Cytotoxicity of 125I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells, MDA-231 and oestrogen receptor-expressing MCF-7 cells.

PURPOSE: To compare the cytotoxicity of 125I-oestrogen (E-17alpha[125I]iodovinyl-11betamethoxyoestradiol or 125IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). MATERIALS AND METHODS: MDA-231 cells were labelled with 125IVME2 or [125I]iododeoxyuridine (125IdU), frozen for decay accumulation, thawed and then plated for colony formation. gamma-irradiation survival was also determined. A whole-cell 3H-oestrogen-binding assay and a specific-binding assay were used to detect ER. RESULTS: No MDA-231 cell killing by accumulated 125IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125IVME2 (D(o)=28 dpc). MDA-231 cells were not significantly more radioresistant to gamma-rays (D(o)=1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125IdU decays (D(o)= 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. CONCLUSIONS: ER-negative cells, MDA-231, are not killed by 125IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125IVME2 to its nuclear target), formation of the 125IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.

Relative biological effectiveness of accumulated 125IdU and 125I-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells.

The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by gamma radiation or accumulation of (125)I-iododeoxyuridine ((125)IdU) or E-17alpha[(125)I]iodovinyl-11betamethoxyestradiol ((125)IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of (125)IdU (D(0) = 30 decays per cell) and (125)IVME2 decays (D(0) = 28 decays per cell). DNA DSBs were induced by the accumulation of (125)IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or (125)IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after gamma irradiation, the D(0) was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by (125)IVME2 relative to gamma radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by (125)IdU relative to gamma radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by (125)IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.

DNA damage induction by 125I-estrogen.

DNA damage induced by the radioactive decay of 125I-estrogen (125I-VME2) in an estrogen receptor expressing CHO cell line, CHO-ER, was measured. 125I-VME2 targeted 125I atoms proximal to DNA estrogen response elements (EREs). 125I decays were accumulated at -135 degrees C, and thereafter assayed by alkaline and neutral filter elution techniques to measure DNA single strand break (ssb) and double strand break (dsb) induction respectively. Increasing DNA damage (both ssbs and dsbs) was detected after exposure of cells to increasing concentrations of 125I-VME2. DNA ssb and dsb dose-response curves for 125I-VME2 were multiphasic. The rates of DNA damage induction by the decay of 125I-VME2 was determined by comparing slopes of all data or by comparing initial slopes. DNA ssb induction per 125I-VME2 decay was approximately 2 times greater compared with DNA dsb induction. 125I-VME2 decay induced approximately 4-8 times more DNA dsbs than 125IUdR decay.

Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5.

Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. The rate of DNase I digestion of unirradiated CHO K1 and xrs-5 was not significantly different, but the extent of digestion was greater in unirradiated CHO K1 cells that in xrs-5 cells suggesting that less xrs-5 cell chromatin at DNA attachment points is accessible to the enzyme DNase I. The extents of digestion in irradiated (1 Gy) CHO K1 and xrs-5 cell nuclei also differed but the relationship was reversed. The irradiated xrs-5 cell samples were digested to a greater extent compared with CHO K1 cells. These chromatin digestion data suggest that the matrix attachment regions in xrs-5 cells are different from CHO K1 cells. The different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity.

Morphological characterization of the radiation sensitive cell line, xrs-5.

Morphometric analysis was performed on the radiation sensitive Chinese hamster ovary (CHO) xrs-5 cell line, reverting xrs-5 cells and parental K1 cells. Several ultrastructural parameters (increased nuclear envelope membrane separation, cell and nuclear volume, nuclear to cytoplasmic ratio, and the nuclear surface area per unit volume of the cell) were measured and correlated with radiation sensitivity. A trend in increased cell size and radiosensitivity was observed. However, only the substantially increased nuclear envelope membrane separation in sensitive xrs-5 cells significantly correlated with radiation sensitivity. The maximal nuclear envelope membrane separation in sensitive xrs-5 cells was 270.8 nm. The maximal K1 cell nuclear envelope membrane separation was 134.8 nm, although, on average the K1 cell nuclear envelope membrane separation was 36.8 nm. The reverted xrs-5 cells had a smaller nuclear envelope membrane separation (maximal 83.6 nm), but the measured space did not completely revert to that for K1 cells. Therefore, we conclude that the nuclear envelope membrane separation is correlated with radiation sensitivity of xrs-5 cells, but it cannot be considered as the only defect correlatable with the radiation sensitivity.

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5.

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.

