title: 123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1).
abstract: OBJECTIVE
  To evaluate the internalization and nuclear translocation of (123)I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1).
  
  
  METHODS
  Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21(WAF-1/Cip-1) antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21(WAF-1/Cip-1). Internalization and nuclear translocation were measured. The ability of tat-anti-p21(WAF-1/Cip-1) to block G(1)-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21(WAF-1/Cip-1) was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF.
  
  
  RESULTS
  There was 13.4+/-0.2% of radioactivity internalized by MDA-MB-468 cells incubated with (123)I-tat-anti-p21(WAF-1/Cip-1) and 34.6+/-3.1% imported into the nucleus. Tat-anti-p21(WAF-1/Cip-1)(8 muM) decreased the proportion of EGF-treated cells in G(1) phase from 81.9+/-0.7% to 46.1+/-0.7% (p<0.001), almost restoring the G(1) phase fraction to that of unexposed cells (25.8+/-0.2%). Non-specific tat-mouse IgG did not block EGF-induced G(1)-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21(WAF-1/Cip-1) than in mice not receiving EGF (3.1+/-0.4% versus 1.8+/-0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21(WAF-1/Cip-1)/beta-actin ratio.
  
  
  CONCLUSIONS
  We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible.
authors.affiliation: Division of Nuclear Medicine, University Health Network, Toronto, ON, Canada.
authors.forename: Meiduo
authors.initials: M
authors.lastname: Hu
authors.forename: Paul
authors.initials: P
authors.lastname: Chen
authors.forename: Judy
authors.initials: J
authors.lastname: Wang
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Scollard
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Vallis
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics*; D007553:Isotope Labeling / Q000379:methods; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009928:Organ Specificity; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D015696:Gene Products, tat; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals