title: Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells.
abstract: BACKGROUND
  Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy.
  
  
  METHODS
  The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR).
  
  
  RESULTS
  Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells, suggesting a synergistic effect of the Mcl-1L siRNA with IR on radiosensitivity. Interestingly, during the development of radioresistant sublines using FIR, high expression of Mcl-1L was observed.
  
  
  CONCLUSIONS
  Our studies suggest that Mcl-1L isoform has an important role in the survival and radioresistance of OSCC and may be a promising therapeutic target in oral cancers.
authors.affiliation: Teni Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, India.
authors.forename: Vinayak C
authors.initials: VC
authors.lastname: Palve
authors.forename: Tanuja R
authors.initials: TR
authors.lastname: Teni
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D049109:Cell Proliferation / Q000528:radiation effects; D003037:Cobalt Radioisotopes / Q000009:adverse effects*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D009062:Mouth Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000502:physiology*; D011839:Radiation, Ionizing; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003037:Cobalt Radioisotopes; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein