Chromatin modifications associated with N-methylformamide-induced radiosensitization of clone A cells.

Exposure of certain cell lines to the differentiation-inducing agent N-methylformamide (NMF) enhances their radiosensitivity. As part of an attempt to elucidate the mechanism of NMF-induced radiosensitization, we examined the effects of NMF on chromatin structure, as reflected by changes in DNA-protein cross-links (DPCs) and the chromatin protein/DNA ratio, in two cell lines, clone A and HCA-1. Both lines form a better-differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. Ionizing radiation induced DPCs in a linear manner beginning at about 10 Gy and continuing to at least 50 Gy in both cell types. NMF treatment of HCA-1 cells did not affect the background level of DPCs, but it enhanced the formation of radiation-induced DPCs at each dose tested. In clone A cells, NMF exposure elevated the DPC background level more than two-fold, and modified radiation-induced DPCs. The dose response for radiation-induced DPCs in NMF-treated clone A cells consisted of a linear increase up to 12.5 Gy, which was greater than in untreated cells, followed by a plateau level of DPCs out to 50 Gy, the highest dose tested. NMF treatment of clone A, but not HCA-1, cells also increased the chromatin protein/DNA ratio by about 30-35%. In clone A cells, the increases in DPC background level and chromatin protein/DNA ratio as a function of NMF exposure time followed a pattern similar to that of the enhancement of radiosensitivity. These data suggested that modifications of chromatin structure, not involved in differentiation, may be associated with the radiosensitizing actions of NMF.

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.

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.

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.

Effects of nucleoside analogs and sodium butyrate on recovery from potentially lethal X ray damage in human colon tumor cells.

The effects of 5-azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza-CdR) both alone and in combination with sodium butyrate (NaB) on intrinsic radiation sensitivity and ability to recover from potentially lethal damage (PLDR) were studied in two subpopulations of cells (clones A and D) from a heterogeneous human colon adenocarcinoma (DLD-1). Growth for three passages in medium containing 1 mM NaB alone enhanced radiation cell killing in the low dose ("shoulder") region of the survival curve for both cell lines. Neither 1.0 microM 5-aza-CR nor 0.25 microM 5-aza-CdR alone enhanced cell killing. However, treatment of these cells with a combination of either 5-aza-CR or 5-aza-CdR and NaB enhanced radiation cell killing at a clinically relevant dose level of 2.0 Gy by approximately 25% for both clone A and clone D cells. Also, while exposure to these differentiation-inducing agents separately enhanced the expression of PLDR in both tumor subpopulations, treatment with either of the combinations reversed this increase in PLDR. These results indicate that the gene-activating agents 5-aza-CR, 5-aza-CdR, and NaB may interact to modify the radiation sensitivity of two human tumor cell lines. Such combinations may prove useful clinically, if enhanced X ray cell killing of tumor cells can be achieved without a concomitant enhancement of recovery from potentially lethal X ray damage.

Spontaneous transformation to anchorage-independent growth of a xeroderma pigmentosum fibroblast cell strain.

A skin fibroblast cell strain (GM2995) derived from a patient with xeroderma pigmentosum was received at low passage. As the cells were serially passaged (1:4 dilution), their size and growth characteristics changed. By passage 13, approximately 90% of the colonies produced by cells seeded at low density were composed of small, densely packed cells. Cells capable of anchorage-independent growth were observed after passage 7; they formed round, smooth-edged colonies in soft agar. The frequency of cells exhibiting anchorage-independent growth increased rapidly at subsequent passages, reaching 35-50% of the population by passage 20. This phenomenon was accompanied by the appearance of aneuploidy. These cells are still proliferating actively at passage 35. These late-passage GM2995 cells retain the extreme hypersensitivity to the cytotoxic effect of UV radiation characteristic of early-passage GM2995 cells.

Response to BCNU of spheroids grown from mixtures of drug-sensitive and drug-resistant cells.

Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R3) to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). As shown previously, after treatment with 3 microM BCNU, percentages of each cell subpopulation in mixed-cell spheroids were estimated with the sister chromatid exchange (SCE) assay and found to be approximately the same as percentages used to initiate spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with BCNU, estimated by changes in the number of SCEs induced by treatment, decreased as the percentage of R3 cells increased. When spheroids were disaggregated into single cells before treatment, however, the number of SCEs induced in the 9L population did not decrease but remained at levels similar to those found for spheroids grown from 9L cells only. These data suggest that the cell-cell interactions that influence BCNU sensitivity in mixed cell spheroids depend on three-dimensional intercellular contact. The response of purely 9L, purely R3, and mixed-cell spheroids to BCNU was also determined using the cell survival and spheroid growth delay assays. The surviving fractions of individual spheroids treated with 40 microM BCNU were slightly greater than expected; growth delays found for mixed-cell spheroids were 2-3 days less than expected. These findings suggest that cells in mixed-cell spheroids are more resistant to BCNU than would be predicted from the sensitivities of purely 9L and R3 spheroids.

