DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

Int J Radiat Oncol Biol Phys. 1999 Jan 1;43(1):191-8. doi: 10.1016/s0360-3016(98)00362-9.

Abstract

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation.

Methods and materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting.

Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines.

Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Squamous Cell / genetics*
  • Cell Survival / genetics
  • DNA / genetics*
  • DNA / radiation effects
  • DNA Damage*
  • DNA Ligases / physiology*
  • DNA Ligases / radiation effects
  • DNA Repair*
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins*
  • Humans
  • Nuclear Proteins
  • Protein-Serine-Threonine Kinases / physiology*
  • Protein-Serine-Threonine Kinases / radiation effects
  • Radiation Dosage
  • Radiation Tolerance
  • S Phase
  • Tumor Cells, Cultured / radiation effects

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • DNA
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • DNA Ligases