John T. Leith

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.

Radiation myelopathy in the rat: an interpretation of dose effect relationships

Abstract Data were collected on the production of overt paralysis in the rat after low linear energy transfer (LET) ionizing radiation exposure of different regions of the spinal cord. Data from both single and multiple radiation exposure schedules were interpreted using the ED 50 value (estimated dose needed to produce 50% paralysis in a group of irradiated animals) as the isoeffect comparison dose. Plots were made of the reciprocal of the ED 50 total dose versus the size of the dose per fraction used. The use of multifractionation data in such a way allows implications to be made about the nature of the in vivo response curve. These reciprocal dose plots indicate that the spinal cord normal tissue system shows two responses that may be specifically characterized by the ration of the intercept (α) of the linear regression fit of the reciprocal total dose versus dose per fraction curve to the slope (β) of the curve (α/β). For rat spinal cord, this value is about 3.8 ± 0.4 (standard error) for the thoraco-lumbar region and about 2.0 ± 0.3 (standard error) for the cervical region. While absolute dose response data vary somewhat among investigators, all of the data on production of paralysis in rats show similar trends with respect to the (α/β). ratio. We feel that this ratio may uniquely characterize this (and other) normal tissue systems. Knowledge of this parameter and how it varies after different treatments (e.g., high LET radiation exposure) may be important.

Selective modification of the X ray survival response of two mouse mammary adenocarcinoma sublines by N,N-dimethylformamide

Abstract The modification of the X ray survival response of two mammary adenocarcinoma tumor cell sublines by the polar solvent N,N-dimethylformamide (DMF) has been studied. The sublines (termed lines 66 and 67) differ significantly in their single dose survival responses to x-irradiation. Survival responses were fitted to the single-hit, multi-target, and linear-quadratic equations. For non-DMF treated cells, line 67 radiation parameters were: D 0 ( Gy ) = 1.41, D q ( Gy ) = 1.41, n (extrapolation number) = 2.73, α (10 −1 Gy −1 ) = 2.83, and β (10 −2 Gy −2 ) = 4.17. For line 66, these values were D 0 = 1.63, D g = 3.01, n = 6.4, α = 0.45, and β = 4.28. For investigation of DMF effects, cells were seeded at 1 x 10 4 per 25 cm 2 tissue culture flask in a DMF concentration of 0.8 % (0.10 M) and allowed to multiply for 4 days. For line 67, this increased the cell culture doubling time by about 27 %, and for line 66, by about 22 %. Cells were still in exponential growth at the time of irradiation. Immediately prior to irradiation, the media was removed, fresh media added and flasks were exposed to 100 kVp X rays. After exposure, cells were removed by trypsinization, counted and replated at appropriate dilution in fresh media. The survival parameters for line 67 (the radiosensitive line) then became D 0) = 1.56, D q = 0.89, n = 1.77 , α = 3.39, and β = 3.49. For line 66 (the radioresistant line), the survival parameters were D 0 = 1.56, D q , = 0.94, n = 1.83, α = 4.14, and β = 2.41. These results indicate that DMF may have the potential for modifying the low dose “shoulder” region of survival, particularly for cell lines that possess large shoulders.

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.

X RAY RESPONSES OF A HUMAN COLON TUMOR CELL LINE AFTER EXPOSURE TO THE DIFFERENTIATION-INDUCING AGENT N-METHYLFORMAMIDE: CONCENTRATION DEPENDENCE AND REVERSIBILITY CHARACTERISTICS

The combination of differentiation-inducing agents with conventional cytotoxic agents has been suggested as a potential cancer therapeutic strategy. In this regard, we have chronically exposed (3 passages) a human colon tumor line (clone A) to varying concentrations (0-170 mM) of N-methylformamide and examined the change in sensitivity to ionizing radiation in vitro. The linear-quadratic formalism of survival was used to characterize the single graded dose survival curves. This equation yields two constants (alpha and beta) relating to cellular inactivation produced by either single events (alpha) or by the combination of two events (beta). As the N-methylformamide concentration increased, the alpha parameter increased while the beta parameter concomitantly decreased, yielding a concentration dependent radiosensitization which was most marked in the low dose region of the survival curve. Upon removal of NMF, the original radiation resistance was regained within 2-3 cell culture doubling times.

