Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling.

OBJECTIVES: DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. METHODS: We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. RESULTS: A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). CONCLUSIONS: To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Targeted RNA sequencing with touch imprint cytology samples for non-small cell lung cancer patients.

BACKGROUND: RNA-based sequencing is considered ideal for detecting pathogenic fusion-genes compared to DNA-based assays and provides valuable information about the relative expression of driver genes. However, RNA from formalin-fixed paraffin-embedded tissue has issues with both quantity and quality, making RNA-based sequencing difficult in clinical practice. Analyzing stamp-derived RNA with next-generation sequencing (NGS) can address the above-mentioned obstacles. In this study, we validated the analytical specifications and clinical performance of our custom panel for RNA-based assays on the Ion Torrent platform. METHODS: To evaluate our custom RNA lung panel, we first examined the gene sequences of RNA derived from 35 NSCLC tissues with diverse backgrounds by conventional methods and NGS. Next, we moved to the clinical phase, where clinical samples (all stamp-derived RNA) were used to examine variants. In the clinical phase we conducted an NGS analysis while simultaneously applying conventional approaches to assess the feasibility and validity of the panel in clinical practice. RESULTS: In the prerun phase, all of the variants confirmed with conventional methods were detected by NGS. In the clinical phase, a total of 80 patients were enrolled and 80 tumor specimens were sequenced from February 2018 to December 2018. There were 66 cases in which the RNA concentration was too low to be measured, but sequencing was successful in the vast majority of cases. The concordance between NGS and conventional methods was 95.0%. CONCLUSIONS: RNA extraction using stamp specimens and panel sequencing by NGS were considered applicable in clinical settings. KEY POINTS: Significant findings of the study Next-generation sequencing using RNA from stamp specimens was able to detect driver gene changes in non-small cell lung cancer including fusion genes with the same accuracy as conventional methods. What this study adds Using RNA from stamp specimens obtained from biopsy increases the number of candidate cases for RNA sequencing in clinical settings.

Negative reactions of BRAF mutation-specific immunohistochemistry to non-V600E mutations of BRAF.

BRAF mutations are rare driver mutations in non-small cell lung cancer (NSCLC), accounting for 1%-2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non-V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non-V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation-specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non-V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non-V600E mutations, using BRAF mutation-specific IHC. None of the tumors with non-V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation-specific IHC is specific without any cross-reactions to non-V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.

Fibrolamellar Hepatocellular Carcinoma with Multiple Lung Metastases Treated with Multidisciplinary Therapy.

A 20-year old man was diagnosed with fibrolamellar hepatocellular carcinoma (FLHCC) with multiple lung metastases, and chemotherapy with FOLFOX was administered. Contrast enhanced CT after 3 cycles of FOLFOX showed no disease progression. We therefore performed surgical resection and radiofrequency ablation of the liver lesions and lung metastases, after obtaining the patient's informed consent. The liver lesions and lung metastases tested positive for DNAJB1-PRKACA. The treatment for FLHCC with extrahepatic metastasis has not been established; however, in a few cases, good long-term prognoses were obtained with multidisciplinary therapy. We herein report a case of FLHCC with multiple lung metastases that was treated with multidisciplinary therapies.

A proliferation-inducing ligand sustains the proliferation of human naïve (CD27⁻) B cells and mediates their differentiation into long-lived plasma cells in vitro via transmembrane activator and calcium modulator and cyclophilin ligand interactor and B-cell mature antigen.

Long-lived plasma cells (PCs) contribute to humoral immunity through an undefined mechanism. Memory B cells, but not human naïve B cells, can be induced to differentiate into long-lived PCs in vitro. Because evidence links a proliferation-inducing ligand (APRIL), a tumor necrosis factor family member, to the ability of bone marrow to mediate long-term PC survival, we reasoned that APRIL influences the proliferation and differentiation of naïve B cells. We describe here the development of a simple cell culture system that allowed us to show that APRIL sustained the proliferation of naïve human B cells and induced them to differentiate into long-lived PCs. Blocking the transmembrane activator and calcium modulator and cyclophilin ligand interactor or B-cell mature antigen shows they were required for the differentiation of naïve B cells into long-lived PCs in vitro. Our in vitro culture system will reveal new insights into the biology of long-lived PCs.

Functional difference between membrane-bound and soluble human thrombomodulin.

