Journal of Biomedical Science

, Volume 13, Issue 3, pp 357–371 | Cite as

JWA, a novel signaling molecule, involved in all-trans retinoic acid induced differentiation of HL-60 cells

  • Shu Huang
  • Qun Shen
  • Wen-Ge Mao
  • Ai-Ping Li
  • Jian Ye
  • Qi-Zhan Liu
  • Chang-Ping Zou
  • Jian-Wei Zhou


JWA (AF070523) was originally identified as a novel all-trans retinoic acid (ATRA) responsible gene in primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in ATRA-mediated differentiation of the human myeloid leukemia HL-60 cells. We found that concomitant with the progressive cell differentiation, JWA expression was up-regulated by ATRA in a dose- and time-dependent manner. Inhibition of JWA expression by RNA interference partially blocked ATRA-induced differentiation and growth inhibition of HL-60 cells. Pre-treatment of phorbol-12-myristate-13-acetate (TPA), a PKC activator, decreased ATRA-mediated differentiation, companied with the down-regulation of JWA expression. Arsenic trioxide (As2O3, 0.5  \(\upmu\)M) enhanced the cellular differentiation induced by 0.01  \(\upmu\)M ATRA, but had no noticeable effect on the differentiation induced by 0.1 \(\upmu\)M ATRA. Concurrent with the enhancement, JWA expression was up-regulated. All the data suggest that up-regulation of JWA expression is essential for ATRA-induced differentiation of HL-60 cells. And JWA, associated with PKC, is involved in its signal pathways. Ideal therapeutic efficacy with low toxicity may be obtained if low doses of ATRA (0.01 \(\upmu\)M) and As2O3 (0.5  \(\upmu \)M) are combined. These findings may present a novel mechanism that cellular differentiation and growth inhibition induced by ATRA are mediated at least in part through regulation of JWA expression. JWA may be a novel molecular marker for ATRA-induced HL-60 cell differentiation. ATRA up-regulates JWA expression by stimulating the transcriptional activity of JWA gene promoter.