Changes in the nuclear structure in the radiation-sensitive CHO mutant cell, xrs-5.

The structural organization of the cell nucleus was investigated by transmission electron microscopy in the radiosensitive Chinese hamster ovary (CHO) cell mutant, xrs-5 (D0 = 45 cGy), relative to parental K1 cells (D0 = 200 cGy). In 99% of all xrs-5 cells, the outer layer of the nuclear envelope was separated from the inner layer, while 96% of K1 cells had closely apposed layers. This separation of the inner and outer layers of the nuclear envelope in xrs-5 cells was not explained by an increased susceptibility of xrs-5 cells to osmotically induced changes because (1) xrs-5 cells retained the altered nuclear periphery even when several different fixation protocols were used and (2) xrs-5 cells were not more susceptible to cell lysis as measured by trypan blue dye exclusion or by the extracellular presence of lactate dehydrogenase. The difference in the morphological organization in the nuclear periphery of xrs-5 cells correlated with the radiation sensitivity of the cells; xrs-5 cells which spontaneously reverted to a radiation sensitivity similar to that of K1 cells also reverted to a nuclear morphology similar to that of K1 cells. The inner and outer layers of the nuclear envelope were retained in nuclear scaffolds isolated from K1 and xrs-5 cells, indicating that components of the nuclear periphery are part of the nuclear scaffold. These data show that xrs-5 cells have an altered nuclear periphery which correlates with the radiation sensitivity of the cells. The separation of the layers of the nuclear envelope may represent an altered template for repair of DNA damage at the nuclear scaffold and thus may play a role in the defective repair of X-ray-induced DNA double-strand breaks in xrs-5 cells.

Thermal sensitivity of CHO cells with different shapes.

Confirming previous reports, we observed that rounded Chinese hamster ovary (CHO) cells from suspension culture were more resistant to heat kill than flattened CHO cells from monolayer culture. Cell survival was quantitatively described by determining full cell survival curves for flattened versus rounded CHO cells after exposure to 43, 44, 45 and 46 degrees C and calculating the To values. The cell survival responses of the rounded cells were significantly different from those of the flattened cells. Based on the ratio of the To values, the rounded cells were consistently twice as resistant to heat-induced cell death than flattened cells. This difference in cell survival was not explained by a trypsin effect in monolayer cells, or by the amount of time rounded cells were maintained in suspension culture, or technical differences in heat-up and cool-down kinetics, or differences in extracellular pH. The two groups also showed no differences in cell generation times or whole cell protein contents. However, we found that the rounded cells had a lower percentage of cells in the S phase of the cell cycle (40% versus 52%) than the flattened cells, which could account for part of the differential heat-induced cell kill in the flattened versus rounded cells. These data suggest there is a significant association between cell shape and heat-induced cell killing.

Measurement of radiation-induced DNA damage using gel electrophoresis or neutral filter elution shows an increased frequency of DNA strand breaks after exposure to pH 9.6.

The filter elution technique using nondenaturing conditions is widely used to assay DNA double-strand break (DSB) induction and repair. It has been reported that in the measurement of strand breaks higher rates of elution and of initial rejoining are obtained at pH 9.6 compared to pH 7.2. In the present experiments neutral elution at pH 7.2 and 9.6 were compared in the assay of damage to DNA induced by X rays, 125I decay, and restriction enzyme digestion, in an effort to explain this discrepancy and to determine whether the higher rate of elution observed at pH 9.6 corresponds to a greater number of DSBs. X-ray damage to cellular DNA resulted in significantly different elution profiles at the two pH values. In contrast the elution profiles of the DSB induced by intragenomic 125I decays or restriction endonuclease were independent of the pH of the elution buffer. When gamma-irradiated SV40 DNA was exposed to pH 7.2 or 9.6 elution buffer prior to analysis by gel electrophoresis, a significantly greater number of DNA DSBs were detected in the DNA exposed to pH 9.6. We conclude that X and gamma radiation produce lesions (pH 9.6-labile lesions), in proportion to dose, that have the potential of becoming measurable DSBs following incubation under the mildly alkaline condition of pH 9.6. The data suggest that these lesions may result from single-hit events.

The effect of chromatin decondensation on DNA damage and repair.

