Modification of the hypoxic fraction of a xenografted human colon tumor by differentiation-inducing agents.

Xenografted tumors were produced in nude mice by injection of HCT-15 human colon tumor cells. The hypoxic fractions of control tumors as determined from x-ray survival curves were approximately 18%. Other tumors were treated (every day X 9) with daily injections of N-methylformamide (150 mg/kg) or sodium butyrate (2,000 mg/kg). For both agents, it was found that the hypoxic fractions were less than 0.05% and less than 1.7%, respectively. These data indicate that selected differentiation-inducing agents could be of value for treatment of human solid tumors that contain hypoxic cells.

X-ray responses of human colon tumor cells grown in artificial capillary culture.

Clone A human colon adenocarcinoma cells were grown in three-dimensional artificial capillary culture (ACC) to determine responses of capillaries treated 3 weeks after tumor cell inoculation with a specific, easily quantifiable cytotoxic agent, ionizing radiation. The high-density growth of tumor cells in ACC can be considered to be an in vitro analogue of a solid tumor. Changes in extracapillary space (ECS) fluid concentrations of lactate dehydrogenase (LDH) and aspartate aminotransferase (GOT) and the utilization of glucose in circulating medium were monitored after a supralethal radiation dose (90 Gy) of X-rays. Immediately after irradiation, increased levels of LDH and GOT were found that reached maximum levels about four to five times those found in nonirradiated control capillaries at 10-13 days post irradiation and then declined. Patterns of enzyme production appeared to correlate with the numbers of nonviable tumor cells collected from the ECS of the artificial capillaries. In contrast, glucose utilization showed little correlation with either enzyme concentration or dead cell production. It was determined that, while capillaries grown and treated in this manner appear to respond in a dose-dependent manner to ionizing radiation (as indicated by changes in LDH and GOT levels), these particular end points are relatively insensitive and are not suitable for studies in which therapeutic levels of X-radiation might be given. In other studies, tumor cells were removed from unirradiated capillaries by trypsinization and used to obtain complete survival curves after graded doses of X-radiation. The dose-response curves obtained indicate that clone A colon tumor cells grown in ACC show a marked decrease in their ability to accumulate sublethal radiation injury as compared to responses of these cells growing exponentially in asynchronous monolayer cultures, to synchronized mid-G1 tumor cells, or to tumor cells in stationary growth phase. These data suggest that ACC is a potentially useful model to study the effects of cytotoxic agents on human tumor cells.

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.

Effect of preirradiation of transplantation site on growth characteristics and hypoxic fractions in human colon tumor xenografts.

The volumetric growth curves and hypoxic fractions of seven different human colon tumor lines (clone A, clone D, WiDR, SW480, SW620, DLD-2, and HCT-8) xenografted into the flank regions of either unirradiated nude mice or mice that had received 17.5 Gy of 250-kVp X-rays 1 day prior to implantation were biomathematically analyzed using the Verhulstian equation. Significant variation was found among tumors with respect to both initial growth rates (r, days-1) and theoretical final volumes (carrying capacities, K, mm3). In radiation-damaged normal tissue, tumors grew relatively well for about the first 2 wk postimplantation, attaining volumes of about 70 to 155 mm3. Then, tumor growth rates altered. This effect varied from relatively minor effects on growth rate (tumors of clones A and D) to inhibition of growth, with actual decreases in tumor volume (e.g., WiDr, SW480, SW620, HCT-8, and DLD-2). After this short-term transience in growth kinetics, neoplasms began to steadily regrow at about 3 wk postimplantation, albeit at a slower rate than that seen in controls. Tumor bed effect values were calculated using the ratio of times at which control tumors and tumors growing in the radiation-injured tissue reached a volume of 7.5% of the K values derived from the respective control growth curves. Values for clone D, clone A, and WiDR, SW480, SW620, DLD-2, and HCT-8 tumors were, respectively, 1.89, 2.41, 3.48, 3.62, 2.82, 3.66, and 3.65, indicating that tumor bed effect responses varied by almost 100%, even for cancers of the same neoplastic class. Also, the hypoxic fractions of all tumors growing in radiation-damaged sites were increased as compared with levels in controls.

Hypoxic fractions in xenografted human colon tumors.

We investigated the percentage of radiobiologically hypoxic cells within 11 different xenografted human colon tumors using an in vivo-in vitro excision assay technique. Tumors were excised at average volumes of 750 mm3, and it was found that hypoxic fractions varied from less than 1% (clone D) to over 80% (HCT-8). The geometric mean hypoxic percentage was 10.4% (95% confidence interval, 4.9 to 22.1%). Comparison of the percentage of hypoxia results from the xenografted human colon tumors to published data from xenografted melanomas suggests that transplanted colorectal tumors as a class contain significantly less hypoxia than do the melanomas.