JWA ATRA HL-60 cells differentiation RNA interference 


  1. 1.
    Zauli G., Visani G., Bassini A., Caramelli E., Ottaviani E., Bertolaso L., Bertagnolo V., Borgatti P., Capitani S. (1996) Nuclear translocation of protein kinase C-alpha and -zeta isoforms in HL-60 cells induced to differentiate along the granulocytic lineage by all-trans retinoic acid. Br. J. Haematol. 93: 542–550PubMedCrossRefGoogle Scholar
  2. 2.
    Tallman M.S., Nabhan C., Feusner J.H., Rowe J.M. (2002) Acute promyelocytic leukemia: evolving therapeutic strategies. Blood 99: 759–767PubMedCrossRefGoogle Scholar
  3. 3.
    Idres N., Benoit G., Flexor M.A., Lanotte M., Chabot G.G. (2001) Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites. Cancer Res. 6: 700–705Google Scholar
  4. 4.
    Launay S., Gianni M., Diomede L., Machesky L.M., Enouf J., Papp B. (2003) Enhancement of ATRA-induced cell differentiation by inhibition of calcium accumulation into the endoplasmic reticulum: cross-talk between RAR \(\alpha\) and calcium-dependent signaling. Blood 101: 3220–3228PubMedCrossRefGoogle Scholar
  5. 5.
    Chambon P. (1996) A decade of molecular biology of retinoic acid receptors. FASEB J. 10: 940–954PubMedGoogle Scholar
  6. 6.
    Zhou J.W., Di Y.P. and Zhao Y.H. A novel cytoskeleton associate gene-cloning, identification, sequencing, regulation of expression and tissue distribution of JWA. In: Ye X.S., Shen B.F. and Tang X.F. (Eds), Investigation on Cell Modulation: Signal Transduction, Apoptosis and Gene Expression. Beijing, 1999, pp. 110–119Google Scholar
  7. 7.
    Lin C.I., Orlov I., Ruggiero A.M., Dykes-Hoberg M., Lee A., Jackson M., Rothstein J.D. (2001) Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3-18. Nature 410: 84–88PubMedCrossRefGoogle Scholar
  8. 8.
    Butchbach M.E., Lai L., Lin C.L. (2002) Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18. Gene 292: 81–90PubMedCrossRefGoogle Scholar
  9. 9.
    Butchbach M.E., Guo H., Lin C.L. (2003) Methyl-beta-cyclodextrin but not retinoic acid reduces EAAT3-mediated glutamate uptake and increases GTRAP3-18 expression. J. Neurochem. 84: 891–894PubMedCrossRefGoogle Scholar
  10. 10.
    Schweneker M., Bachmann A.S., Moelling K. (2005) JM4 is a four-transmembrane protein binding to the CCR5 receptor. FEBS Lett. 579: 1751–1758PubMedCrossRefGoogle Scholar
  11. 11.
    Ingley E., Williams J.H., Walker C.E., Tsai S., Colley S., Sayer M.S., Tilbrook P.A., Sarna M., Beaumont J.G., Klinken S.P. (1999) A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit. FEBS Lett. 459: 69–74PubMedCrossRefGoogle Scholar
  12. 12.
    Matsuda A., Suzuki Y., Honda G., Muramatsu S., Matsuzaki O., Nagano Y., Doi T., Shimotohno K., Harada T., Nishida E., Hayashi H., Sugano S. (2003) Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways. Oncogene 22: 3307–3318PubMedCrossRefGoogle Scholar
  13. 13.
    Campbell S.L., Khosravi-Far R., Rossman K.L., Clark G.J., Der C.J. (1998) Increasing complexity of Ras signaling. Oncogene 17: 1395–1413PubMedCrossRefGoogle Scholar
  14. 14.
    Bar-Sagi D., Hall A. (2000) Ras and Rho GTPases: a family reunion. Cell 103: 227–238PubMedCrossRefGoogle Scholar
  15. 15.
    Kyriakis J.M., Avruch J. (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81: 807–869PubMedGoogle Scholar
  16. 16.
    Shen Q., Zhou J.W., Shen R.L., Lu H., Cao H.X., Xia W. (2001). The significance of expression of JWA gene in acute non-lymphocytic leukemia. Acta Nanjing Med. Univ. 21:406–407. (in Chinese)Google Scholar
  17. 17.
    Shen Q., Zhou J.W., Shen R.L., Lu H., Cao H.X., Xia W. (2001). To detect the expression of JWA gene by semi-quantity RT-PCR in chronic myelogenous leukemia. Jiangsu Med. J. 27:1–3; (in Chinese)Google Scholar
  18. 18.
    Xia W., Zhou J.W., Cao H.X. (2001) The relationship between structure and function of JWA in the modulation of cell differentiation. Chinese Sci. Bull. 46:2063–2067CrossRefGoogle Scholar
  19. 19.
    Cao H.X., Xia W., Zhou J.W. (2002) The role of JWA in acute promyelocytic leukemia cell differentiation and apoptosis triggered by retinoic acid, 12-tetradecanoylphorbol-13-acetate and arsenic trioxide. Chinese Sci. Bull. 47:834–838CrossRefGoogle Scholar
  20. 20.
    Savickiene J., Gineitis A., Stigbrand T. (1999) Modulation of apoptosis of proliferating and differentiating HL-60 cells by protein kinase inhibitors: suppression of PKC or PKA differently affects cell differentiation and apoptosis. Cell Death Differ 6:698–709PubMedCrossRefGoogle Scholar
  21. 21.
    Paquette R.L., Koeffler H.P. (1992) Differentiation therapy. Hematol. Oncol. Clin. N. Am. 6: 687–706Google Scholar
  22. 22.
    Scott R.E. (1995) The differentiation therapy of cancer: past, present, and future. Mol. Cell Differ 3: 157–173Google Scholar
  23. 23.
    Chou T.C., Talalay P. (1984) Quantitative analysis of dose effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22: 27–55PubMedCrossRefGoogle Scholar
  24. 24.
    Berenbaum M.C. (1981) Criteria for analyzing interactions between biologically active agents. Cancer Res. 35: 269–335CrossRefGoogle Scholar
  25. 25.
    Fontana J.A., Reppucci A., Durham J.P., Miranda D. (1986) Correlation between the induction of leukemic cell differentiation by various retinoids and modulation of protein kinases. Cancer Res. 46:2468–2473PubMedGoogle Scholar
  26. 26.
    Carter C.A., Parham G.P., Chambers T. (1998) Cytoskeletal reorganization induced by retinoic acid treatment of human endometrial adenocarcinoma (RL95-2) cells is correlated with alterations in protein kinase C-alpha. Pathobiology 66:284–292PubMedCrossRefGoogle Scholar
