title: The antiretroviral agent saquinavir enhances hTERT expression and telomerase activity in human T leukaemia cells in vitro.
abstract: BACKGROUND
  Saquinavir, a protease inhibitor utilized in HIV infection, shows antitumor activity in various experimental models. In previous studies performed in our laboratory the drug was found to induce a substantial increase of telomerase activity in normal peripheral blood mononuclear cells. Aim of the present investigation was to test whether saquinavir was able to increase telomerase activity and the expression of the catalytic subunit of telomerase, hTERT, in human malignant hematopoietic cells.
  
  
  METHODS
  Human Jurkat CD4+ T cell leukaemia cell line was used throughout the present study. The antiproliferative effect of saquinavir was tested by the MTT assay. Telomerase activity was determined according to the telomeric repeat amplification protocol. The expression of hTERT mRNA was semi-quantitative evaluated by RT-PCR amplification and quantitative Real Time PCR. The binding of the transcription factor c-Myc to its specific E-Box DNA binding-site of hTERT promoter was analyzed by Electophoretic Mobility Shift Assay (EMSA). The amount of c-Myc in cytoplasm and nucleus of leukemia cells was determined by Western Blot analysis, and c-Myc down-regulation was obtained by siRNA transfection.
  
  
  RESULTS
  Saquinavir produced a substantial increase of telomerase activity in Jurkat cells in vitro without increasing but rather reducing target cell proliferation rate. Telomerase up-regulation appeared to be the result of enhanced expression of hTERT. Saquinavir-mediated up-regulation of hTERT gene was the result of the increased binding of proteins to the E-Box sequence of the promoter. Moreover, saquinavir amplified the expression of c-Myc especially in the nuclear cell fraction. The direct influence of saquinavir on this transcription factor was also demonstrated by the antagonistic effect of the drug on siRNA induced c-Myc suppression. Since c-Myc is the main responsible for hTERT transcription, these findings suggest that the main mechanism underlying saquinavir-induced telomerase activation is mediated by c-Myc up-regulation.
  
  
  CONCLUSIONS
  Saquinavir augments hTERT expression while inhibiting leukemic cell growth. Experimental evidences show that this effect is mediated by saquinavir-influenced increase of c-Myc levels. This could have relevance in terms of enhanced hTERT-dependent tumor cell immunogenicity and suggests new paharmacological approaches interfering with c-Myc dependent pathways.
authors.affiliation: Department of Systems Medicine, Pharmacology Section, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Adamo
authors.forename: Alessandro
authors.initials: A
authors.lastname: Comandini
authors.forename: Angelo
authors.initials: A
authors.lastname: Aquino
authors.forename: Laura
authors.initials: L
authors.lastname: Bonmassar
authors.forename: Loredana
authors.initials: L
authors.lastname: Guglielmi
authors.forename: Enzo
authors.initials: E
authors.lastname: Bonmassar
authors.forename: Ornella
authors.initials: O
authors.lastname: Franzese
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D017320:HIV Protease Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D019169:Jurkat Cells; D015458:Leukemia, T-Cell / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D019258:Saquinavir / Q000494:pharmacology* / Q000627:therapeutic use; D019098:Telomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D017320:HIV Protease Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D019098:Telomerase; D019258:Saquinavir