Compositional stability of artificial heterogeneous tumors in vivo: use of mitomycin C as a cytotoxic probe.

A major part of the overall response of solid cancers to cytotoxic treatments will be due to the differential sensitivities of the neoplastic cell subpopulations present. To quantitatively investigate this, artificial heterogeneous human colon xenograft tumors were constructed using two clonally related cell lines (A and D) which were mixed to create compositions of approximately 9:1 or 1:9 A:D cells. Then, the pure A, D, or admixed tumors were challenged with mitomycin C which kills A cells more efficiently than D cells by a factor of about 2.3 as determined by in vitro survival curve inactivation slopes. This difference was also exhibited in vivo by the post-mitomycin C cell survival responses determined by excision assay and by the shapes of the regrowth curves of the pure A and D or admixed tumors. By approximately 30 days after treatment, artificial heterogeneous tumors had reached a new stable cellular composition which could be quantitatively predicted based on the individual survival of A and D cells from pure clonal tumors measured 24 h after treatment. Thus, in this model system, cytotoxic treatment of heterogeneous neoplasms produced predictable and stable, albeit altered, percentage admixtures of subpopulations. This result is consistent with the observed decreased clinical responsivity of primary tumors to sequential courses of therapy due to selection of preexisting resistant subpopulations.

Growth properties of artificial heterogeneous human colon tumors.

Two clonal cell lines (designated as clones A and D), originally isolated from the heterogeneous DLD-1 human colon adenocarcinoma, were used to produce xenograft tumors in nude mice. Neoplasms produced from either A or D cells alone were compared to those produced from a range of percentage admixtures of the two subpopulations. Then, Gompertzian growth parameters (initial growth rates, retardation rates) were determined, along with estimation of the final asymptotic volumes. It was found that the growth kinetics of the various artificial heterogeneous tumors could not be predicted from knowledge of the growth parameters of the pure clonal xenograft tumors. Additionally, both pure clonal and artificial heterogeneous tumors were enzymatically disaggregated as a function of time postinjection, and it was found that the admixed tumors became more zonal in composition as time progressed. Further, admixtures of extreme composition (i.e., 9% A plus 91% D or 88% A plus 12% D) remained stable with time, while those of intermediate initial composition (i.e., 50% A plus 50% D) did not. All of these data (growth kinetics, zonality, compositional stability) indicate that the growth properties of heterogeneous tumors are very complex.

Modification of the growth rates and hypoxic fractions of xenografted A431 tumors by sialoadenectomy or exogenously supplied epidermal growth factor.

We studied A431 epidermoid carcinomas xenografted into male nude mice either in the unperturbed state or after either surgical removal of the salivary glands or i.p. injection of exogenous epidermal growth factor (0.2 mg/kg daily for 7 days). The percentage of hypoxic cells in unperturbed tumors was 10.5% (95% confidence limits, 6.6-16.8%). In mice that received epidermal growth factor injections, hypoxic percentages decreased to 3.7% (1.7-7.8%), and tumor growth rates increased. In sialoadenectomized mice, hypoxic percentages increased to 35.2% (27.1-45.6%), and tumor growth rates decreased. These data indicate that the biology of solid tumors can be significantly modified by the host status.

Intrinsic and extrinsic heterogeneity in the responses of parent and clonal human colon carcinoma xenografts to photon irradiation.

Responses to photon irradiation of xenografted human colon tumors derived from the heterogeneous DLD-1 line or its derivative A and D subpopulations were determined using excision assay and tumor regrowth delay assays. Differential responses among the three xenografted carcinomas were demonstrated. Clone A tumors treated with up to 17.5 Gy showed no actual regression below pretreatment volume. In contrast, clone D tumors were sensitive to doses as low as 3.5 Gy, and tumor volumes were reduced by 65% with a dose of 17.5 Gy. The responses of DLD-1 tumors were intermediate between the clone A and clone D tumor responses. The survival parameters obtained in the excision assay studies for the DLD-1, clone A, and clone D tumors were, respectively: n = 3.3, 1.4, and 1.0; D0 (Gy) = 2.1, 2.2, and 2.7; and DQ (Gy) = 2.6, 0.6, and 0.0. These data indicate that the DLD-1 tumors were the most resistant, with clone A of intermediate sensitivity, clone D being the most sensitive tumor. In addition to the interclonal diversity among xenograft lines, intraclonal variation was also observed with clone A (but not clone D or DLD-1) tumors. A biphasic survival curve of cells from clone A xenografts irradiated in air-breathing hosts clearly indicated a minority (approximately 3%) subpopulation of hypoxic cells. Similar results indicating a small percentage of hypoxic cells in clone A solid tumors were obtained from the tumor regrowth delay studies. Also, excision assay data from experiments in which the heterografted carcinomas were irradiated under anoxic conditions support the interpretation that clone A tumors contain a small fraction of hypoxic cells. This study indicates that: (a) heterogeneity in vivo to ionizing radiation exists in the DLD-1 system; and (b) intraclonal variation occurs in vivo due to extrinsic (e.g., environmental hypoxia) factors, such that the intrinsic radioresistance of a subpopulation (clone A) of a heterogeneous human tumor can be further increased.