  27. 27.
    Newton A.C. (1997) Regulation of protein kinase C. Curr. Opin. Cell Biol. 9: 161–167PubMedCrossRefGoogle Scholar
  28. 28.
    Yang K.D., Mizobuchi T., Kharbanda S.M., Datta R., Huberman E., Kufe D.W., Stone R.M. (1994) All-trans retinoic acid reverses phorbol ester resistance in a human myeloid leukemia cell line. Blood 83:490–496PubMedGoogle Scholar
  29. 29.
    Bertolaso L., Gibellini D., Secchiero P., Previati M., Falgione D., Visani G., Rizzoli R., Capitani S., Zauli G. (1998) Accumulation of catalytically active PKC-zeta into the nucleus of HL-60 cell line plays a key role in the induction of granulocytic differentiation mediated by all-trans retinoic acid. Br. J. Haematol. 100:541–549PubMedCrossRefGoogle Scholar
  30. 30.
    Kikkawa U., Takai Y., Tanaka Y., Miyake R., Nishizuka Y. (1983) Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J. Biol. Chem. 258:11442–11445PubMedGoogle Scholar
  31. 31.
    Shen Z.X., Chen G.Q., Ni J.H., Li X.S., Xiong S.M., Qiu Q.Y., Zhu J., Tang W., Sun G.L., Yang K.Q., Chen Y., Zhou L., Fang Z.W., Wang Y.T., Ma J., Zhang P., Zhang T.D., Chen S.J., Chen Z., Wang Z.Y. (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): □ Clinical efficacy and pharmacokinetics in relapsed patients. Blood 89:3354–3360PubMedGoogle Scholar
  32. 32.
    Soignet S.L., Maslak P., Wang Z.G., Jhanwar S., Calleja E., Dardashti L.J., Corso D., DeBlasio A., Gabrilove J., Scheinberg D.A., Pandolfi P.P., Warrell Jr. R.P. (1998) Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N. Engl. J. Med. 339:1341–1348PubMedCrossRefGoogle Scholar
  33. 33.
    Hannon G.J. (2002) RNA interference. Nature 418:244–251PubMedCrossRefGoogle Scholar
  34. 34.
    Elbashir S.M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498PubMedCrossRefGoogle Scholar
  35. 35.
    Castanotto D., Li H., Rossi J.J. (2002) Functional siRNA expression from transfected PCR products. RNA 8:1454–1460PubMedCrossRefGoogle Scholar
  36. 36.
    Gou D., Jin N., Liu L. (2003) Gene silencing in mammalian cells by PCR-based short hairpin RNA. FEBS Lett. 548:113–118PubMedCrossRefGoogle Scholar
  37. 37.
    Zheng L., Liu J., Batalov S., Zhou D., Orth A., Ding S., Schultz P.G. (2004) An approach to genome wide screens of expressed small interfering RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 101:135–140PubMedCrossRefGoogle Scholar
  38. 38.
    Valverde M.A., Parker M.G. (2002) Classical and novel steroid actions: a unified but complex view. Trends Biochem. Sci. 27:172–173PubMedCrossRefGoogle Scholar
  39. 39.
    Liu T.X., Zhang J.W., Tao J., Zhang R.B., Zhang Q.H., Zhao C.J., Tong J.H., Lanotte M., Waxman S., Chen S.J., Mao M., Hu G.X., Zhu L., Chen Z. (2000) Gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells. Blood 96:1496–1504PubMedGoogle Scholar
  40. 40.
    Radominska-Pandya A., Chen G., Czernik P.J., Little J.M., Samokyszyn V.M., Carter C.A., Nowak G. (2000) Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). J. Biol. Chem. 275:22324–22330PubMedCrossRefGoogle Scholar
  41. 41.
    Jing Y., Dai J., Chalmers-Redman R.M., Tatton W.G., Waxman S. (1999) Arsenic Trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 94:2102–2111PubMedGoogle Scholar
  42. 42.
    Cai X., Shen Y.L., Zhu Q., Jia P.M., Yu Y., Zhou L., Huang Y., Zhang J.W., Xiong S.M., Chen S.J., Wang Z.Y., Chen Z., Chen G.Q. (2000) Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia 14:262–270PubMedCrossRefGoogle Scholar
  43. 43.
    Jing Y., Wang L., Xia L., Chen G.Q., Chen Z., Miller W.H., Waxman S. (2001) Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo. Blood 97:264–269PubMedCrossRefGoogle Scholar
  44. 44.
    Andre C., Guillemin M.C., Zhu J. (1996) The PML and PML/RARalpha domains: from autoimmunity to molecular oncology and from retinoic acid to arsenic. Exp. Cell Res. 229:253–260PubMedCrossRefGoogle Scholar
  45. 45.
    Leid M.P., Kastner R., Lyons H. (1992) Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68:377–395PubMedCrossRefGoogle Scholar
  46. 46.
    Kliewer S.A., Umesono K., Mangelsdorf D.J., Evans R.M. (1992) Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446–449PubMedCrossRefGoogle Scholar
  47. 47.
    Gery S., Park D.J., Vuong P.T., Chih D.Y., Lemp N., Koeffler H.P. (2004) Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes. Blood 104:3911–3917PubMedCrossRefGoogle Scholar
  48. 48.
    Tenen D.G., Hromas R., Licht J.D., Zhang D.E. (1997) Transcription factors, normal myeloid development, and leukemia. Blood 90:489–519PubMedGoogle Scholar
  49. 49.
    Ubeda M., Wang X.Z., Zinszner H., Wu I., Habener J.F., Ron D. (1996) Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element. Mol. Cell Biol. 16:1479–1489PubMedGoogle Scholar

Copyright information

© National Science Council Taipei 2006

Authors and Affiliations

  • Shu Huang
    • 1
  • Qun Shen
    • 1
    • 2
  • Wen-Ge Mao
    • 1
  • Ai-Ping Li
    • 1
  • Jian Ye
    • 1
  • Qi-Zhan Liu
    • 1
  • Chang-Ping Zou
    • 3
  • Jian-Wei Zhou
    • 1
  1. 1.Department of Molecular Cell Biology and Toxicology, Jiangsu Provincial Key Laboratory of Human Functional Genomics and Applied Toxicology, School of Public HealthNanjing Medical UniversityNanjingChina
  2. 2.Department of HematologyThe Traditional Chinese Medicine Hospital of Jiangsu ProvinceNanjingChina
  3. 3.Department of OB/Gyn and Department of UrologyThe University of ArizonaTucsonUSA

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