Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 May 9;92(10):4234–4238. doi: 10.1073/pnas.92.10.4234

Study of the role of retinoblastoma protein in terminal differentiation of murine erythroleukemia cells.

S Zhuo 1, S Fan 1, S Huang 1, S Kaufman 1
PMCID: PMC41918  PMID: 7753788

Abstract

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.

Full text

PDF
4234

Images in this article

4235Fig. 1
on p.4235
4236Fig. 2
on p.4236
4236Fig. 3
on p.4236

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberts A. S., Thorburn A. M., Shenolikar S., Mumby M. C., Feramisco J. R. Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):388–392. doi: 10.1073/pnas.90.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagchi S., Weinmann R., Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell. 1991 Jun 14;65(6):1063–1072. doi: 10.1016/0092-8674(91)90558-g. [DOI] [PubMed] [Google Scholar]
  3. Bandara L. R., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. doi: 10.1038/351494a0. [DOI] [PubMed] [Google Scholar]
  4. Bernards R., Shackleford G. M., Schackleford G. M., Gerber M. R., Horowitz J. M., Friend S. H., Schartl M., Bogenmann E., Rapaport J. M., McGee T. Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6474–6478. doi: 10.1073/pnas.86.17.6474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buchkovich K., Duffy L. A., Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989 Sep 22;58(6):1097–1105. doi: 10.1016/0092-8674(89)90508-4. [DOI] [PubMed] [Google Scholar]
  6. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  7. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  8. Chittenden T., Livingston D. M., Kaelin W. G., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. doi: 10.1016/0092-8674(91)90559-h. [DOI] [PubMed] [Google Scholar]
  9. Christoffersen J., Smeland E. B., Stokke T., Taskén K., Andersson K. B., Blomhoff H. K. Retinoblastoma protein is rapidly dephosphorylated by elevated cyclic adenosine monophosphate levels in human B-lymphoid cells. Cancer Res. 1994 Apr 15;54(8):2245–2250. [PubMed] [Google Scholar]
  10. Clarke A. R., Maandag E. R., van Roon M., van der Lugt N. M., van der Valk M., Hooper M. L., Berns A., te Riele H. Requirement for a functional Rb-1 gene in murine development. Nature. 1992 Sep 24;359(6393):328–330. doi: 10.1038/359328a0. [DOI] [PubMed] [Google Scholar]
  11. Dunaief J. L., Strober B. E., Guha S., Khavari P. A., Alin K., Luban J., Begemann M., Crabtree G. R., Goff S. P. The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest. Cell. 1994 Oct 7;79(1):119–130. doi: 10.1016/0092-8674(94)90405-7. [DOI] [PubMed] [Google Scholar]
  12. Egorin M. J., Sigman L. M., Van Echo D. A., Forrest A., Whitacre M. Y., Aisner J. Phase I clinical and pharmacokinetic study of hexamethylene bisacetamide (NSC 95580) administered as a five-day continuous infusion. Cancer Res. 1987 Jan 15;47(2):617–623. [PubMed] [Google Scholar]
  13. Fan S., el-Deiry W. S., Bae I., Freeman J., Jondle D., Bhatia K., Fornace A. J., Jr, Magrath I., Kohn K. W., O'Connor P. M. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res. 1994 Nov 15;54(22):5824–5830. [PubMed] [Google Scholar]
  14. Friend C., Patuleia M. C., De Harven E. Erythrocytic maturation in vitro of murine (Friend) virus-induced leukemic cells. Natl Cancer Inst Monogr. 1966 Sep;22:505–522. [PubMed] [Google Scholar]
  15. Gambari R., Marks P. A., Rifkind R. A. Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4511–4515. doi: 10.1073/pnas.76.9.4511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goodrich D. W., Wang N. P., Qian Y. W., Lee E. Y., Lee W. H. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell. 1991 Oct 18;67(2):293–302. doi: 10.1016/0092-8674(91)90181-w. [DOI] [PubMed] [Google Scholar]
  17. Gu W., Schneider J. W., Condorelli G., Kaushal S., Mahdavi V., Nadal-Ginard B. Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell. 1993 Feb 12;72(3):309–324. doi: 10.1016/0092-8674(93)90110-c. [DOI] [PubMed] [Google Scholar]
  18. Harlow E. Retinoblastoma. For our eyes only. Nature. 1992 Sep 24;359(6393):270–271. doi: 10.1038/359270a0. [DOI] [PubMed] [Google Scholar]
