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[CANCER RESEARCH 57, 943-947, March I, 19971 Role of Double-Stranded RNA-activated Protein Kinase in Human Hematological Malignancies' S. Basu, P. Panayiotidis, S. M. Hart, L-Z. He, A. Man, A. V. HotThrand, and K. Ganeshaguru2 Department of Haematology. Royal Free Hospital School of Medicine. Rowland Hill Street. London NW3 2PF, United Kingdom (S. B., P. P.. S. M. H., L-Z H.. A. V. H.. K. G.J: and Department of International Clinical Research, Ciba, Basel, Switzerland (A. M.J ABSTRACT The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is one of many genes induced by IFN. The PKR sequentially undergoes autophosphorylation and activation on binding to dSRNA.Previous stud ies have shown that PKR may be an important factor in the regulation of viral and cellular protein synthesis. Recent studies suggest that PKR may function as a tumor suppressor gene. The role of PKR in various human leukemic cells was therefore investigated. PKR mRNA levels by reverse transcription-PCR, protein expression by Western blot and FACSCaII analysis, and activity by phosphorylation status were studied. The expres sion ofa known inhibitor ofPKR, p58, was also investigated at mRNA and protein levels. A total of 24 samples from normal mononuclear cells (MNCs), 26 samples of acute lymphoblastic leukemia, 26 samples of acute myelogenous leukemia, 32 samples of chronic lymphocytic leukemia, and 5 samples of hairy cell leukemia was investigated. Mean mRNA levels were increased in acute lymphoblastic leukemia and acute myelogenous leukemia and decreased in chronic lymphocytic leukemia compared to normal MNCs. The mRNA levels in hairy cell leukemia were similar to those of normal MNCs. PKR protein was detectable in normal MNCs and leukemic cell extracts, and on FACSCananalysis, more than 70% of cells stained positive for PKR. PKR activity was detectable in all samples investigated and was enhanced 4—23-foldin the presence of the synthetic dSRNA, poly(I)poly(C). Protein expression of a known PKR inhibitor, PS8, was barely detectable in normal MNCs and leukemic cells, with high expression in the HeLa cell line. These findings provide no evidence to support the hypothesis that PKR acts as a tumor suppressor in human leukemic cells. INTRODUCTION The dsRNA3-activated protein kinase, PKR, also known as p68 kinase, DAI, ds-RNA-PK, is an IFN-activated serine/threonine kinase and is 1 of more than 30 genes induced by IFN (1, 2). IFN in the presence of dsRNA can inhibit protein synthesis in a cell-free system made from rabbit reticulocytes (3, 4). An analogous enzyme has been found in rabbit reticulocytes, various mouse tissues (p65 kinase), and human peripheral blood mononuclear cells (5—8).In general, PKR is activated by binding to dsRNA (9), although some single-stranded RNA species due to their stem-loop structure can also bind and activate PKR (10). Once activated PKR exhibits two distinct protein kinase activities in the presence of ATP autophosphorylation and phosphorylation of exogenous substrate of which the well-known physiological substrate is the a subunit of the eIF-2 (11, 12). Other substrates include the NF-scB inhibitor 1KB (13). Evidence that PKR is implicated in the antiviral and antiprolifera tive effects of IFN has been provided by expressing recombinant PKR in murine cells and in Saccharomyces cerevisiae. Expression of hu man PKR in murine cells mediated a partial resistance to encephalo myocarditis virus growth (14), and in yeast it caused inhibition of cell growth (15). Two groups have reported the tumorigenic potential of murine NIH3T3 clones expressing mutant, inactive forms of human PKR. When injected into nude mice, both a point-mutated form of inactive PKR (catalytic MATERIALS study was supported by Hoffmann-La whom requests for reprints should Fax: 0171-794-0645; E-mail: abbreviations used are: ds, double stranded; PKR, dsRNA-activated protein Lys —@Pro) and a blood, 15 bone marrow, 2 tonsils), 26 patients with ALL (9 T-ALL, 11 B-lineage ALL), 26 patients with AML [FAB patients with (27), 1 patient M3, 8 patients with M@, 1 patient with with M1, 3 patients M4, 2 patients with M5, with M2, 2 1 patient with secondary, 1 patient with biphenotypic, and 7 patients with AML and dyspla sia), 32 patients with CLL (stage A, 17 patients; stage B, 6 patients; and stage C, 9 patients),and 5 patientswith HCL. Diagnosis was based on cell mor phology according to the FAB classification for acute leukemia and by im munophenotyping and bone marrow histology for CLL and HCL. Disease [email protected]. 