Carla M. Arundel

Enhancement of radiation injury in human colon tumor cells by the maturational agent sodium butyrate (NaB).

Two subpopulations of human colon tumor cells (clones A and D) which differ in their intrinsic sensitivity to X irradiation were grown for several passages in tissue culture medium containing the differentiation-inducing agent sodium butyrate (NaB, 2 mM). Values of the single-hit, multitarget survival curve parameters for non-NaB-treated clone A cells were n = 17.1, D0(Gy) = 0.81, and DQ(Gy) = 2.31; corresponding parameters for NaB-treated cells were 1.04, 1.16, and 0.05. For non-NaB-treated clone D cells, the survival parameters were n = 4.27, D0 = 1.05, and DQ = 1.52; corresponding parameters for NaB-treated cells were 1.19, 1.15, and 0.20. The large reduction in the DQ parameters of both clone A and D cells after NaB treatment indicates that sodium butyrate-induced cell maturation is accompanied by increase in radiation cell kill, particularly in the low-dose region of the survival curve.

Effects of alpha-difluoromethylornithine-induced polyamine depletion on the radiosensitivity of a human colon carcinoma cell line.

The effect of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) on the in vitro radiation response of Clone A human colon adenocarcinoma cells was investigated. Analysis of intracellular polyamine levels showed that exposure of Clone A cells to 1 mM DFMO for 96 h reduced putrescine and spermidine to nondetectable levels, while spermine was decreased by approximately 50%. This DFMO treatment protocol enhanced the radiosensitivity of Clone A cells, which was reflected by a decrease in both the Do and Dq. The addition of putrescine (1 mM) for the final 48 h of DFMO exposure restored polyamine levels and returned clone A radiosensitivity to that of control cells. These results indicate that polyamine depletion by DFMO sensitizes Clone A tumor cells to ionizing radiation.

Polar solvent modification of X ray induced potentially lethal damage in heterogeneous human colon tumor cells in vitro

Two subpopulations of tumor cells (clones A and D) obtained from a human colon adenocarcinoma were examined for their sensitivities to x-irradiation as unfed, early plateau phase cultures. Both the single dose survival curves and the kinetics of potentially lethal damage recovery (PLDR) were determined for the two tumor lines. Also, possible modification of PLDR by N,N-dimethylformamide (DMF), which has previously been shown to enhance the radiosensitivity of exponentially growing tumor cells, was investigated by adding DMF (0.8% v/v) to plateau phase cultures immediately after irradiation, and determining effects on the extent of PLDR. For non-DMF treated cells, the survival curve parameters of the diploid (clone D) and aneuploid (clone A) lines were very similar. The single-hit, multitarget values for n, Do (Gy), and Dq (Gy) were: 7.9, 0.82, and 1.70 for clone D; and 10.6, 0.83, and 1.96 for clone A. Using initial survival levels of 3.5% (clone D) or 5.5% (clone A) to investigate PLDR, it was found that the increase in survival (surviving fraction ratio or SFR) for clone D was 2.2, while the SFR for clone A was 1.6. DMF did not change either the kinetics or extent of PLDR in these two tumor lines when added to cultures immediately after irradiation. Our results indicate that significant heterogeneity in PLDR exists between these closely related tumor subpopulations.

Changes in X-Ray Sensitivity and Glutathione Content of Human Colon Tumor Cells after Exposure to the Differentiation-Inducing Agent Sodium Butyrate

Clone A human colon cancer cells were exposed to concentrations of sodium butyrate (NAB, 0-2 mM) for three passages in vitro, and responses to either graded single doses or split doses of 250 kVp X rays were determined. The survival data were fit to the single-hit, multitarget model of inactivation. For the graded single dose experiments, we found that NAB produced a decrease in the magnitude of the quasi-threshold (Dq) parameter after a concentration of about 0.9 mM was exceeded. Similarly, in split dose experiments, the amount of sublethal damage recovery (SLDR) was reduced in a concentration-dependent manner as shown by a decrease in the Dq parameter. However, the inhibition of SLDR occurred with no apparent threshold NAB concentration. NAB did not affect potentially lethal damage recovery. Paradoxically, increasing concentrations of NAB produced an exponential increase in the intracellular glutathione content, which could be blocked by exposure of the cells to buthionine sulfoximine (BSO). BSO treatment of NAB-adapted cells led to additional cell killing, again most noted by changes in the Dq parameter. We postulate that these responses are associated with NAB-induced changes in chromatin structure, particularly the association between DNA and nucleosomal histones H3 and H4.

Potentiation of in vitro cytotoxic effects of misonidazole on human colon tumor cells by the differentiation-inducing agent N-methylformamide

Human colon tumor cells (clone A) were studied in vitro with regard to modification of dose-dependent cytotoxicity to misonidazole (MISO) treatment by pre-exposure growth in medium containing the differentiation-inducing agent N-methylformamide (NMF). Cells were grown as exponential cultures and were exposed for 2 passages to 170 mM NMF before exposure to graded doses of MISO (0-100 mM, 3 hours at 37 degrees C, oxic or hypoxic). Both oxic and hypoxic cells could be sensitized to MISO cell killing. Using the 10% level of survival for comparison, the calculated MISO doses (mM) were: 105, 37, 50, and 10 for oxic control cells, hypoxic control cells, oxic-NMF treated cells, and hypoxic-NMF treated cells, respectively. Therefore, for NMF treated oxic cells, cell killing was increased by a factor of about 2.1, while for NMF treated hypoxic cells, cell killing increased by a factor of about 3.7. These data indicate that NMF treatment, while potentiating effects on both oxic and hypoxic cells, appears to have selectivity towards hypoxic cells. NMF may therefore have use in combined modality radiation therapy of solid tumors with electron-affinic radiosensitizers.

Enhancement of Radiation-Induced DNA-Protein Crosslinking by N-Methylformamide

The effects of the differentiating agent N-methylformamide (NMF) on radiation-induced DNA damage and repair in vitro were investigated using the alkaline elution assay. Two tumor cell lines were examined: Clone A, a human colon adenocarcinoma, and HCA-1, a murine hepatocarcinoma. Both cell lines showed changes suggestive of a better differentiated phenotype when exposed to NMF. Treatment with NMF enhanced the radiation sensitivity of Clone A cells but had no effect on the radiation response of HCA-1 cells. Irradiation of NMF-treated cells, both Clone A and HCA-1, induced the formation of DNA-protein crosslinks (DPCs). The level of DPCs induced increased linearly as a function of increasing gamma-ray dose. The DPCs did not seem to be the result of NMF exposure alone, but rather an NMF-mediated modification of the spectrum of gamma-ray-induced DNA lesions. When the DPCs were removed by proteolytic digestion, no NMF effect was observed on either strand-break formation or repair.