Effect of Radiation and Gamma Linolenic Acid on Cell Cycle Progression of TK6 Lymphoblastic and U937 Myelomonocytic Cells in Vitro.

The aim of the current work is to investigate how g-radiation might affect progression of TK6 and U937 cells through the cell cycle, and whether the addition of g linolenic acid (GLA), which has been shown to induce apoptosis, may cause any additional effects.

Exponentially growing cells were exposed to 0, 0.1 and 0.3 Gy ⁶⁰Co g-radiation, then at intervals cells were fixed and stained with propidium iodide and cell cycle distributions analysed by flow cytometry. In some of the experiments cells were exposed to 20 m g GLA /ml for 24 hours prior to as well as after radiation until they were fixed.

In the case of TK6 cells we observed an accumulation in the G2/M phase by 3 hours after radiation with a maximum accumulation at 4 and 8 hours. The degree of accumulation was slightly greater for cells exposed to 0.3 Gy. At 16 hours after radiation the percentage of cells in the G2/M phase had reverted to that observed for the controls. The presence of GLA did not appear to have any additional effect on the flow cytometric patterns observed for irradiated cells. Increased DNA degradation as evidenced by signals below that of the G0/G1 peak was observed in cells exposed to GLA for up to 32 hours, although longer times of exposure indicated less DNA degradation.

By contrast, in the U937 cells, radiation doses of 0.1 and 0.3 Gy did not result in any change in the flow cytometric profiles at any time after radiation. About 10% of the cells showed DNA degradation, regardless of whether they had been irradiated or exposed to GLA.

The demonstration of accumulation of TK6 cells in the G2/M phase after doses as low as 0.1 Gy may indicate that a checkpoint is operational at the G2/M border, whereas this checkpoint is not operational in the U937 cells because there was no accumulation of cells in G2/M in this cell line. The checkpoint may be p53 related, because TK6 cells have normal p53, whereas U937 cells have have mutant inactive p53. The relevance of our findings to clinical radiotherapy will be addressed.
back...

The dose dependence of G2 delay

The cell cycle delay at the G2/M transition is generally thought to facilitate repair and prevent the propagation of defective DNA. At this stage no relationship exists between the duration of the G2/M delay and the level of DNA damage. We have examined this question in a number of p53 mutant cell lines of different radiosensitivity. 

The SF2 of HT29 colon carcinoma cells, L132 lung epithelial cells and ATs4 (Ataxia telangiectasia) was found to be 0.48, 0.83 and 0.18 respectively. Hence these cells differ widely in radiosensitivity. Irradiation with 4 Gy, 6 Gy and 2 Gy of ⁶⁰Co gamma radiation induced the same survival level of 0.165 in all three cell lines. Using flow cytometry to determine the length of G2 delay, it was found that all three cell lines stayed in the G2 block for ±41 hours before restoring normal cell cycle progression.

These results suggest that the time spent in G2 block by cells after gamma irradiation is an indication of the amount of DNA damage. Repair studies are underway to confirm this impression.
back...

Flow cytometric analysis of p53 protein expression in gamma irradiated wild type and mutant tumour cells.

The tumour suppressor protein p53 is a conformationally flexible sequence-specific DNA binding protein. Mutant p53 proteins fail to bind to DNA and is inactive in cell regulation. This may be one reason for enhanced cell proliferation in tumours. The gene encoding the p53 protein is mutated in 60% of human tumours. Whereas wild-type p53 protein has a short half-life, the mutated p53 protein is not degraded and accumulates in the nucleus. We measured p53 accumulation by multiparameter flow cytometry. To this end we have evaluated 4 commercially available, monoclonal antibodies, MAbs 421, 240, 1620 and DO1, in 7 tumour cell lines with known p53 status: A549 and 4917 (wild type p53), BE11, MeWo and 4451 (mutated p53), HeLa (p53 null) and VA13 (overexpression of p53). We found that MAb DO1 effectively discriminated p53 positive and p53 negative cells, whereas MAb 1620 was negative in all cell lines tested.

Exposure to ionising radiation induces p53. In wild type p53 cells, the subsequent activation of p21 inhibits the cyclin dependent kinase. This leads to a block at the G₁/S transition and generates adequate time for repair. In tumour cells which express mutant p53, the G₁ checkpoint response is absent and this may lead to acquired radioresistance, accumulation of mutations and conversion to a cancerous state. Treatment of cells with 7 Gy ionising radiation and analysis of p53 levels 1, 4 hours and at the time of maximum G₂ delay postirradiation showed that MAb 421 and MAb 240 or Mab DO1 effectively detect changing p53 levels. p53 mutant cells exhibit altered damage responses amongst which absence of the G₁ block and endoreplication at the stage of the G₂ block are significant. The altered damage responses in tumours may be harnessed clinically and the p53 status would be a major indicator.
back...

Assessment of Proton Radiobiology using Established Neuronal Cell Lines and Peripheral Blood Lymphocytes

Treatment of malignant disease by standard dose-fractionation induces a wide variety of local and systemic responses in normal tissue. More often than not, malignant tissues are more radioresistant than the surrounding normal tissue. The degree to which normal tissue is spared in radiation treatment would therefore, be influenced by the radiosensitivity of the malignant tissue. The high intrinsic radioresistance and poor tumour control of neuronal cells are of great concern. In clinical radiotherapy, it would therefore be very desirable that the radiosensitivity of tumour tissues be accurately predicted. Reconstruction of the extent of radiation damage from biomarkers has been found to be a sensitive tool in the assessment of tissue radiosensitivity. The levels of radiation-induced micronuclei, dicentric chromosomes, reciprocal translocations and apoptosis observed in vitro may be a reflection of the cell-specific differences in radiosensitivity, and these may also be manifested at the level of the chromosome. In this study, the assessment of the radiobiological responses of neuronal cells, as well as normal blood lymphocytes, to 200 MeV protons, neutrons and ⁶⁰Co g -rays is proposed. Histological and molecular biological techniques like flow cytometry, micronuclei assay, fluorescence in situ hybridisation (FISH) and fluorescence microscopy would be used.
back...