3 The or Patients. Peripheralblood or bone marrowwas collected from 24 patients Roche, Basel, Switzerland. be addressed. —@Arg AND METHODS with normal MNCs (7 peripheral 18 U.S.C. Section 1734 solely to indicate this fact I Lys The possibility that suppression of PKR through its underexpres sion, dysfunction, or inhibition could contribute to the development of human hematological malignancies was investigated by studying the expression and activity of PKR in a wide range of leukemic cells. classification 2 To II, (26). Received 8/12/96; accepted 1/2/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with @ subdomain six-amino acid deletion mutant form of PKR (catalytic subdomains V and VI) produced rapidly growing tumors (16, 17). Although the exact mechanism by which PKR exerts its tumor-suppressive action is not known, it is believed that the inactive mutant PKR behaves as a dominant negative inhibitor Of endogenous mouse p65 kinase and to function as a tumor suppressor it needs functional PKR activity (17). Conceivably under physiological conditions, mutations causing mac tivation of PKR activity or factors inhibiting its expression may allow normal cells to transform into tumors. PKR has also been associated with tumor growth and antitumoral action (18, 19). It is known, for example, that malignant embryonal carcinoma stem cells do not express this kinase even after treatment with IFN (20). The gene for PKR has recently been mapped to chromosome 2p2l—22.Chromosomal breakpoints involving 2p2l have been asso ciated with lipoma and large cell lymphoma, while translocation and altered 2p2l has also been reported in T-cell leukemia, myelodysplas tic syndromes, and possibly acute myeloid leukemia (21, 22). Certain viruses are known to down-regulate PKR to enable their continued replication in a host (23). Purification and cloning of this dsRNA-activated PKR inhibitor protein, p58. resulted in a cellular and not a viral protein (24, 25). They also show that p58 is readily expressed in some tumor cell lines, including HeLa. The mechanism of PKR inhibition by p58 is unknown, but it is suggested that it may block the autophosphorylation through direct interaction with PKR stage in acute leukemia was further classified as presentation kinase; MNC, mononuclear cell; ALL, acute lymphoblastic leukemia; AML, acute my (not previously treated with cytotoxic drugs) and relapse (or resistant) disease. In CLL the stage of the disease was classified as described by Binet et a!. (28). Samples were collected in preservative-free heparin and leukemic cells were elogenous leukemia; CLL, chronic lymphocytic leukemia; HCL, hairy cell leukemia; PHA, phytohemagglutinin; PFGE, pulse-field gel electrophoresis; eIF-2, eukaryotic mi tiation factor 2; FAB, French-American-British; f32M, f32-microglobulin; moab, mono cionalantibody. separated by gradient centrifugation on Lymphoprep (Nyegaard). The MNCs 943 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. PKR IN LEUKEMICCELLS were washed twice with HBSS, and further purification with monocyte deple over 48 h. The fractionated gels were transferred and probed in a manner tion by plastic adherence and T-cell depletion by sheep RBC rosetting were similar carried out when required. In all samples the final leukemic/blast cell count varied from 71 to 98% in acute leukemia, >70% in HCL, and >96% in CLL described using May-Grunwald-Geimsa-stained cytospin Semiquantitative Reverse as described was used for cDNA Transcnption-PCR. previously synthesis using a commercial cellular RNA was ng of total cellular kit (Boehringer RNA homologous products, to other (3,M or (3-actin, kinases. which For the semiquantitation are constitutively 1.25 mM deoxyribonucleotides expressed, were also am (Pharmacia), 100 bromide, Dynamics photographed, Personal and the negatives Densitometer. The results scanned were @M each of the Protein content as a ratio of of the supematant was esti mated using a modified Lowry method (33). Total cell lysate or immunopre cipitated protein from