Thermal survival characteristics of cell subpopulations isolated from a heterogeneous human colon tumor.

Responses of a heterogeneous human colon adenocarcinoma model tumor system to in vitro hyperthermic treatment at various temperatures have been studied. This model tumor system consists of an original tumor line (DLD-1) obtained from surgical biopsy, and two derivative subpopulations termed clones A and D. These 3 tumor cell populations differ in many properties, including karyotype and DNA content, production of specific antigens, and sensitivities to other cytotoxic agents such as chemotherapeutic drugs and X-irradiation. In these experiments, exponentially growing tumor cells were exposed to hyperthermia (42.2, 42.5, 43.0, 44.0, or 45.0 degrees) for graded time periods. A single-hit, multitarget equation was used to express the dependence of survival on time at a given temperature, and values for extrapolation numbers, quasi-threshold time (min), and T0 (mean lethal time; min) were obtained for the initial regions of survival. At the lower temperatures of 42.2 and 42.5 degrees, biphasic survival curves were obtained for all three tumor lines and, as a consequence, a second mean lethal time (T0,f) was also determined for the final thermal-resistant portion of the survival curves. Using the T0 values as an index of relative resistance, values at 42.2 and 42.5 degrees indicated that, in this temperature region, the parent (DLD-1) line was the most sensitive, the clone A line showed intermediate sensitivity, and the clone D line was the most resistant. In the thermally resistant portion of the survival curve, T0 values indicated that the clone A subpopulation was the most sensitive, the DLD-1 line showed intermediate sensitivity, and the clone D tumor subpopulation remained the most resistant. At the higher temperatures of 43, 44, and 45 degrees, in which thermotolerance is not observed during heat treatment, values for T0 indicated the parent (DLD-1) tumor line was still the most sensitive tumor line, and the clone A and clone D lines showed approximately equal resistance. These data indicate that significant differences may exist among subpopulations of heterogeneous tumors in their survival responses to hyperthermia.

Enhancement by N-methylformamide of the effect of ionizing radiation on a human colon tumor xenografted in nude mice.

Polar solvents, which induce differentiation in murine and human tumor cells, enhance the effect of ionizing radiation on cultured mouse mammary and human colon cancer cells. To determine whether this enhancement occurs in vivo, DLD-2 human colon carcinoma xenografts in nude mice were treated with combinations of 6 MV photon irradiation, the polar solvent N-methylformamide (NMF), or combinations of the two agents. Nude mice bearing 300-mg s.c. implants of DLD-2 tumors were treated i.p. with 150 mg NMF/kg daily for 19 days. Local tumor irradiations were administered as graded single doses or as fractionated doses, daily for 4 days, following the third NMF injection. The growth-inhibiting effect of the radiation treatment for both single dose and fractionation protocols was enhanced by the polar solvent. NMF alone increased the time required for a doubling of initial tumor volume by 1.7 days, compared to control tumors. Initial tumor volume doubling times compared to untreated controls were increased by 3.6 and 7.6 days by photon doses of 10.0 and 13.75 Gy, respectively, whereas NMF plus 10.0 or 13.75 Gy increased the DLD-2 regrowth delay time by 7.5 or 12.9 days. NMF caused essentially equivalent enhancements, whether split-dose schedules of 2.5 Gy daily for 4 days, and 3.44 Gy daily for 4 days, or single doses of 10.0 and 13.75 Gy were used; therefore, radiation enhancement was not due to effects on sublethal damage repair. The results support the use of NMF, currently in Phase 1-Phase 2 clinical trials, with radiation in the therapy of selected human neoplasms.

Differential responses to x-irradiation of subpopulations of two heterogeneous human carcinomas in vitro.