BACKGROUND: For successful xenotransplantation, in addition to α1,3-galactosyltransferase gene-knockout and human complement regulatory protein (CD46, CD55, CD59) gene insertion, cloned pigs expressing human thrombomodulin (hTM) have been produced to solve the problem of molecular incompatibility in their coagulation system. Recombinant soluble hTM (S-hTM) which has been recently approved for treatment of disseminated intravascular coagulation might be potentially available. The purpose of this study is to examine the functional difference in endothelial cells between membrane-bound hTM (MB-hTM) and S-hTM and to elucidate effective strategy using both types of hTM. METHODS: The following factors regarding coagulation and inflammation were compared between hTM-expressing pig aortic endothelial cells (PAEC) derived from cloned pig and nontransgenic PAEC in the presence of S-hTM under tumor necrosis factor-α-activated conditions; (i) clotting time (ii) pig tissue factor (TF), (iii) pig E-selectin, (iv) direct prothrombinase activity, (v) activated protein C (APC), and (vi) prothrombinase activity. RESULTS: The MB-hTM significantly suppressed the expression of pig TF and E-selectin and direct prothrombinase activity in tumor necrosis factor-α-activated PAEC, suggesting strong anti-inflammatory effect, compared to S-hTM. In contrast, S-hTM had more potent capacity to inhibit thrombin generation and to produce APC than MB-hTM, although MB-hTM had the same level of capacity as human endothelial cells. CONCLUSIONS: It was speculated that S-hTM treatment would be of assistance during high-risk periods for excessive thrombin formation (e.g., ischemia reperfusion injury or severe infection/rejection). Considering the properties of MB-hTM exhibiting anti-inflammatory function as well as APC production, hTM-expressing cloned pigs might be indispensible to long-term stabilization of graft endothelial cells.

AMP-activated protein kinase as a promoting factor, but complement and thrombin as limiting factors for acquisition of cytoprotection: implications for induction of accommodation.

Accommodation has been termed as a condition without graft rejection even in the presence of antidonor antibody. We previously reported an in vitro accommodation model, which demonstrated that preincubation of A/B antigen-expressing endothelial cells with anti-A/B antibody resulted in ERK inactivation followed by resistance to complement-mediated cytotoxicity through the induction of complement regulatory genes. However, under the in vivo condition, the effects of complement and coagulation system cannot be ignored. The purpose of this study is to find effective ways to navigate accommodation by exploring the relevant signal transduction. Preincubation with a low level of complement or thrombin failed to induce resistance to complement-mediated cytotoxicity. AMP-activated protein kinase (AMPK) activators such as resveratrol, AICAR and metformin protected endothelial cells against complement-mediated cytotoxicity through the increase in CD55, CD59, haem oxygenase-1 (HO-1) and ferritin heavy chain (ferritin H) genes, all of which were attenuated by AMPKα knock-down. Resveratrol counteracted the inhibitory effect of pretreated complement and thrombin on acquisition of resistance to complement-mediated cytotoxicity through AMPKα. AMPK regulation in endothelial cells could become the potential strategy to induce accommodation in clinical pro-inflammation and pro-coagulation.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

BACKGROUND: Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. METHODS: Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. RESULTS: Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. CONCLUSION: Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

Adipose-derived stromal cells cultured in a low-serum medium, but not bone marrow-derived stromal cells, impede xenoantibody production.

BACKGROUND: Although the immunomodulatory effects of mesenchymal stromal cells (MSC) on T cells have been elucidated, little is known about their effects on B cells. Recently, we have established a novel culture method for adipose-derived MSC (ASC) using low (2%) serum medium containing fibroblast growth factor-2. We showed that low serum-cultured ASC (LASC) was superior to high (20%) serum-cultured ASC (HASC) when used in regenerative therapy. The aim of this study was to compare the action of LASC, HASC, and bone marrow-derived MSC (BM-MSC), on xenoantibody production by B cells. METHODS: Adipose-derived mesenchymal stromal cells and BM-MSC were obtained from humans or F344 rats and expanded in a low-serum or a high-serum culture medium. Proliferation of human peripheral mononuclear cells (PBMC) or rat splenocytes was induced by phytohemagglutinin (PHA) or anti-IgM-antibody. These cells were then co-cultured with LASC, HASC, or BM-MSC, and cell proliferation was studied. Porcine red blood cells (pRBC) were intraperitoneally injected into Lewis rats, and LASC, HASC, or BM-MSC obtained from F344 rats were injected intravenously or intraperitoneally. The levels of antibodies (IgM and IgG) against pRBC were examined using flow cytometry. RESULTS: Human LASC suppressed PBMC proliferation more effectively than human HASC. Human LASC suppressed both T-cell and B-cell proliferation when incubated with PHA (a T-cell stimulus). However, human LASC did not suppress B-cell proliferation after incubation with anti-IgM-antibody (a T-cell-independent stimulus). Rat LASC suppressed PHA-stimulated splenocyte proliferation more effectively than rat HASC or rat BM-MSC. In vivo studies showed that intravenous injection of rat LASC significantly reduced the levels of IgG antibodies against pRBC, while intravenous administration of the other two types of MSC (rat HASC or rat BM-MSC) or intraperitoneal administration of rat LASC did not impede IgG production. A significant number of LASC were observed in the spleen when injected intravenously while only a few LASC were observed when given intraperitoneally. CONCLUSIONS: Administration of LASC effectively impeded xenoantibody production by B cells through the inhibition of T-cell function, while HASC or BM-MSC showed less promising effects. These results suggest that intravenous injection of LASC may be useful in attenuating antibody-mediated rejection.

