Severe combined immune deficiency (scid) mice fail to produce mature B and T cells and are sensitive to ionizing radiation. They contain a mutation in the 460-kDa catalytic subunit of the DNA-dependent protein kinase that is involved in both V(D)J rejoining and DNA double-strand break (DSB) repair. The kinetics of DSB rejoining was quantified in both scid cells and the parental C.B-17 cells after three different doses of X irradiation: 3, 7.5 and 10 Gy. Repair of DNA DSBs was determined using pulsed-field gel electrophoresis, Southern hybridization and phosphor image analysis. After X irradiation, the cells were allowed to repair at 37 degrees C for up to 1 h or up to 24 h. The most profound difference between the two cell lines was the greatly reduced rate of the slow component of DSB repair in scid cells. C.B-17 cells repaired most of the damage within 1 h, whereas scid cells required 4 to 6 h to reach a similar level after the same dose. No residual or unrepairable DSBs were detected in either cell line 24 h after doses as high as 10 Gy. The scid cells subjected to two doses of 1.5 Gy separated by increasing amounts of time showed no ability to repair sublethal damage between doses, whereas C.B-17 cells receiving two doses of 3.75 Gy separated by increasing periods did show increased levels of survival. These results indicate that scid cells can repair radiation-induced DNA DSBs, although at a reduced rate, but they lack the ability to undergo repair of sublethal damage.

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.

To determine whether microscopically visible double-minute chromosomes (DMs)

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.