total lysate, followed by 2.5 polyacrylamide with and without I p@g/ml poly(I)'poly(C), @Ciof l'y-32PIATP incubation was subjected to a 10% gel and electrotransferred onto nylon membranes. For total protein studies, the blots after initial blocking were incubated with moab to PKR (17) or p58 (24) at 4°Covernight followed by incubation with a secondary antibody and detection using chemiluminescence (enhanced chemi luminescence; Amersham). Blots from immunoprecipitated samples were au toradiographed (Kodak S-OMAT), signal intensity was analyzed in a laser densitometer as on each blot. (Molecular Dynamics), and phosphorylation purification kit (Qiagen, Inc.) and ligated to SmaI-cut mpl 8/19 vectors. Single-strand DNA from insert-containing plaques were prepared and sequenced using the dideoxy termination method (Sequenase 2.0; United States Biochemical Corp.). Heparin Study. Since heparmnhas been shown to induce PKR in cell heparmn-induced PKR mRNA levels were excluded. PHAStudy. To studytheeffectof cell proliferation on PKRexpression, normal MNCs from two volunteers were cultured with PHA (I @zg/ml) for 24 to 72 h, and PKR mRNA levels measured showed a sharp rise in PKR mRNA levels at 24 h and a return to basal levels by 72 h. Statistical Analysis. The mean values of the PKR:32M ratio or PKR:actin ratio in different leukemias and their SDs were calculated and Student's RESULTS tion, cells were lysed in high salt buffer containing protease and phosphatase for total cell protein. scanned and normal unpaired t test was applied in each instance to calculate the significance PKR:f3,Mor p58:f3-actin. Protein and Phosphorylation. For Western blots and immunoprecipita inhibitors were between the means. using a Molecular expressed using a QlAquick-spin artifactual primers, and 4 units of Taq polymerase (Amersham). PCR was carried out on a Hybaid thermal reactor with a I-mm denaturation at 94°C,1-mm annealing, and 2-mm DNA synthesis at 72°C.The primers used with the annealing temperature (T°)and cycle numbers are listed in Table I. The PCR products were separated on a 2% agarose gel, stained with ethidium DNA samples Autoradiographs the data were quantitated anticoagulant. Normal MNCs derived from two volunteers showed no differ ence in PKR mRNA levels either at 0 h or after 18—24h of culture, and of the PCR plified and the products run on the same gel. When studies commenced on PKR,the laboratorywas set upto studysemiquantitationof PCRproductswith f3,M. With the developmentof other studies, includingthose with IFN, the laboratory switched to amplifying j3-actin for quantitation. The reaction mix ture for PCR in 50 @.dcontained 25 ng of cDNA, 5 pA of lOX buffer (Amersham), above. extracts, our preliminary study included a comparison of PKR mRNA levels in normal MNCs from blood collected using either EDTA or heparmn as the Mannheim). The primers were designed from the published cDNA sequence, avoiding regions described and for gene dosage, Sequencing of PCR Products. Amplified PCR products were purified Total (29). Five hundred above, ized to control slides. Cell Cultures. Cell culturereagentswereobtainedfromLife Technologies, Inc. All cell cultures were carried out in RPMI 1640 with 10% FCS at 37°Cin a 5% CO1 incubator. PHA (1 j.@g/ml; Wellcome) was used for treatment of cells where indicated. extracted to the method evaluated. Flow cytometry analysis was performed on fresh cells fixed overnight at 4°Cin 0.25% buffered paraformaldehyde, incubated with either PKR or p58 moab, followed by FITC-conjugated secondary antibody, and analyzed on a Becton Dickinson FACScan. Gene Dosage, Rearrangement, and Translocation. High molecular Specificity of the PCR Product. The specificity of the PKR product was confirmed by (a) the predicted product size of 249 bp being obtained on agarose gel; (b) Dde! restriction digest of the PCR product yielded the expected fragment sizes of I55 bp and 94 bp as predicted from the restriction map of the cDNA sequence; and (c) amplification of genomic DNA yielded a product of400 bp which was distinct from the 249-bp product of the cDNA, indicating the presence of an intron between the selected primer sequence. The different product size also helped to detect contaminating genomic DNA in the RNA samples. The PCR products were also verified by