Cell lethality after selective irradiation of the DNA replication fork

Summary It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with¹²⁵I-iododeoxyuridine (¹²⁵IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 µg/ml) reduced cellular¹²⁵IUdR incorporation to 3–5% of the control value. The residual¹²⁵I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix.Based on these observations an attempt was made to compare the lethal consequences of¹²⁵I decays at the replication fork to that of¹²⁵I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D₀: 101¹²⁵I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.     

Targets for radiation-induced cell death: when DNA damage doesn't kill

Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[(125)I]-iodo-2'-deoxyuridine ((125)IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (-80 degrees C) for accumulation of (125)I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher (125)I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs-20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.     

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.     

Using Hoechst 33342 to target radioactivity to the cell nucleus

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37 degrees C and accumulating 125I decays at -90 degrees C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (gamma-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 gamma-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against gamma radiation. We conclude that cell killing by 125I-H33342 and the induction of gamma-H2AX foci are highly correlated.     

Cell progression after selective irradiation of DNA during the cell cycle

Chinese hamster ovary cells were labeled with [125I]iododeoxyuridine (125IUdR, 0.1184 MBq/ml for 20 min) and the labeled mitotic cells were collected by selective detachment ("mitotic shake off"). The cells were pooled, plated into replicate flasks, and allowed to progress through the cell cycle. At several times after plating, corresponding to G1, S, late S, and G2 plus M, cells were cooled to stop cell cycle progression and to facilitate accumulation of 125I decays. Evaluation of cell progression into the subsequent mitosis indicated that accumulation of additional 125I decays during G1 or S phase was eight to nine times less effective in inducing progression delay than decays accumulated during G2. The results support our previous hypothesis that DNA damage per se is not responsible for radiation-induced progression delay. Instead, 125I-labeled DNA appears to act as a source of radiation that associates during the G2 phase of the cell cycle with another radiosensitive structure in the cell nucleus, and damage to the latter structure by overlap irradiation is responsible for progression delay (M. H. Schneiderman and K. G. Hofer, Radiat. Res. 84, 462-476 (1980].     

Paradoxical effects of iodine-125 decays in parent and daughter DNA: a new target model for radiation damage

Chinese hamster ovary cells were synchronized at the G(1)/S-phase boundary of the cell cycle and were pulse-labeled with (125)I-iododeoxyuridine 30 min after they entered the S phase. Cell samples were harvested and frozen for accumulation of (125)I decays during the first and second G(2) phase after labeling. Cell aliquots that had accumulated the desired number of decays were thawed and plated for evaluation micronucleus formation and cell death. Cells subjected to (125)I decays during the first G(2) phase after labeling exhibited single-hit kinetics of cell killing (n = 1, D(0) 41 decays/cell). In contrast, decays accumulated during the second G(2) phase killed cells with dual-hit kinetics (n = 1.9, D(0) 81 decays/cell). A similar divergence in the action of (125)I was noted for micronucleus formation. These findings indicate that the effects of (125)I varied depending on whether the decays occurred in daughter DNA (first G(2) phase) or parent DNA (second G(2) phase). Control studies with external X rays showed no such divergence of the action of radiation. To account for this paradox, a model is proposed that invokes higher-order chromatin structures as radiation targets. This model implies differential spatial arrangements for parent and daughter DNA in the genome, with DNA strands organized such that a single (125)I decay originating in daughter DNA damages two targets during the first G(2) phase, but identical decays occurring during the second G(2) phase damage only one of the targets.     

Toxicity of DNA-incorporated iodine-125: quantifying the direct and indirect effects

To understand the biophysical mechanism(s) underlying the induction of cell death by the decay of the Auger electron emitter iodine-125 in DNA, Chinese hamster V79 lung fibroblasts were labeled with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU) for two doubling times and frozen and stored at -135 degrees C in the presence of 0.26-3.0 M dimethyl sulfoxide (DMSO), which acts simultaneously as a cryoprotector and a hydroxyl radical scavenger. After the accumulation of (125)I decays, the cells were defrosted and their survival was determined. Within the range of the number of decays examined (up to 470 disintegrations per cell), the survival curves are exponential. The dependence of the D(37) on DMSO concentration is triphasic and seems to reach a plateau at approximately 1.3 M. By extrapolating to infinite DMSO concentration, we estimate the D(37) for maximal hydroxyl radical scavenging to be 411 +/- 36 disintegrations per cell. To determine the D(37) in the absence of DMSO, we extrapolate the D(37) curve to zero concentration, and a D(37) of 54 +/- 5 disintegrations per cell is obtained. The maximal dose modification factor, calculated as the ratio of the D(37) at infinite DMSO concentration (i.e. direct effects only) to the D(37) at zero DMSO concentration (i.e. direct and indirect effects), is 7.6 +/- 1.0. By inference, approximately 90% of the radiotoxic effects of DNA-incorporated (125)I are due to indirect mechanisms.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

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.     

Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons

To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of (125)I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0-4 MBq/ml) of (125)I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose-response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear (125)I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when (125)I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.     

Killing of EMT-6 cells by decays from isotopes incorporated on sensitizer adducts.

