Polyamine depletion and drug-induced chromosomal damage: new results.
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Carmustine↗
Biomedical subjects
P J Tofilon
Publications and source records attributed to P J Tofilon.
Radiation-induced apoptosis of oligodendrocytes in vitro.
It has been suggested that glial cells and/or their progenitors are the primary target cells for radiation-induced demyelination. Cultures of terminally differentiated oligodendrocytes, immature oligodendrocytes, and O-2A progenitor cells were generated from the cerebral cortex and spinal cord of perinatal rat pups. Irradiation of cultures of terminally differentiated oligodendrocytes resulted in a significant increase in the percentage of apoptotic cells from 15% in control to 30% in irradiated samples, with the maximum increase induced by 10 Gy. This increase in apoptosis could be observed by 1 h after irradiation with the maximum level reached at 3-6 h. Apoptotic cells were not detected before or after irradiation of cultures of O-2A progenitor cells or immature oligodendrocytes. These data suggest that radiation-induced apoptosis of terminally differentiated oligodendrocytes may be involved in early demyelination.
Animals↗
Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.
Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.
3T3 Cells↗
Enhancement of radiation-induced cell killing and DNA double-strand breaks in a human tumor cell line using nanomolar concentrations of aclacinomycin A.
Several agents that induce differentiation have previously been shown to induce the terminal differentiation of leukemic cells and enhance the radiosensitivity of certain solid tumor cell lines in vitro using millimolar concentrations. We now report that aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation in vitro, can significantly enhance the radiosensitivity of a human colon tumor cell line (Clone A) at a concentration of 10 nM. Based on colony-forming efficiency, the maximum increase in radiosensitivity was found using 15 nM ACM for 3 days with a dose enhancement factor of 1.4 at a surviving fraction of 0.10. This treatment increased cell doubling time, but had no effect on cell-cycle phase distribution. To gain insight into the mechanisms responsible for this radiosensitization, gamma-ray-induced DNA single- and double-strand breaks were examined. Aclacinomycin A had no effect on the induction of DNA single-strand breaks but significantly enhanced the formation of gamma-ray-induced DNA double-strand breaks. The rate or extent of repair of the induced double-strand breaks was not influenced by ACM treatment. These data suggest that ACM, at achievable plasma concentrations, can enhance the radiosensitivity of a human tumor cell line by increasing the initial level of radiation-induced DNA double-strand breaks.
Aclarubicin↗
Loss of intragenomic DNA repair heterogeneity with cellular differentiation.
The influence of terminal differentiation on UV-induced DNA damage and its repair in transcriptionally active and inactive genomic sequences was investigated using the murine 3T3-T proadipocyte cell culture system. Actively cycling 3T3-T cells terminally differentiate into adipocytes after exposure to media containing platelet-depleted human plasma. Suitable DNA fragments were analyzed from four genes: beta-actin, adenosine deaminase, dihydrofolate reductase, and lipoprotein lipase. As a result of 3T3-T cell differentiation, lipoprotein lipase and beta-actin expression was modified, whereas adenosine deaminase and dihydrofolate reductase expression was not affected. A DNA fragment representing the transcriptionally inactive locus 70-38 was also evaluated. UV-induced cyclobutane pyrimidine dimers, detected as UV-specific endonuclease-sensitive sites, in each fragment increased linearly as a function of UV dose (0-20 J/m2) independently of gene expression or differentiation. Sequence-specific repair of dimers was measured in stem and terminally differentiated 3T3-T cells after UV irradiation (10 J/m2). For undifferentiated stem cells, the rate and extent of dimer repair was higher in the actively transcribed adenosine deaminase and dihydrofolate reductase genes than in the inactive lipoprotein lipase or 70-38 fragments, the greater difference being observed in the first 8 h post-UV irradiation. In contrast, similar dimer repair rates were found for each DNA fragment in terminally differentiated 3T3-T cells. These data suggest that cellular differentiation is accompanied by a loss of heterogeneity in intragenomic DNA repair.
3T3 Cells↗
Enhancement of the radiosensitivity of two human tumour cell lines by hexamethylene bisacetamide.