Intra-bone marrow bone marrow transplantation rejuvenates the B-cell lineage in aged mice.

Age-related reductions in the frequency and absolute number of early B lineage precursors in the bone marrow of aged mice have been reported. Reversal of B-cell lineage senescence has not been achieved. Age-related impairment of the B-cell lineage is caused by the decreasing functionality of hematopoietic and B lineage precursors, and reduced efficacy of bone marrow stromal cells that constitute the bone marrow microenvironment. To induce rejuvenation of aged B cells, we injected whole bone marrow from young donors to irradiated aged recipients through the tibia and analyzed B-cell development and immune responsiveness. In aged mice, we found significant reductions in the frequencies and absolute numbers of pro-B cells (B220(+)CD43(+)CD24(+)BP-1(-) and B220(+)CD43(+)CD24(int)BP-1(+)) and pre-B cells (B220(+)CD43(+)CD24(high)BP-1(+) and B220(+)CD43(-)IgM(-)IgD(-)). Intra-bone marrow bone marrow transplantation (IBM-BMT) of young marrow cells including both hematopoietic stem cells and bone marrow stromal cells reversed the reduction of pro-B cells and pre-B cells. In the periphery, the frequency and absolute number of marginal zone-B cell were not significantly different between young, old and IBM-BMT group. The frequency of follicular-B cells in the IBM-BMT group was significantly increased compared to old group. The frequency of B1a B cells in the peritoneal cavity was significantly decreased in the IBM-BMT group. Antibody production against T-independent antigens was not different among the young, the aged and IBM-BMT groups.

Significance of HLA class I antibody-induced antioxidant gene expression for endothelial cell protection against complement attack.

It has been observed that a graft organ continues to survive and function normally even in the presence of anti-graft antibodies. However, the mechanisms behind acquirement of this condition remain unknown. Here we report that the anti-HLA ligation on endothelial cells induces PI3K/AKT activation followed by antioxidant gene induction through Nrf2-mediated antioxidant-responsive element (ARE) activation. Activation of PI3K/AKT in endothelial cells by a low concentration of anti-HLA ligation enhances protection from complement attack. A real-time quantitative PCR and flow-cytometry experiment showed that ferritin H and HO-1 mRNAs were induced in a PI3K/AKT-dependent manner, while CD55 and CD59 expression were not enhanced by anti-HLA ligation. Anti-HLA ligation on endothelial cells activates ferritin H ARE and induces Nrf2 binding on its enhancer element. Finally, overexpression of Nrf2 in endothelial cells attenuates complement-mediated cytotoxicity. These experiments suggest that induction of PI3K/AKT-dependent cytoprotective genes by Nrf2 is an important mechanism to prevent complement attack. Thus, a protocol to activate this pathway would be a potential strategy for avoidance of graft rejection in transplantation.

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway.

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.

GADD34 induces p21 expression and cellular senescence.

We previously identified GADD34 (growth arrest and DNA damage protein 34) by screening for genes involved in oncogenic-transformation and/or cellular senescence in Ras-transformed rat F2408 fibroblasts (7EJ-Ras), which exhibit anchorage-independent growth and do not senesce. In the current study, we found that transduction of 7EJ-Ras cells with a retroviral vector expressing GADD34 suppressed their proliferation. Furthermore, we observed that fibroblasts derived from GADD34-knockout mice (GADD34-KO MEFs) did not undergo senescence. Whereas the expression of p21 was decreased in GADD34 KO MEFs, its expression was rescued in these cells by ectopic expression of GADD34 by retroviral transduction. These findings suggest that GADD34 contributes to the regulation of p21 expression, and that it suppresses cellular proliferation through the induction of cellular senescence.

GADD34 inhibits mammalian target of rapamycin signaling via tuberous sclerosis complex and controls cell survival under bioenergetic stress.

Cells regulate the rate of protein synthesis during conditions of cell stress to adapt to environmental changes. However, the molecular interactions between signaling pathways controlling translation and the cellular response to stress remain to be elucidated. Here, we show that the expression of growth arrest and DNA damage protein 34 (GADD34) is induced by energy depletion and that the expression of this protein protects cells from apoptotic cell death. During conditions of cell stress, GADD34 forms a stable complex with tuberous sclerosis complex (TSC) 1/2, causes TSC2 dephosphorylation, and inhibits signaling by mammalian target of the rapamycin (mTOR). These findings demonstrate that crosstalk between GADD34 and the mTOR signaling pathways contributes to the response of the protein synthetic machinery to environmental stress. GADD34 may find clinical potential as a target drug for the treatment of mTOR-associated diseases.