EMT-6 tumor cell killing by decays from 3H and 125I incorporated by adduct formation of radiolabeled sensitizers was studied in vitro. Hypoxic radiosensitizers become covalently bound to cellular molecules after metabolic reduction, and EMT-6 tumor cells can tolerate over 10(9) adducts/cell of misonidazole without loss of colony-forming ability. Cells were incubated under hypoxic conditions in the presence of [3H]misonidazole or [125I]iodoazomycinriboside for various times and the amounts of bound 3H and 125I were determined. Cells were stored as monolayers at 22 degrees C, in suspension culture at 4 degrees C, and frozen in complete medium plus 8% DMSO at -196 degrees C for various times to facilitate the accumulation of radioactive decays before plating in vitro for colony-forming assays at 37 degrees C. At 22 degrees C in monolayer culture, EMT-6 tumor cells tolerated 950 and 1720 decays/cell of 3H and 125I, respectively, without evidence of radiotoxicity. This number of decays/cell over the exposure times used represents 1.54 x 10(6) 3H/cell and 8.4 x 10(4) 125I/cell, respectively. Significant cell killing was detected after similar amounts of isotope decay when cells were held at 4 degrees C. When cells were frozen in the presence of 8% DMSO, they were more resistant to inactivation by isotope decays or by gamma rays than cells in liquid phase at 4 degrees C. These data suggest that selective hypoxic tumor cell suicide by 3H or 125I decays from bound sensitizer at 37 degrees C will be an inefficient process, at least for drugs with specific activities as tested. These data are consistent with data on cell inactivation by isotopes incorporated into cells by other procedures.     

125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 X 10(-12) DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Radiotoxicity of 5-[123I]iodo-2'-deoxyuridine in V79 cells: a comparison with 5-[125I]iodo-2'-deoxyuridine

The toxic effects of the short-lived (T 1/2 = 13.2 h) Auger-electron-emitting isotope 123I, incorporated in the form of 123IUdR into the DNA of V79 cells in vitro, have been investigated and compared to those of 125IUdR. For the concentrations tested, the rate of incorporation of 123IUdR at any time is proportional to the concentration of extracellular radioactivity. The curve for survival of clonogenic cells decreases exponentially and exhibits no shoulder at low doses. The mean lethal dose (D37) to the nucleus is 79 +/- 9 cGy and is about the same as that obtained previously with 125IUdR. However, the total number of decays needed to produce this D37 with 123IUdR is about twice that required with 125IUdR, approximately equal to the ratio of the energy deposited in microscopic volumes by 125I and 123I, respectively. This correlation suggests that nuclear recoil, electronic excitation, and chemical transmutation are probably of minor importance to the observed biological toxicity with either isotope. The results also indicate that there are no saturation effects in the decay of 125IUdR in the DNA of V79 cells (i.e., all of the emitted energy is biologically effective) and that each of the two steps involved in the 125I decay is equally effective in causing biological damage.     

Pathways of DNA double-strand break repair during the mammalian cell cycle

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.     

Efficient mutation induction by 125I and 131I decays in DNA of human cells

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.     

Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis

We examined the ability of Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan synthesis to metabolize 125I-labeled thrombospondin (TSP). Wild type CHO cells bound and degraded 125I-TSP with kinetics similar to those reported for endothelial cells. Both binding and degradation were saturable (half-saturation at 20 micrograms/ml). When the concentration of labeled TSP was 1-5 micrograms/ml, mutant 745, defective in xylosyltransferase, and mutant 761, defective in galactosyltransferase I, bound and degraded 6- to 16-fold less TSP than wild type; mutant 803, which specifically lacks heparan sulfate chains, bound and degraded 5-fold less TSP than wild type; and mutant 677, which lacks heparan sulfate and has increased levels of chondroitin sulfate, bound and degraded 2-fold less TSP than wild type. Binding and degradation of TSP by the mutants were not saturable at TSP concentrations up to 100 micrograms/ml. Bound TSP was localized by immunofluorescence to punctate structures on wild type and, to a lesser extent, 677 cells. Heparitinase pretreatment of wild type cells caused a 2- to 3-fold decrease in binding and degradation, whereas chondroitinase pretreatment had no effect. Chondroitinase pretreatment of the 677 mutant (deficient heparan sulfate and excess chondroitin sulfate) caused a 2-fold decrease in binding and an 8-fold decrease in turnover, whereas heparitinase pretreatment had no effect. Treatment of wild type cells with both heparitinase and chondroitinase resulted in a 6- to 8-fold decrease in binding and turnover. These results indicate that cell surface proteoglycans mediate metabolism of TSP by CHO cells and that the primary effectors of TSP metabolism are heparan sulfate proteoglycans.     

Metabolic interrelations within guanine deoxynucleotide pools for mitochondrial and nuclear DNA maintenance

Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [(3)H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation.     

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

Effect of ploidy on the response of V79 cells to ionizing radiation

The responses of diploid, tetraploid and near-hexaploid V79 cells to X-irradiation or DNA-associated 125I-decay were compared. When cell killing, following X-irradiation, was plotted against the induced level of DNA double-strand breakage (dsb) per unit length of DNA, there was no significant difference between the relationships for each cell line. This suggested that the number of X-ray-induced DNA dsb per cell required to produce a lethal lesion was proportional to ploidy. Consistent with the X-ray results, tetraploid cells required 121 +/- 4 and diploid cells 60 +/- 1 125I-decays to produce a lethal lesion. However, the hexaploid cells deviated from this relationship and required 137 +/- 5 decays. The relationship between relative elution and 125I decays/cell reflected cellular DNA content. It is concluded that current models of radiation action are unable to explain these findings satisfactorily.     

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.