title: Combination of all-trans retinoic acid and interferon-gamma upregulated p27(kip1) and down regulated CDK2 to cause cell cycle arrest leading to differentiation and apoptosis in human glioblastoma LN18 (PTEN-proficient) and U87MG (PTEN-deficient) cells.
abstract: OBJECTIVE
  Deletion or mutation of phosphatase and tensin homolog located on chromosome ten (PTEN) occurs in as high as 80% glioblastoma. All-trans retinoic acid (ATRA) induces differentiation in cancer cells. Interferon-gamma (IFN-gamma) induces apoptosis in many cancers including glioblastoma. We used the combination of ATRA and IFN-gamma to control growth of human glioblastoma LN18 (PTEN-proficient) and U87MG (PTEN-deficient) cells and explored any advantage of having PTEN in the cells.
  
  
  METHODS
  LN18 and U87MG cells were treated with ATRA (1 microM) for 7 days and then IFN-gamma (5 ng/ml) for 1 day. Methylene blue staining indicated astrocytic differentiation. Wright staining and ApopTag assay showed characteristic features of apoptosis. Western blotting demonstrated the levels of specific proteins.
  
  
  RESULTS
  ATRA and IFN-gamma alone and in combination could induce apoptosis in LN18 cells; while ATRA alone induced differentiation only, IFN-gamma alone induced apoptosis, and ATRA plus IFN-gamma increased apoptosis in U87MG cells. The variation in induction of apoptosis by ATRA alone might be attributed to difference in PTEN expression in the two cell lines. Compared with control cells, IFN-gamma alone and ATRA plus IFN-gamma increased PTEN expression in LN18 cells while there was no PTEN expression or induction in U87MG cells after treatments with ATRA alone and ATRA plus IFN-gamma. Apoptosis in both cell lines was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c into the cytosol, and calpain and caspase-3 activities. Treatments elevated p27(kip1) and decreased CDK2 levels in both cell lines, indicating cell cycle arrest at G(1)/S phase.
  
  
  CONCLUSIONS
  The combination of ATRA and IFN-gamma could control the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells by arresting cell division and inducing differentiation and apoptosis. Thus, our study indicated that the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells could effectively be controlled by treatment with the combination of ATRA and IFN-gamma.
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.forename: Naren L
authors.initials: NL
authors.lastname: Banik
authors.forename: Swapan K
authors.initials: SK
authors.lastname: Ray
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D015536:Down-Regulation; D005909:Glioblastoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000008:administration & dosage / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051059:PTEN Phosphohydrolase / Q000096:biosynthesis*; D011994:Recombinant Proteins; D014212:Tretinoin / Q000008:administration & dosage / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014212:Tretinoin; D007371:Interferon-gamma; D045304:Cytochromes c; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human