Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac)

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 μM tetrac (1 h at 37oC) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.     

Repair of potentially lethal radiation damage in confluent and non-confluent cultures of human hybrid cells

The repair of potentially lethal damage (PLDR) in a gamma-irradiated human hybrid cell line (skin fibroblast X HeLa) and its tumourigenic segregant has been studied as a function of cell density at the time of irradiation and during the postirradiation repair period. The data show that PLDR occurs in both non-confluent and confluent cultures of both cell lines. Furthermore, there is evidence that the extent of PLDR is dependent on cell density and that cell-cell contact may be an important factor in this regard.     

The effect of pH on potentially lethal damage recovery in A549 cells

The radiation sensitivity and potentially lethal damage recovery (PLDR) capacity of A549 human lung carcinoma cells have been studied. For unfed monolayer cultures, radiation sensitivity was greater in plateau phase than in log phase of growth. PLDR was observed when plateau-phase cells were held in their own spent medium postirradiation, such that the dose-response curve with 24 h holding was similar to that for log-phase cells plated immediately after irradiation. The high PLDR capacity of A549 plateau-phase cells (recovery factor between 40 and 70 for 24 h holding after 10 Gy) was reduced 10-fold or more by alkalinizing the pH of the spent medium immediately after irradiation from a value of 6.5 +/- 0.1 to a value of 7.6. Medium alkalinization resulted in an increase in the rate of glycolysis, with subsequent reacidification to a pH of 7.3 within 2 h of the pH adjustment. No change in cell cycle distribution was observed in the plateau-phase cultures up to 32 h after change of medium pH, and no increase in cell density was found after 48 h. A slight increase in the rate of incorporation of radiolabeled thymidine into acid-precipitable material was observed at 4 and 24 h after alkalinization of the medium. While it is not possible at present to define a mechanism for this pH effect, our results demonstrate that, at least for this cell line, variables such as medium pH and glucose concentration can profoundly influence the observation of PLDR.     

In vitro holding and PLD-repair: II. A flow cytometric and electron microscopic analysis of some mammalian cell lines

Summary The repair of potentially lethal damage (PLDR) in mammalian cells is expected to be better in quiescent cultures since PLD is supposedly fixed during cycle progression. Plateau phase cultures, therefore, serve as models because of assumed mitotic quiescence. Four established cell lines (V79, CHO, L5178Y and HELA) and one euploid cell strain IMR-90 have been analysed by flow cytometry and electron microscopy to address questions on quiescence in the plateau phase and the effect of holding (induction of quiescence by nutrient privation). In contrast to commonly held views, our results indicate that the quiescent fraction in cultures from transformed cells is exceedingly low (1% or less). Plateau phase cultures of transformed cells are constantly turning over. Euploid cells like the IMR-90 show true quiescence in the plateau phase. Holding causes typical cytopathological changes. These changes have been ultrastructurely characterised. Resistant sub-populations of cells can be selected out under holding-conditions. Such selected cells show completely different radiobiological characteristics, which raise questions on the interpretation of data on PLDR.Dedicated to Prof. Dr. Johannes Meißner, Borstel on the occasion of his 75th birthday     

Repair of potentially lethal X-ray damage in fibroblasts derived from patients with hereditary and D-deletion retinoblastoma

We examined X-ray induced potentially lethal damage repair (PLDR) in density inhibited plateau phase cultures of six fibroblast strains derived from patients with hereditary retinoblastoma and two patients with D-deletion retinoblastoma and compared them to three normal controls. PLD was measured in hereditary retinoblastoma (7 Gy exposure) and normal cells (7 and 9 Gy exposure) after 24 h repair time. PLD survival curves were performed at 2-9 Gy on six retinoblastoma and three normal control cell strains. Thus, PLDR was compared at equitoxic survival levels as well as after exposure to equal doses of radiation. Some retinoblastoma strains showed normal PLDR whereas others were possibly deficient. Implications of PLDR for susceptibility to radiation-induced and spontaneous tumours in hereditary retinoblastoma patients are discussed.     

FTIR spectroscopy demonstrates biochemical differences in mammalian cell cultures at different growth stages

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.     

Radiation response characteristics of human cells in vitro

Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant. The most recent addition to the list are endothelial cells from umbilical cord veins. Interesting results in radiosensitivity studies of these human cells have been obtained, some of which may have implications in radiation therapy. (i) Repair of potentially lethal damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness. (ii) No apparent correlation can be made between the extent of PLDR and the traditional radioresponsiveness of a particular tumor type. Indeed, if anything, it could appear to have an inverse correlation since the most resistant tumor cells show the smallest amount of PLD repair. (iii) Dose-rate effects appear to be better predictors of radiosensitivity than PLDR capacity. (iv) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested. Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast).     