blotting and probing with labeled K- I PKR cDNA. Finally, PCR products from one patient with normal MNCs, one patient with CLL and a low mRNA level, and one patient with AML and a high mRNA level were cloned and sequenced. No sequence abnormality in the region ampli fled was detectable in any of these samples. PKR mRNA Levels in Normal MNCs and Leukemic Cells. The mean PKR:@7M mRNA ratio in seven normal MNCs was 0.42 ±0.23 [mean ± 2 (SD); range, 0.22—0.8]. The mean PKR value in 31 patients with CLL was 0.26 ±0.21 (range, 0.005—1.0).This mean weight DNA from normal MNC and leukemic samples was subjected to two level was lower than that of normal cells but this was not statistically restriction endonuclease digests (Hindlll and EcoRI), fractionated on 1% significant (P = 0.07). There was no apparent correlation among agarose gels, transferred onto nylon membranes, and probed with [32PJdCTP WBC count, previous treatment status, or stage of the disease in these labeled K1 eDNA for PKR (30). To assess gene dosage, blots were simulta patients with PKR mRNA levels. The mean PKR mRNA levels in neously probed with a [32PJdCTP-labeledeDNA for the APC gene (34). blast cells from 26 patients with AML was 0.77 ±0.39 (range, For PFGE, whole cells were embedded in low melting agarose and sub 0.14—I.6). This mean level was significantly higher than that of jected to two rare cutting restriction endonuclease digests (BssHII and BbrPI), normal MNCs (P = 0.03). There was no correlation among FAB type, fractionated on a cooled PFGE system, with pulse time ranging from 25 to 60 s WBC count, and the PKR mRNA levels. PKR mRNA levels were significantly higher in relapsed/resistant disease (0.88 ±0.4) com [email protected]'-gAACAgTgTgCATCgggggT-3'58'C3030AS5'-ATFCAgAAgCgAgTgTgCTg-3'p58S5'-TgCAgTACgAAggTgCTgAA-3'59'C2824AS5'-gATgAgCTCTfCAgC Table I Primers and cycle pared with presentation samples (0.52 ± 0.34, P = 0.05). PKR mRNA levels in blast cells of 20 patients with ALL (0.87 ±0.61 with a range of 0.03—1.8) were higher than those in normal MNCs (P = 0.07). There was no correlation among PKR mRNA levels and immunophenotype, disease status, or WBC count. The mean PKR mRNA levels in the hairy cells of five patients with HCL was 0.46 ±0.26 (range, 0.02—0.7)and was not significantly different from those in normal MNCs (P = 0.76; Fig. I). PKR Protein Levels. PKRproteinlevels were studiedusing West ern blot in two samples of normal MNCs, seven samples of CLL, and two samples of AML cell extracts. The cell extracts were from 944 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. PKR IN LEUKEMICCELLS a 0. 11—0.38),respectively. There was a statistically significant eleva tion in the p58 mRNA levels in CLL (P = 0.03) and none between the AML (P = 0.4) or ALL (P = 0.7 1) groups and normal MNCs (Fig. 4). There was no difference in the p58 mRNA levels between disease stages in CLL. In the cell line HeLa, p58 mRNA levels were consis tently higher than those detected from most of the normal MNCs and clinical samples (data not shown). 1.8 1.6 1.4 1.2 0 p58 Protein. In the seven CLL andtwo AML total proteinextracts C,' @ a 0.8 a. (50;.Lg)studiedwith Westernblot,p58proteinwasbarelydetectable and in most not detected. It was readily detected in the HeLa cell extract (Fig. 2a). When studied with flow cytometry, p58-positive cells ranged from 8 to 10% in two normal MNCs, 10—15%in four 0.6 8 0.4 Q CLL, and 0 HeLa analyzed 10—20% in three AML samples. Cells from the cell line 0.2 on six separate occasions showed 41—76% positivity for p58 (Fig. 2b). 0 CLI AMI ALL HCL Normal DISCUSSION b 1.6 1.4 1.2 This study shows that PKR mRNA levels are higher in cells patients with acute leukemia (AML or ALL) and lower in compared to normal MNCs. This seems paradoxical if PKR tumor suppressor function, implying that rapidly proliferating (i.e., ALL) should have a lower PKR mRNA level which 0 @ @ from CLL has a cells would allow more rapid protein synthesis. It was important therefore to 1 establish whether the cells in patients with acute leukemia ex C,' 0 pressed abnormal, mutated, or dysfunctional PKR protein. The 0.8 representative PCR products that were sequenced failed to show a. 0.6 any abnormality. The amplified fragment in this study was selected from a region not homologous to other kinases but did not include .80 0.4 the sequences from the kinase conserved regions used by Koromi las et a!. (16) and Meurs et a!. (17) for their substitution and 0.2 deletion studies in nude mice. The existence of mutations outside the amplified region of this study has therefore not been excluded. 