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.

Contrasting effects of the differentiating agent sodium butyrate on recovery processes after x-irradiation in heterogeneous human colon tumor cells.

The X ray survival responses of two clonal subpopulations of cells (clones A and D) from a heterogeneous human colon adenocarcinoma (DLD-l) were studied in unfed plateau phase cultures either as control cultures or in cultures grown for three passages in medium containing the differentiating agent sodium butyrate (NaB, 2 mM). Specifically, the cultures were studied with regard to their ability to express both potentially lethal and sublethal damage recovery (PLDR and SLDR). Growth in NaB-containing medium enhanced the radiation sensitivity of both cell lines in the low dose ("shoulder") region of the survival curve. For clone A, the Dq value was reduced by 59%, and for clone D, the Dq value was reduced by 96%. NaB treatment increased both the rate and the extent of PLDR in both cell lines as assessed by single dose kinetic studies. However, when split dose experiments are performed to assess the expression of SLDR, NaB pretreatment was shown to totally inhibit the expression of SLDR, and also to alter the expression of PLDR under these conditions. These data suggest that PLDR and SLDR are separate, yet related, cellular recovery processes. In addition, NaB may be useful as an adjunct to radiotherapy by virtue of its ability to sensitize tumor cells in the dose range conventionally used for therapy, as well as by inhibition of sublethal damage recovery.

Effects of nutritional state on the expression of radiation injury in two tumor subpopulations obtained from a heterogeneous human colon carcinoma.

Environmental modification of the intrinsic ionizing radiation sensitivity of two tumor cell subpopulations obtained from a single heterogeneous human colon adenocarcinoma was studied. The two subpopulations (termed clones A and D) were grown as monolayers to produce plateau phase cultures. Two different plateau phase growth states were produced by either changing the culture medium daily until maximum density was achieved ("fed" cultures), or by not changing the original medium for the entire duration of growth ("unfed" cultures). Cells were irradiated with graded single doses of X rays to determine survival responses. Using conventional single-hit, multi-target terminology, the extrapolation numbers (n), mean lethal doses (D0, Gy), and quasi-threshold doses (Dq, Gy) for fed and unfed cultures of clone A cells were 23.7, 0.80, and 2.54 (fed); and 9.65, 0.85, and 1.93 (unfed). For fed and unfed cultures of clone D cells these values were: 13.0, 0.76, and 1.94 (fed); and 23.8, 0.73, and 2.30 (unfed). No significant difference was found between the radiation responses of these two tumor subpopulations in the plateau phase of growth, either as fed or unfed cultures. The D0 value for clone D, but not for clone A, was significantly less than the D0 value for cells in exponential growth, indicating that the change from exponential growth to a plateau growth state is accompanied by a change in the intrinsic sensitivity of clone D tumor cells. After establishment of the single dose response curves, we then investigated the ability of clone A and D cells to recover from radiation injury (potentially lethal damage recovery, PLDR) by irradiating plateau phase cultures with a single radiation dose sufficient to reduce survival to about 1%, and then subculturing the cells into fresh medium at times varying from 0-24 hours postirradiation. We found that the maximum increase in survival was seen at 12-24 hours postirradiation, and that the ratio of this maximum survival to the cell survival when subculturing was done immediately after irradiation was 2.7 (fed) and 2.5 (unfed) for clone A cells, and 3.6 (fed) and 3.0 (unfed) for clone D cells. Therefore, whereas clone D cells have a greater ability to express PLDR (by a factor of about 1.3) than clone A cells, the modification of this recovery by the environment was identical, with survival in the fed state being greater than in the unfed state by a factor of 1.2 and 1.1 for clone D and clone A tumor subpopulations, respectively.

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.

In vitro effects of N,N-dimethylformamide on sublethal and potentially lethal damage recovery processes after X-irradiation in heterogeneous human colon tumor cells.

The responses of unfed plateau-phase cultures of two clonal subpopulations of cells (clones A and D) from a human colon adenocarcinoma (DLD-1) to X-irradiation were examined in detail either as control cultures or after growth in medium containing the differentiating agent N,N-dimethylformamide (DMF, 0.8%, three passages). Specifically, the cultures were studied with regard to their ability to express both potentially lethal and sublethal damage recovery (PLDR and SLDR, respectively). In PLDR studies on control cells, clone D expressed more PLDR than clone A, although recovery half-times were the same. DMF treatment increased the expression of PLDR in both cell lines and decreased the half-times for recovery. When recovery from sublethal radiation injury was assessed, the rate and extent of SLDR in non-DMF-treated clone A and D cells were identical. In contrast to the PLDR results, DMF treatment had no significant effect on SLDR in either cell line. These studies show that, while DMF treatment of human colon tumor cells increases cell killing in the clinically relevant, low-dose ("shoulder") region of the X-ray survival curve, this increase in cytotoxicity is not due to an inhibition of the repair of sublethal damage.

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.