The responses of two heterogeneous human cancer cell lines and their derivative clones to graded single doses of X-rays were examined in vitro. One system consisted of the human colon carcinoma line DLD-1 and two subpopulations (clones A and D). The second system consisted of the human lung carcinoma line (LX1) and four subpopulations (LX1-1, LX1-2, LX1-3, and LX1-9). These subpopulations have previously been shown to be markedly heterogeneous in terms of such characteristics as karyotype, morphology, drug sensitivity, tumorigenicity, and expression of membrane glycoproteins (such as carcinoembryonic antigen and tumor colonic mucoprotein antigen). Exponentially growing cultures were irradiated with graded single doses of 100-kVp X-rays. Survival was assessed using colony formation as the end point, and responses from multiple experiments were fitted to the single-hit, multitarget equation of cell survival. Values for the mean lethal dose (D0, grays), quasithreshold dose (Dq, grays), and extrapolation number (n) were obtained. For the human colon adenocarcinoma system, these values for the three tumor lines were: DLD-1, 0.95, 2.34, and 11.7; clone A, 1.06, 2.23 and 8.20; and clone D, 1.08, 1.89, and 5.80. For the human lung carcinoma system, these values for the five sublines were: LX1, 1.14, 0.19, and 1.20; LX1-1, 0.96, 2.06, and 8.54; LX1-2, 0.98, 0.88, and 2.48; LX1-3, 0.68, 2.05, and 20.3; and LX1-9, 1.12, 0.00, and 1.00. These two human tumor systems therefore exhibit variability in their intrinsic sensitivity to X-irradiation. The data indicate that failure of some human carcinomas to respond to physical treatment modalities can be due to preexisting resistant subpopulations.

Tumor heterogeneity and drug resistance.

Drug resistance has long been identified as a major reason for therapy failure in cancer patients. Concurrently, work from many laboratories in the past 10 years has established tumor heterogeneity as a phenomenon of critical importance in the natural history of individual neoplasms. The two most sinister aspects of intraneoplastic diversity in human solid tumors are the genesis of clones with metastatic potential, and the existence of drug-resistant variants in primary cancers and their metastases. Thus, recent investigations on drug resistance and on tumor heterogeneity have converged to focus attention on the clonal organization of primary tumors and their metastases as the underlying basis for anticancer drug resistance. This review examines the degree of heterogeneity observed within tumors and the relationship of this diversity to resistance that might be anticipated for any given agent. A question critical to our discussion is "How many subpopulations are there?" The impact of multiple tumor clones on therapy is next discussed in relationship to normal tissue tolerance, the barrier clinicians face regardless of the specific agent used in treatment. Finally, laboratory and clinical approaches are presented for addressing a drug resistance problem that is seemingly overwhelming because of its complex biological roots.

Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration.

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37 degrees C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O2) or the 0.01% O2 mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10(6) cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O2 were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10(6) cells/h) in 21% O2 was 62.5. This rate increased to 428.8 pg/10(6) cells/h at 0.01% O2, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O2 were too low to be measured by ELISA. However, between 10% and 0.01% O2, secretion rates increased from 5.0 to 376.0 pg/10(6) cells/h, a 75-fold increase. Therefore, at very low O2 levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm3.

Levels of selected growth factors in viable and necrotic regions of xenografted HCT-8 human colon tumours.

Xenografted tumours were produced in nude mice by injection of HCT-8 human colon tumour cells. At average volumes of about 750 mm3, animals were injected with fast green vital dye, and 20 min later, tumours were excised and dissected into viable (stained) and necrotic portions (unstained). Viable and necrotic regions were then examined for cell yields, colony forming efficiencies, and levels of basic fibroblast growth factor (FGF-2), transforming growth factors-beta 1 and -alpha (TGF-beta 1, TGF-alpha), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) using enzyme-linked immunoassay (ELISA) procedures. Levels in the viable and necrotic regions were compared to levels in unseparated tumours. The average extent of necrosis in HCT-8 tumours of this size was 64%. The data for cell yields, colony forming efficiencies FGF-2, VEGF, TGF-beta 1 and TGF-alpha indicated that values determined in the unseparated tumours could be understood on the basis of the weighted average between viable and necrotic tissue, with the higher values occurring in the viable tissue. Low levels of FGF-2 and VEGF were found in the necrotic portions of the tumour while no measurable levels of TGF-beta 1 and TGF-alpha could be determined. PDGF levels were, however, equivalent in both the viable and necrotic regions indicating that necrotic tissue could be an important reservoir for this growth factor.

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice.

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.

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.

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.

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.

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.