0 Autophosphorylation studies of PKR in representative leukemic Presentation Relapse cells, however, failed to detect any functional abnormality. Since Fig. I. PKR mRNA levelsas a ratioof PKR:f32Min CLL, AML ALL HCL, and eIF-2 phosphorylation studies were not done, the possibility of a normal MNC samples (a) and in presentation and relapsed AML cells (b). @, AML with dysplasia; bar, mean. dysfunctional PKR cannot be totally ruled out. Southern blot and PFGE analysis failed to show any gene ampli fication or chromosomal abnormality in the samples studied. The relapsed/resistant samples from AML showed higher PKR mRNA representative samples showing normal, high, and low PKR mRNA levels than those at presentation. This could reflect a selection phe levels. PKR protein was detectable in all normal MNCs and leukemic cell extracts studied (Fig. 2a). PKR protein was also studied with flow nomenon as noted by Meurs et a!. (17). Two patients with AML had a clone with 2p2 translocation associated with other abnormalities. cytometry in 3 normal MNCs, 16 CLL, and 5 AML samples. Most cells stained positive for PKR, ranging from 86 to 97% in normal PKR is mapped to chromosome 2p2l—22.Their PKR values however were 0.76 and 1.6 (i.e., high compared to normal MNC). MNCs, 73—100%in CLL, and 75—95%in AML samples (Fig. 2b). The possibility of inhibition of the expressed PKR protein and its PKR Activity (Autophosphorylation). PKR kinase baseline ac activity were investigated by studying the PKR inhibitor p58. An tivity was observed from 100 @g of total protein immunoprecipitated with PKR moab in two of two normal MNCs, six of seven CLL, and elevation in the mRNA levels of p58 in CLL cells with no statistically two of two AML samples. In all samples, the baseline PKR activity significant difference in the other leukemias from normal MNCs was could be enhanced 4—23-fold after activation with 1 @ig/ml found. The elevated mRNA levels in CLL did not translate into poly(I)-poly(C) in the different groups of samples. High baseline increased p58 protein expression. Although high expression of p58 activity of PKR was observed in the control cell line Daudi, and this protein can be detected in HeLa cells, its expression in leukemic cells activity was further enhanced with poly(I)poly(C) (Fig. 3). was either undetectable (using Western blots) or very low (using PKR Gene Dosage. Gene dosage studies in a representativenum fluorescence-activated cell-sorting analysis). Furthermore, despite re ber of samples from CLL (six samples), AML (three samples), and ports that p58 inhibits autophosphorylation of PKR, this was not ALL (three samples) showed no deletions of the PKR gene. Similarly, observed in leukemic cells when treated with poly(I)poly(C). It could no translocations were detectable in these samples by either Southern be concluded that PKR autophosphorylation activity is not inhibited blotting or PFGE (data not shown). by p58 in leukemic cells. p58 mRNA Levels in Normal MNCs and Leukemia. The mean The purity of cells in the CLL group was >96% and contaminating p58:actin ratio in 21 normal MNC samples was 0.16 ± 0.30 cells were not a problem. In the case of the acute leukemias, the purity (mean ±2 SD), with a range of 0.03—1.03.The p58:actin ratio in 32 varied from 71 to 98% leukemic cells. It is possible that contaminating CLL, 9 AML, and 12 ALL samples was 0.44 ±0.24 (range, 0.13- residual nonleukemic cells may affect the final result. However, the 0.94), 0.47 ± 0.17 (range, 0.14—0.56), and 0.29 ±0.02 (range, PKR mRNA levels in the acute leukemias were significantly greater 945 0 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. PKR IN LEUKEMIC CELLS a kDa 4@.. @ @- — PKR @- . 66 @ .-.c,. 46 ‘4 @m' AML CLL Fig. 2. PKR and p58 protein expression by Western blot (a). Lanes 1—4and 6 representative of CLL, Lane 7 representative of AML, and Lane 8 representative of HeLa cell extracts. Left' size markers. b, FACScan; MNC, normal MNCs. ‘p58 HeLa ANt. @@J@CL@L . @ b ¶PKR CONT P58 @ PKR CONT poly(I).poly(C) @ + P-PKR - + ..