The effect of the maturation-inducing polar solvent, hexamethylene bisacetamide (HMBA), on the radiosensitivity of two human tumour cell lines (clone A, a colon carcinoma; and EJ, a bladder carcinoma) was investigated. Exposure of clone A or EJ cells to HMBA resulted in a concentration-dependent increase in doubling time, a decreased plating efficiency and changes in cell morphology, which are consistent with the formation of a better-differentiated phenotype. Growth of clone A cells in 2 or 3 mM HMBA, followed by irradiation and plating into HMBA-free medium, resulted in a significant enhancement in radiosensitivity, as determined by colony-forming ability. A similar increase in radiosensitivity was detected for EJ cells; however, for these cells a concentration of 7 mM HMBA was required. The increased radiosensitivity caused by HMBA was observed primarily in the low-dose, shoulder region of the gamma-ray cell survival curves for both cell lines, which is reflected by an increase in the alpha component of the survival curve with essentially no effect on beta. These data indicate that HMBA can radiosensitise human tumour cells at concentrations and for exposure periods that can be achieved in the clinic.
Acetamides↗
Combination of N-methylformamide with cis-diamminedichloroplatinum (II) in murine mammary carcinoma: importance of timing.
The maturational agent N-methylformamide (NMF) is an antitumor agent that also enhances the response of tumor cells in vitro to chemotherapeutic agents. Here, we tested whether NMF can improve therapy of the murine MCA-K mammary carcinoma with cis-diamminedichloroplatinum(II) (cis-DDP). Although the in vitro cell cultures of MCA-K tumor cells exhibited increased sensitivity to cis-DDP cytotoxicity when they were first treated with NMF, administration of NMF to mice bearing MCA-K tumors did not enhance cis-DDP-induced tumor growth delay. However, when NMF treatment was begun after cis-DDP administration, the growth delays were significantly greater than those induced by the individual treatment, with an increase in temporary tumor regression and a small proportion of cures. These results indicate that therapeutic benefit can be achieved in this experimental tumor system when NMF is administered after cis-DDP. In addition, they demonstrate the significance of the timing of administration in combined protocols involving NMF.
Animals↗
Measurement of sister chromatid exchanges and their relationship to DNA damage, repair and cell killing.
We have concentrated on how SCE is measured, how it correlates with other measures of cellular damage, and how SCE can be used as a tool to study cell cycle kinetics. We have not discussed the biological significance of SCEs, i.e. why they occur spontaneously, and what role, if any, in cellular preservation they play. Similarly, our discussion of the mechanisms of formation of SCE has been deliberately brief. These are important considerations, but the answers to them are still largely only speculative. What is clear is that SCE is an extremely interesting biological phenomenon from a variety of points of view, and even though the mechanisms underlying SCE are not yet well understood, measurement of these events has produced much useful and provocative information.
Animals↗
Heterogeneity in radiation sensitivity within human primary tumour cell cultures as detected by the SCE assay.
The ability of the sister chromatid exchange (SCE) assay to detect heterogeneity in intrinsic radiation sensitivity was investigated. In order to identify tumour cell subpopulations, frequency histograms of cis-diamminedichloroplatinum (II) (cPt)-induced SCEs were generated and compared to those from cultures that had been irradiated 96 h before drug treatment. The results suggested that subpopulations with different radiosensitivities were present in nine of 18 human primary tumour cell cultures evaluated. When the effects of prior irradiation on the subsequent X-ray survival response and on cPt-induced SCE frequency histograms were compared, a good correlation was obtained between the two assays regarding the prediction of heterogeneity in radioresponse. These results suggest that primary cultures can contain both radiation-sensitive and radiation-resistant cells, and thus heterogeneity in intrinsic radiosensitivity may exist in human solid tumours.
Animals↗
Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide.
The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.
Adenocarcinoma↗
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.
Adenocarcinoma↗
Reduction in DNA repair capacity following differentiation of murine proadipocytes.