  19. Huang S., Wang N. P., Tseng B. Y., Lee W. H., Lee E. H. Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. EMBO J. 1990 Jun;9(6):1815–1822. doi: 10.1002/j.1460-2075.1990.tb08306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacks T., Fazeli A., Schmitt E. M., Bronson R. T., Goodell M. A., Weinberg R. A. Effects of an Rb mutation in the mouse. Nature. 1992 Sep 24;359(6393):295–300. doi: 10.1038/359295a0. [DOI] [PubMed] [Google Scholar]
  21. Kato J. Y., Matsuoka M., Polyak K., Massagué J., Sherr C. J. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell. 1994 Nov 4;79(3):487–496. doi: 10.1016/0092-8674(94)90257-7. [DOI] [PubMed] [Google Scholar]
  22. Kim S. J., Wagner S., Liu F., O'Reilly M. A., Robbins P. D., Green M. R. Retinoblastoma gene product activates expression of the human TGF-beta 2 gene through transcription factor ATF-2. Nature. 1992 Jul 23;358(6384):331–334. doi: 10.1038/358331a0. [DOI] [PubMed] [Google Scholar]
  23. Kiyokawa H., Richon V. M., Venta-Perez G., Rifkind R. A., Marks P. A. Hexamethylenebisacetamide-induced erythroleukemia cell differentiation involves modulation of events required for cell cycle progression through G1. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6746–6750. doi: 10.1073/pnas.90.14.6746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee E. Y., Chang C. Y., Hu N., Wang Y. C., Lai C. C., Herrup K., Lee W. H., Bradley A. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature. 1992 Sep 24;359(6393):288–294. doi: 10.1038/359288a0. [DOI] [PubMed] [Google Scholar]
  25. Lees J. A., Buchkovich K. J., Marshak D. R., Anderson C. W., Harlow E. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 1991 Dec;10(13):4279–4290. doi: 10.1002/j.1460-2075.1991.tb05006.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lin B. T., Gruenwald S., Morla A. O., Lee W. H., Wang J. Y. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. EMBO J. 1991 Apr;10(4):857–864. doi: 10.1002/j.1460-2075.1991.tb08018.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ludlow J. W., Glendening C. L., Livingston D. M., DeCarprio J. A. Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol. 1993 Jan;13(1):367–372. doi: 10.1128/mcb.13.1.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ludlow J. W., Shon J., Pipas J. M., Livingston D. M., DeCaprio J. A. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell. 1990 Feb 9;60(3):387–396. doi: 10.1016/0092-8674(90)90590-b. [DOI] [PubMed] [Google Scholar]
  29. Marks P. A., Richon V. M., Kiyokawa H., Rifkind R. A. Inducing differentiation of transformed cells with hybrid polar compounds: a cell cycle-dependent process. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10251–10254. doi: 10.1073/pnas.91.22.10251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Melloni E., Pontremoli S., Michetti M., Sacco O., Cakiroglu A. G., Jackson J. F., Rifkind R. A., Marks P. A. Protein kinase C activity and hexamethylenebisacetamide-induced erythroleukemia cell differentiation. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5282–5286. doi: 10.1073/pnas.84.15.5282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  32. Richon V. M., Rifkind R. A., Marks P. A. Expression and phosphorylation of the retinoblastoma protein during induced differentiation of murine erythroleukemia cells. Cell Growth Differ. 1992 Jul;3(7):413–420. [PubMed] [Google Scholar]
  33. Rustgi A. K., Dyson N., Bernards R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature. 1991 Aug 8;352(6335):541–544. doi: 10.1038/352541a0. [DOI] [PubMed] [Google Scholar]
  34. Terada M., Fried J., Nudel U., Rifkind R. A., Marks P. A. Transient inhibition of initiation of S-phase associated with dimethyl sulfoxide induction of murine erythroleukemia cells to erythroid differentiation. Proc Natl Acad Sci U S A. 1977 Jan;74(1):248–252. doi: 10.1073/pnas.74.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Welch P. J., Wang J. Y. A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle. Cell. 1993 Nov 19;75(4):779–790. doi: 10.1016/0092-8674(93)90497-e. [DOI] [PubMed] [Google Scholar]
  36. Zhou Y., Li J., Xu K., Hu S. X., Benedict W. F., Xu H. J. Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4165–4169. doi: 10.1073/pnas.91.10.4165. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES