Gene expression of double-stranded RNA-dependent protein kinase in the human endometrium and decidua

To clarify the biological significance of double-stranded RNA-dependent protein kinase (PKR), an interferon (INF)-inducible substance, we investigated (1) PKR gene expression and the (2) effect of IFN-gamma on PKR gene expression in human endometrium. By Northern blot analysis, PKR mRNA was detected as a 2.5 kb band in human endometrium throughout the menstrual cycle and decidua in early pregnancy. The addition of IFN-gamma to culture medium increased the PKR mRNA level in a dose-dependent manner in cultured endometrial stromal cells. These results suggest that IFN-gamma, which is reported to have an inhibitory effect on cell proliferation, plays an important role in human endometrial function by mediating PKR gene expression.     

Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells.

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations have associated protein kinase activity--i.e., addition of [gamma-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35S]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.     

Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells.

We have previously reported that cultured mouse 3T3-F442A cells exhibit a transient, double-stranded RNA (dsRNA)-dependent phosphorylation of the dsRNA-dependent eIF-2 alpha kinase (eIF-2 alpha, alpha-subunit of the eukaryotic initiation factor 2) (dsI). When dsI is activated by low levels of dsRNA, it is a potent inhibitor of protein synthesis. The transient expression of dsI is due to an autocrine effect of interferon at specific stages of growth and differentiation, and it may represent a mechanism for regulating cell growth and differentiation in 3T3-F442A cells. In this report, the purification of dsI from 3T3-F442A cell cultures by a two-step procedure is described. A specific immune serum to dsI was prepared by immunizing a rabbit with highly pure preparations. Immune precipitation studies demonstrate that the serum reacts with phosphorylated dsI both in vitro and in vivo and with de novo synthesized dsI after induction with interferon. We find that dsI of 3T3 cells can undergo phosphorylation in vitro without the addition of dsRNA and in vivo in the absence of viral infection. These results are consistent with a physiologic role for dsI in the growth and differentiation of these cells.     

Apoptosis and p53 protein expression in human hepatoma cells induced by etoposide,mitomycin C and thapsigargin

We found that the anti-cancer drugs of mitomycin C and etoposide will induce chromatin decondensation in human PLC/PRF/5 hepatoma cells, while the tumor promoter thapsigargin instead induces chromatin condensation. Both of these changes are subsequently followed by internucleosomal DNA cleavage and cell death characteristic to apoptosis. The p53 protein, which is known to induce apoptosis, increased in the anti-cancer drug-treated cells but not in thapsigargin-treated cells, thereby strongly suggesting that p53-dependent and -independent pathways linked to apoptosis exist in this hepatoma cell line.     

Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA

Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.     

Ribavirin up-regulates the activity of double-stranded RNA-activated protein kinase and enhances the action of interferon-alpha against hepatitis C virus

BACKGROUND: Ribavirin's mechanism of action in the treatment of chronic hepatitis C remains to be clarified. Double-stranded RNA-activated protein kinase (PKR) plays a role in cell defense against virus infection. This study investigated whether PKR is a mediator of the effectiveness of ribavirin, used either alone or in combination with interferon (IFN)- alpha , against hepatitis C virus (HCV) infection. METHODS: Primary human hepatocytes and HCV-replicon cells were treated with ribavirin and/or IFN- alpha . PKR activity was assayed by immunoblotting. A pulse-chase assay of the half-life of PKR protein was performed to study whether ribavirin decreases PKR degradation. We used small-interference RNA (siRNA) to knock down PKR to assess its importance in the suppression of HCV-RNA replication in the replicon system. RESULTS: Ribavirin was able to up-regulate the levels of phosphorylated PKR and phosphorylated eIF2 alpha , leading to suppression of HCV-RNA replication. The effects that treatment with ribavirin plus IFN- alpha had on PKR activity were greater than those observed for treatment with either ribavirin alone or IFN- alpha alone. Knockdown of PKR increased HCV-RNA replication, supporting the importance of PKR in the control of HCV-RNA replication. The pulse-chase experiment showed that ribavirin can reduce the degradation rate of PKR protein. CONCLUSION: These results suggest that the anti-HCV action of ribavirin is partly attributable to its ability to up-regulate PKR activity.     

Sodium butyrate enhances Fas-mediated apoptosis of human hepatoma cells

BACKGROUND/AIMS: Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells. METHODS: In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs. RESULTS: Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed. CONCLUSIONS: SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.     

Tyrosine protein kinase activity in normal and leukaemic human blood cells

Tyrosine protein kinase (TPK) activity was measured in subcellular fractions of normal granulocytes, lymphocytes and monocytes, and acute and chronic myeloid and lymphoid leukaemic cells. Of several tested tyrosine-containing substrates, poly (glutamic acid: tyrosine = 4:1) (S1) proved to be the best synthetic substrate. High cytosolic TPK activity was found in every cell type. Different TPKs may exist in various cell fractions, as was indicated by the difference in Km values for S1 in cell fractions of normal granulocytes and lymphocytes. No significant difference was found in total TPK activity between normal and leukaemic cells, indicating that total TPK activity is not related to the leukaemic process itself. A highly significant difference was found in membrane fractions in normal granulocytes and M1-M2 AML cells versus normal monocytes and M4-M5 AML cells, suggesting an association between TPK activity and monocytic differentiation in these cell fractions.     

Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells

BACKGROUND & AIMS: The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)-mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. METHODS: The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. RESULTS: RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal-regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I-mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. CONCLUSIONS: Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.     

Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.     

Increased expression of p53 protein in human leukemia cells.

We examined synthesis of the cellular phosphoprotein p53 in fresh bone marrow or peripheral blood cells from normal donors and from patients with leukemia, preleukemia, or other hematopoietic disorders. Lysates of cells labeled with [35S]methionine were immunoprecipitated with monoclonal antibodies to p53, and the immunoprecipitates were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Bone marrow or peripheral blood cells from 8 of 33 patients with hematopoietic disorders showed increased p53, seven of the eight occurring in cells of patients with preleukemia or acute myelogenous leukemia. Increased p53 synthesis was not associated with p53 gene amplification, as shown by Southern blot analysis. Synthesis of p53 was not increased in any of nine normal human bone marrow samples or eight normal human peripheral blood granulocyte, macrophage, and lymphocyte samples. The hematopoietic cells of patients in remission or with chronic forms of leukemia did not generally synthesize elevated levels of p53. In addition, we found negligible p53 mRNA and protein expression in a variety of human myeloid leukemia lines blocked at different stages of differentiation. Southern blot analysis showed that, except for the HL-60 cells, the p53 gene of the myeloid cell lines was intact. In view of recent evidence implicating p53 in transformation of cultured cells, our results using fresh leukemia cells suggest that p53 may contribute to the phenotype of certain leukemias in vivo.     

Analysis of gene expression in human bronchial epithelial cells upon influenza virus infection and regulation by p38 mitogen-activated protein kinase and c-Jun-N-terminal kinase

Background and objective:   Airway epithelial cells, which are the initial site of influenza virus (IV) infection, participate in the inflammatory process through the expression of various genes. In this process, mitogen-activated protein kinase (MAPK) may be associated with the expression of many genes, but its precise role remains unknown.
Methods:   A comprehensive analysis was performed of gene expression in human bronchial epithelial cells upon IV infection, using an Affymetrix gene chip containing 12 000 genes. Regulation of gene expression by MAPK was also analysed.
Results:   A total of 5998 genes were detected. Upon IV infection, 165 genes were upregulated and 49 of these were interferon-stimulated genes. The functions of 129 genes, including 14 apoptosis-related genes and 6 antiviral genes, were well characterized; however, those of 36 genes were unknown. The expression of 29 genes was inhibited either by SB 203580, a specific inhibitor of p38 MAPK, or by CEP-11004, a specific inhibitor of the c-Jun-N-terminal kinase (JNK) cascade, and the percentage inhibition by SB 203580 correlated with that by CEP-11004, suggesting that p38 and JNK participate in a common downstream pathway involved in the regulation of gene expression. p38 MAPK- or JNK-dependent genes were functionally classified into diverse categories.
Conclusions:   Although further studies are needed to obtain a more complete understanding of gene expression and the role of MAPK in gene expression, the present results are important in understanding the molecular mechanisms involved in the response of bronchial epithelial cells to IV infection.     

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.     

Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis.

BACKGROUND/AIMS: Fas is a cell surface antigen, that triggers apoptosis upon specific ligand or antibody binding. The proto-oncogene bcl-2 prevents apoptosis induced by various treatments. The aim of our study was to evaluate whether Bcl-2 protects hepatoma cells from Fas-mediated apoptosis. METHODS: Two human cell lines, HCC-T and HepG2 were used. Expression of Fas antigen and Bcl-2 was detected by flow cytometry and Western blotting. Cell viability and apoptotic change were examined after anti-Fas- and antisense oligodeoxynucleotide treatments. Apoptotic cells were detected by nick-end labelling and the TUNEL method. To test if Bcl-2 expression can protect HepG2 cells from Fas-mediated apoptosis, the cells were transduced using retroviral vector, LZBC, designed to coexpress E. coli beta-galactosidase and human Bcl-2. To further confirm the protective effect of Bcl-2 expression against Fas-mediated apoptosis in HepG2, Bcl-2 expressing plasmid vector was produced and a cell line stably expressing Bcl-2 was cloned. RESULTS: Western blot analysis showed constitutive Bcl-2 expression in HCC-T cells, but not in HepG2 cells. HCC-T was resistant to apoptosis after treatment with an agonist anti-Fas antibody (1 microg/ml for 3 days), whereas 33% of the HepG2 cells were killed by this treatment. Inhibition of Bcl-2 expression by transfection of antisense oligodeoxynucleotides caused spontaneous apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is essential for survival of HCC-T cells, whereas other proteins may substitute for it in HepG2 cells. Following LZBC infection, 10% HepG2 cells were beta-galactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviving after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fold, indicating that Bcl-2 expression protected the cells from Fas-mediated apoptosis. In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expression. CONCLUSION: Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.     

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3'' untranslated regions of human alpha-tropomyosin.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.