It has been suggested that terminally differentiated mammalian cells have a decreased DNA repair capacity, compared with proliferating stem cells. To investigate this hypothesis, we have examined gamma-ray-induced DNA strand breaks and their repair in the murine proadipocyte stem cell line 3T3-T. By exposure to human plasma, 3T3-T cells can be induced to undergo nonterminal and then terminal differentiation. DNA strand breaks were evaluated using the technique of alkaline elution. No difference was detected among stem, nonterminally differentiated, and terminally differentiated cells in the initial levels of radiation-induced DNA strand breaks. Each of the strand break dose response increased as a linear function of gamma-ray dose. The strand breaks induced by 4 Gy rejoined following biphasic kinetics for each cell type. At each time point examined after irradiation, however, the percentage of strand breaks that had not rejoined in terminally differentiated cells was three to six times greater than in stem cells. The rate of strand break rejoining in nonterminally differentiated cells was of an intermediate value between that of the stem and of the terminally differentiated cells. These results indicate that, at least for 3T3-T cells, differentiated cells have a reduced capacity for DNA repair.
Adipose Tissue↗
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.
Cell Line↗
Influence of cellular differentiation on repair of ultraviolet-induced DNA damage in murine proadipocytes.
The effects of cellular differentiation on the repair of DNA damage induced by uv radiation were investigated in the murine 3T3-T proadipocyte cell culture system. Upon exposure to human plasma, actively cycling 3T3-T cells (stem cells) undergo growth arrest, which is followed by terminal differentiation into lipid-laden adipocytes. In response to uv irradiation, the level of unscheduled DNA synthesis is significantly lower in adipocytes as compared to stem cells. The alkaline elution assay was used to monitor the appearance of repair-induced strand breaks in 3T3-T cells after uv irradiation. DNA strand breaks were detected in stem cells by 4 min post-uv with essentially no further increase after 8 min. When terminally differentiated adipocytes were irradiated and allowed to repair, however, more strand breaks were present at 4 min and, in marked contrast to stem cells, continued to accumulate in adipocytes for at least 16 min post-uv. Inhibition of repair-replication with hydroxyurea and cytosine arabinoside significantly increased accumulation of repair-induced strand breaks in stem cells, yet had little effect on this accumulation in adipocytes. For stem cells and adipocytes, incision activity was linear out to at least 10 Jm-2 without saturation. These data suggested that 3T3-T cell differentiation is accompanied by a defect in some postincision process of the excision-repair pathway.
Adipose Tissue↗
Failure to produce greater than additive sister chromatid exchange induction with X rays and BCNU.
In 1984, Tofilon and Deen reported that X irradiation of 9L cells immediately after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) produced greater than additive induction of sister chromatid exchanges (SCEs) compared to the sum of SCEs induced by each agent alone [Radiat. Res. 97, 171-177 (1984)]. We (C.P.S., D.F.D.) repeated experiments conducted by Tofilon and Deen, scored SCEs in a blind manner, and were unable to reproduce the original findings. Instead, we found that X rays and BCNU induced SCEs in an additive manner. The slides prepared in the original study are extant; they were coded and recounted blind. Data obtained in this reevaluation do not substantiate the previous report. We conclude that the original findings were apparently the result of bias introduced in the SCE scoring process. Our experience with the SCE assay emphasizes the need to recode SCE slide preparations and count SCEs blind.
Animals↗
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.
Animals↗
Modification of tumor and normal tissue radioresponse in mice by N-methylformamide.
The effects of the differentiation-inducing agent N-methylformamide (NMF) on the in vivo response of the murine tumor FSA and its pulmonary metastases to ionizing radiation were investigated. In addition, the radioresponse of acutely responding normal tissues was determined in mice receiving systemic NMF. A dosage of 300 mg/kg administered for 8 days had little effect on the FSA tumor growth, yet enhanced the growth inhibitory actions of ionizing radiation with dose enhancement factors ranging from 1.5 to 1.7. Administration of NMF also enhanced the radiation response of FSA micrometastases. The response to irradiation of hematopoietic tissue, jejunum, and testes in mice receiving NMF was also investigated. NMF administered before or before and after radiation enhanced the formation of endogenous spleen colonies, yet did not influence the LD50/30 for radiation. Jejunal crypt cell survival after radiation was slightly increased in mice receiving NMF, but the survival of spermatogonia after radiation was not affected. These data indicate that NMF administration results in an increase in the radiosensitivity of the FSA tumor and its metastases with no concomitant increase in the radiation response of the normal tissue tested. Thus, at least in this model system, a therapeutic gain is achieved through the combination of NMF and ionizing radiation.
Animals↗