‘, - mRNA level at the time zone of peak DNA synthesis (36—60h after PHA) and cell cycle. The initial increase in PKR mRNA level in PHA-stimulated lymphocytes is not understood but can be attributed + I to multiple gene induction by PHA. Additionally, the variable PKR mRNA levels in leukemic cells do not translate into significant @ @ I -PKR . L. I w@e@ic@@ Daudi ow'-. CLL changesin PKRproteinexpression. Thesefindingsdo notsuggesta — role for PKR in human leukemogenesis and do not provide evidence for a tumor suppressor activity by PKR in these hematological ma lignancies. AML Fig. 3. PKR activity (autophosphorylation) in imrnunoprecipitated Daudi. representa tive CLL. and representative AML cell extracts with (+) and without (—) 10 @zWml poly(I)poly(C). P-PKR, phosphorylation; l-PKR, immunoprecipitated PKR. than those of the normal MNCs, and the lack of contaminating nonleukemic cells would only have enhanced the statistical signifi cance. Furthermore, there was no relationship between increased purity of leukemic cells (71—98%)and increased mRNA levels for PKR and p58. It should be noted that each group of leukemias was compared to MNCs from normal samples and not to their equiv alent normal cells. Recent studies on PKR knockout mice show that they are phe notypically normal and the induction of type I !FN genes by poly(I)poly(C) was unimpaired. Additionally, injection of PKR°'° mouse embryo fibroblasts into nude mice did not give rise to tumors, and the study concluded that PKR does not play an essential role in growth control (35). More recently, Haines 1.2 0.8 0 B 0 C 8 @0.6 8 aIt) 0.4 0I et a!. (36) report the inability of PKR to inhibit cellular proliferation in human breast cancer. These observations raise doubts about the role of PKR in growth control and tumor suppression. The present findings show no abnormal structural or functional changes in PKR in different types of human leukemia. The high and low levels of PKR mRNA in acute and chronic leukemias, respec lively, probably reflect their different proliferative rates, although in the studies with PHA, there was no significant change in the PKR 8 0.2 AML ALL CLI Normal Fig. 4. p58 mRNA levels as a ratio of p58:actin in AML, ALL, CLL, and normal MNCs. 0. bone marrow; •,peripheral blood; A, tonsils. Bar, mean. 946 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. PKR IN LEUKEMIC CELLS ACKNOWLEDGMENTS Tumor-suppressor We are grateful to Dr. A. G. Hovanessian (Pasteur Institute, Paris, France) for advice and a generous supply of moab and cDNA for PKR, Dr. M. G. Katie (University of Washington, Seattle, function of the interferon induced double stranded RNA activated protein kinase. Proc. NatI. Acad. Sci. USA, 90: 232—236,1993. WA) for moab to p58, and Professor M. J. Clemens (St. George's Hospital Medical School, London, United Kingdom) for helpful discussions. REFERENCES I. Lengyel, P. Biochemistry of interferons and their actions. Annu. Rev. Biochem., 51: 251—282, 1982. 2. Berry, M. I., Knutson, G. S., Lasky, S. R., Munemitsu, S. M., and Samuel, C. E. Mechanism of interferon action: purification and substrate specificities of the ds-RNA dependent protein kinase from untreated and interferon treated mouse fibroblasts. J.Biol.Chem.,260: 11240—1 1247,1985. 3. Ehrenfeld, E., and Hant, T. Double stranded polio RNA inhibits initiation of protein synthesis by reticulocyte lysate. Proc. NatI. Aced. Sci. USA, 68: 1075—1078,1971. 4. Kerr, I. M., Brown, R. E., and Ball, L. A. Increased sensitivity of cell free protein synthesis to dsRNA after interferon treatment. Nature (Lond.), 250: 57—59,1974. 5. Farrell, P. i., Balkow, K., Hunt, T., Jackson, J., and Trachsel, H. Phosphorylation of 18. Riviere, Y., and Hovanessian, A. G. 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Expression of the double-stranded RNA-dependent human breast tissues. Tumor Biol., 17: 5—12,1996. protein kinase (p68) in 947 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. Role of Double-Stranded RNA-activated Protein Kinase in Human Hematological Malignancies S. Basu, P. Panayiotidis, S. M. Hart, et al. Cancer Res 1997;57:943-947. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/5/943 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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