), binding proteins (e.g. 14-3-3 theta) and signal transduction. These changes may be important for progression of carcinogenesis; they may be used as the molecular-support for future diagnostic markers.     

Endoplasmic reticulum stress response is involved in the pathogenesis of stress induced gastric lesions in rats

Stress gastric ulcer is a serious complication, but the mechanism involved is not fully clarified. It is well known that mucosal cell apoptosis plays a crucial role in the pathogenesis of gastric ulceration. Recent studies have shown that endoplasmic reticulum (ER) stress is an important pathway leading to cellular apoptosis. To investigate the role of ER stress in the pathogenesis of stress gastric ulcer, we studied the alteration in the expression of ER stress markers GRP78 (glucose-regulated protein 78) and caspase-12 (an ER stress-specific proapoptotic molecule) and their relations with gastric mucosal apoptosis during development of stress gastric lesions in the water-immersion and restraint stress (WRS) model in rats. Rats developed severe gastric lesions after 6 h of WRS. Typical apoptosis was observed at the edge cells of WRS induced gastric lesions. Western blot analysis showed that GRP78 and activated caspase-12 were over-expressed in the gastric tissues of WRS rats. Immunohistochemical analysis demonstrated that increased GRP78 and caspase-12 were distributed only under the lesions. In addition, dithiothreitol and tunicamycin (ER stress inducers), which increased the expression of GRP78 and activated caspase-12, caused gastric mucosal injury and mucosal cell apoptosis in vitro. These findings suggest that ER stress might be involved in the development of stress gastric ulcer through an apoptotic mechanism.     

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P = 0.043, odds ratio [OR] = 0.805, 95% confidence intervals [95% CI] = 0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P = 0.021, OR = 2.882, 95% CI = 1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P = 0.004), alcohol drinking (P < 0.001), tumor location (P = 0.007), histological type (P = 0.005) and differentiation (P = 0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.     

Antibody performance in western blot applications is context‐dependent

An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well‐designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application‐specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full‐length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full‐length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context‐ and application‐dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross‐reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.