The effects of chromatin compaction on X-radiation-induced cell killing and the induction and repair of DNA damage were studied in Chinese hamster ovary cells deprived of isoleucine for 24 h (Ile- cells) and compared to untreated controls. The results show that chromatin is decondensed in Ile- cells; i.e., in Ile- cells the nuclear area occupied by heterochromatin decreased 30-fold over control cells, both the rate and limit of micrococcal nuclease digestion were greater for Ile- cells, and 14.2% more propidium iodide was intercalated into the Ile- cell chromatin. The X-ray-induced cytotoxicity did not change in Ile- cells versus the control cells (D0 = 0.99 Gy) nor did the X-ray-induced DNA damage. However, the repair of DNA damage produced by 10 Gy proceeded with different kinetics in Ile- cells when compared to the controls. The initial rate of DNA damage repair was slower in Ile- cells by a factor of 2 compared to controls (the time required to rejoin 50% of the lesions was 6 versus 3 min, respectively). However, after 2 h of repair no DNA damage was detected in either group. Therefore, we conclude that this decondensation of chromatin, per se, does not directly modify the induction or ultimate repair of DNA damage by X radiation or cell clonogenicity and thus does not appear to be a primary factor in cell survival.

Morphometric changes as a function of the proliferative status of murine mammary carcinoma cells.

Ultrastructural analysis was performed to determine morphological changes in the 67 murine mammary tumor cells grown in four defined metabolic states in vitro, i.e., proliferating cells (P), cells in transition towards quiescence (T), nutrient-deprived quiescent cells (QI), and QI cells stimulated to reenter the cell cycle (St4) by refeeding for 4h in situ with complete medium. Also, these documented changes were evaluated as a function of the radio-sensitivity of the various cell types. The average number of lipid body and mitochondrial profiles per cell was significantly higher in QI and St4 cells versus P cells. Also, greater variability was observed in the number of lipid bodies and mitochondria per cell section in the T, QI and St4 cells relative to P cells. Nuclear alterations involved little change in nuclear area occupied by heterochromatin in QI cells compared to P cells but the number of heterochromatin patches decreased in QI cells compared to P cells indicating a change in higher order chromatin packaging. The nucleolar organization was lost in QI cells as measured by the almost complete lack of nuclear area occupied by nucleoli in QI cells. In addition, nuclear diameter decreased in QI cells compared to P and T cells, but not St4 cells. The multiple changes in the morphological organization suggest a shift in the metabolic functioning of the cells relative to the proliferative status of the cell; however, there was no apparent correlation between these described changes and the respective radiation responses as measured by cell-survival analysis.

Role of mitochondrial DNA in cell death induced by 125I decay.

The role of mitochondrial DNA in radiation-induced cell death was determined by selective [125I]iododeoxyuridine (125IUdR) incorporation into exclusively nuclear sites compared to labelling in both nuclear and mitochondrial DNA of Chinese hamster cells. Such selectivity was achieved by using berenil (25 micrograms/ml for 24 h), a drug which inhibits mitochondrial DNA synthesis without affecting incorporation of 125IUdR into nuclear DNA but does not result in reduced clonogenicity or cell cycle perturbations or alteration in the X-ray response of cells. There was no difference in cell killing between cells with nuclear labelling alone compared with nuclear plus mitochondrial labelling. The absence of decays in mitochondrial DNA does not affect the ability of 125I to induce lethal cell damage. The two treatment groups have superimposable curves with a D0 of 96 decays/cell. These findings indicate that mitochondrial DNA is not the most sensitive target for radiation-induced cell death from 125I decay.

Inhomogeneity of the nucleus to 125IUdR cytotoxicity.

Synchronized suspension cultures of Chinese hamster ovary (CHO) cells were used to determine the lethal effects produced by the decay of 125I incorporated into different subfractions of the nuclear genome. Such a shift in nuclear incorporation pattern was achieved by using the drug aphidicolin, which inhibits 95% of all nuclear DNA synthesis, is nontoxic to cells in a colony-forming assay, and does not modify the radiation response of CHO cells to X irradiation. In addition to shifting incorporation of 125I to only 5% of the nuclear genome, both nuclease digestions to characterize the molecular location of 125I and electron microscope autoradiography show an inhomogeneous distribution of sites of 125I incorporation in the presence of 5 micrograms/ml aphidicolin. These data in combination with survival curves of CHO cells labeled with 125I-iododeoxyuridine (125IUdR) either with or without aphidicolin showed a dramatic change in the survival response (DO: 30 decays/cell and 96 decays/cell, respectively). It is concluded, therefore, that the nucleus is not a homogeneous target for radiation-induced cell death because when subfractions of the nuclear genome are labeled, radically different levels in cell survival are obtained.