Modification of the response of mouse skin to X-irradiation by a polar solvent, N,N-dimethylformamide

The modification of the response of mouse skin to either single or split (24 hrs) graded doses of X rays by topically applied N,N-dimethylformamide (DMF) was investigated. DMF was applied daily for 5 days prior to irradiation. At a radiation dose level producing dry desquamation, DMF enhanced the X ray response by a factor of 1.3. Also, at the same level of response, the fraction of X ray dose repaired in 24 hours was 0.57, whereas for the DMF-treated and irradiated skin, this factor was 0.41, indicating a reduction of about 28% in subeffective damage repair. The times of maximal involvement of the skin reactions were not different in the X ray plus DMF treated mice versus mice receiving x-irradiation only. The data indicate that DMF is able to modify intrinsic radiation sensitivity of mouse skin epithelial cells, possibly through a reduction in the magnitude of the shoulder region of the survival curve.

Potentiation of x ray sensitivity by combinations of sodium butyrate and buthionine sulfoximine

The ability of various concentrations of the differentiation-inducing agent sodium butyrate (NAB, 0-2 mM) to produce radiosensitization in human colon tumor cells when combined with varying concentrations of the irreversible inhibitor of gamma-glutamyl cysteine synthetase, buthionine sulfoximine (BSO, 0-0.75 mM) was studied. We have previously shown that high concentrations of each agent in combination (2 mM NAB + 0.5 mM BSO) produced a supra-additive effect in terms of radiosensitization as indicated by a decrease in the quasi-threshold value (Dq) of the single dose survival curve; we wished to define responses at other concentrations. Cells were adapted in vitro to growth in medium containing NAB for 3 passages prior to x-irradiation and BSO was given acutely 24 hrs before the x-irradiations. The most effective combination was 0.3 mM NAB + 0.75 mM BSO. These data suggest that adaptation of tumor cells to chronic low levels of a differentiation-inducing agent such as NAB followed by administration of BSO just prior to irradiation might be an effective combination in producing increased response of solid tumors.

Modification of the Effects of Ionizing Radiation by Differentiation-Inductiona

The use of differentiation-inducing agents (DIAs) has been proposed as a possible means of altering the phenotypic expression of neoplastic cells; such alterations might then have an impact on the choice of therapeutic strategy.’ We have investigated the effects of two types of such agents (sodium butyrate: NAB; N,N-dimethylformamide or N-methylformamide: DMF or NMF) on the responses of both rodent and human tumor cells to X-irradiation, both in vitro and in v~vo.~-’ These agents do possess significant activity with regard to modification of cellular radiation sensitivity (TABLE 1) . The most important effect in vitro of both NAB and DMF/NMF is the decrease produced in the D, value of the tumor survival curve.’ Since radiotherapy is given in multiple small doses (e.g., 30 fractions), this increased sensitivity is precisely in the right dose region. If such increased killing were carried throughout a clinical treatment, even a small enhancement (e.g., 5 % ) could produce marked effects (about four logarithmic units increase in the number of cells killed). In vivo, an important result has been the observation that chronic administration of NAB or NMF results in essentially complete removal of hypoxic cells.’ Electrode measurements in vitro of NAB-adapted tumor cells suggest that the NAB treatment may be reducing metabolism; this reduction could then lead to effective “ reoxygenation” in vivo (J. Leith, unpublished data). In summary, the increased radiosentization, decreased proliferation, and altered hypoxic cell physiology observed in tumor cells treated with DIAs indicate that continued preclinical research into the combinations of DIAs with conventional cytotoxic agents is worth pursuing. Still, DIAs must be well characterized, as it is likely that individual agents will have unique properties. For example, DMF and NMF produce reductions of intracellular glutathione: while NAB produces a marked increase in such levels5 As glutathione metabolism may be an important factor in the extent of multi-drug resistance which occurs,9 this difference may have clinically important implications for strategies of therapy.