Induction of matrix metalloproteinases (MMP3, MMP12 and MMP13) expression in the microglia by amyloid-beta stimulation via the PI3K/Akt pathway.

Alzheimer's disease is characterized by the presence of senile plaques in the brain composed primarily of amyloid-beta peptide. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. We herein analyzed the process of expression of Matrix metalloproteinases induced by Abeta stimulation. We found that Abeta1-42 induces a high level of MMP3, MMP12 and MMP13 in the microglia. The signal transduction pathway for the expression of these MMPs mRNA induced by Abeta1-42 depends on PI3K/Akt.

Amyloid-beta peptides induce cell proliferation and macrophage colony-stimulating factor expression via the PI3-kinase/Akt pathway in cultured Ra2 microglial cells.

Alzheimer's disease is characterized by numerous amyloid-beta peptide (Abeta) plaques surrounded by microglia. Here we report that Abeta induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony-stimulating factor (M-CSF). We examined signal cascades for Abeta-induced M-CSF mRNA expression. The induction of M-CSF was blocked by a phosphatidylinositol 3 kinase (PI3-kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Abeta enhanced NF-kappaB binding activity to the NF-kappaB site of the mouse M-CSF promoter, which was blocked by LY294002. These results indicate that Abeta induces M-CSF mRNA expression via the PI3-kinase/Akt/NF-kappaB pathway.

Regulation of mouse GADD34 gene transcription after DNA damaging agent methylmethane sulfonate.

The GADD34 gene is transcriptionally induced by growth arrest and DNA damage. However, the mechanisms underlying the transcriptional regulation are still unclear. We analyzed the promoter of mouse GADD34 gene and the methylmethane sulfonate (MMS)-induced transcriptional regulation of this gene. By introducing genome mutants, which were linked to the luciferase reporter, into NIH3T3 cells, we defined a 100-bp fragment upstream of the transcriptional initiating site as the minimal promoter of the GADD34 gene. Subsequent study revealed that CRE-binding site located in this minimal promoter was critical for MMS-induced transcription of the GADD34 gene. In vitro binding experiments showed that phosphorylated c-Jun was contained in the CRE/DNA complex. Overexpression of the dominant negative form of c-Jun led to a decrease of MMS-responsive promoter activity. From these results, we conclude that the CRE site of the GADD34 promoter is indispensable to the MMS-responsive cis-element that c-Jun is the essential transcription factor for MMS-stimulated regulation of GADD34 gene expression and that the upstream signaling is dependent on JNK.

Protein phosphatase 1, but not protein phosphatase 2A, dephosphorylates DNA-damaging stress-induced phospho-serine 15 of p53.

Okadaic acid (OA) is a protein phosphatase (PP) inhibitor and induces hyperphosphorylation of p53. We investigated whether the inhibition of PP1 by OA promotes the phosphorylation of the serine 15 of p53. In vitro dephosphorylation assay showed that PP1 dephosphorylated ultraviolet C (UVC)-induced phospho-ser15 of p53, and that OA treatment inhibited it. One of the PP1 regulators, growth arrest and DNA damage 34 (GADD34), disturbed PP1 binding with p53, interfered with the dephosphorylation of p53 and increased the amount of phospho-p53 after UVC-treatment. This report provides the first evidence that PP1, but not PP2A, dephosphorylates phospho-serine 15 of p53.

GADD34 induces p53 phosphorylation and p21/WAF1 transcription.

Recently, others and we have shown that one of the functions of GADD34 is a recovery from a shutoff of protein synthesis induced by endoplasmic reticulum stress. GADD34 has been shown to induce growth arrest and apoptosis. Main protein of apoptosis is p53, especially phosphorylation of p53. And one of the main proteins of growth arrest is p21/WAF1. Here we analyzed the effects of GADD34 on p53 phosphorylation and p21/WAF1 transcription. Transfected Myc-tagged p53 was dose-dependently phosphorylated at Ser15 by increasing the amount of GADD34. Transfection of GADD34 also induced the endogenous phosphorylation of p53 and enhanced p21 protein expression. Transfection of GADD34 induced p21/WAF1 promoter activity. This activity was dependent on p53, because GADD34 transfection to p53-deficient cells produced only a slight increase of p21/WAF1 promoter activity. The p21/WAF1 promoter activity was greatly enhanced by the transfection of p53. Both GADD34 and p53 transfection induced much higher p21/WAF1 promoter activity. The promoter activity of p21/WAF1 was very low in GADD34 deficient MEF. The transfection of GADD34 increased the p21/WAF1 promoter activity in GADD34 deficient MEF.

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34-/- mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34-/- MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34+/+ or GADD34-/- cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34-/- MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.