Interaction between radiation and drug damage in mammalian cells. III. The effect of adriamycin and actinomycin-D on the repair of potentially lethal radiation damage.

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.     

Insulin receptors in 3T3 fibroblasts : Relationship to growth phase, transformation and differentiation into new cell types

The amount of [¹²⁵I]insulin binding per 2 × 10⁶ cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.     

Properties of the H-4-II-E tumor cell system. II. In vitro characteristics of an experimental tumor cell line.

The growth and cell proliferation characteristics of the H-4-II-E cell line, giving rise to hepatoma H-4-II-E when inoculated into male ACI rats, were studied in vitro. Following seedling of 2 x 10(5) cells into culture dishes, exponential cell growth occurs in cultures fed both at 24 hr and 48 hr intervals with a population doubling time of 18-4 hr. Plateau phase growth conditions are established on day 7 and day 5 for cultures fed at 24 hr and 48 hr intervals respectively. Both the plateau phase cell density and the maintenance of plateau phase appear dependent on the frequency of feeding. For cultures fed daily, the transition from exponetial growth to plateau phase results from both a reduction in the number of proliferating cells (99% v. 35%) as well as an elongation of the cell cycle (17-7 hr v. 128-4 hr). The cell proliferation characteristics of the culture are further discussed in reference to both cell growth and feeding schedules of other cell lines.     

Mutagenicity of the oral carcinogen 4-nitroquinoline-1-oxide in cultured BigBlue rat tongue epithelial cells and fibroblasts

Environmental carcinogen exposures contribute to the development of oral cancer and improved test systems for the analysis of such carcinogens are needed. We have previously isolated and characterized an epithelial cell line from the tongue of a BigBlue rat. Now, we have established an immortalized fibroblast cell line from the same organ. We exposed these cells to 4-nitroquinoline-1-oxide (NQO), a well-known experimental oral carcinogen in the rat and other species, and measured its cytotoxic and genotoxic (cII transgene mutagenesis) effects. Both cell lines were very sensitive to NQO toxicity and showed dose-dependent mutant frequency responses. At the highest NQO dose tested, 70 ng/ml, the mutant frequency was elevated more than eight-fold above background for the epithelial cells and more than 25-fold for the fibroblast cells. We examined cellular parameters which could affect glutathione-dependent detoxication of mutagens. Glutathione (GSH) contents of the two cell lines were similar. Glutathione transferase (GST) activities were measured with several substrates and were generally higher in the epithelial cells. Although multiple biochemical and biological characteristics of individual cell lines are likely to determine responses to mutagens, the greater sensitivity of the fibroblast cells to NQO mutagenicity is in accord with the lower GST activity and the lower DNA content of these cells. These new cell lines are suitable for in vitro testing of chemicals as possible oral mutagens and for studies of their biochemical mechanisms of action.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Incorporation and turnover of phospholipid precursors in normal and tumoral glial cells in culture

The incorporation and turnover of phospholipid precursors in cultured normal and tumoral glial cells was investigated during the plateau phase of growth. Glycerol was incorporated similarly by all cell types, and was renewed with a half-life of 19-37 hr. Acetate had a much longer half-life in primary cultures (50-75 hr) than in proliferative tumor cells (20-40 hr). Phosphate had a more rapid turnover rate in primary cultures (25 hr) than in proliferative tumor cells (50 hr). For all precursors, inositol- and choline phosphoglycerides had a faster turnover rate than other phospholipids.     

Sensitivity of a human hybrid cell line (HeLa X skin fibroblast) to radiation-induced neoplastic transformation in G2, M, and mid-G1 phases of the cell cycle

The dependence of gamma-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa X skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 or for asynchronous cells. However, the data indicate that the transformation frequencies were similar in the different phases of the cell cycle when correlated with surviving fraction. The results indicate that transformation frequency is more sensitive to changes in dose than is cell survival.     

Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay

We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.     

Heat-induced phosphorylation of NBS1 in human skin fibroblast cells

NBS1 is known to be involved in DNA damage-induced cellular responses after exposure to ionizing radiation (IR). Phosphorylation of NBS1 contributes to cell-cycle checkpoints. The aim of this study was to determine whether heat exposure induces or stimulates cellular responses mediated by the phosphorylation of NBS1 in human skin fibroblast cell lines. The results of immunofluorescent staining and Western blot analysis showed that NBS1 proteins are phosphorylated after exposure to heat in the nucleus of a normal skin fibroblast cell line (82-6 cells). This suggests that the NBS1-mediated signal transduction could be induced by heat. We further examined whether a deficiency in the NBS1 protein modifies heat sensitivity in human skin fibroblast cell lines. A skin fibroblast cell line (Gmtert), derived from a Nijmegen breakage syndrome (NBS) patient containing mutant NBS1, showed higher sensitivity to heat than the same cell line transfected with the wild-type copy of the NBS1 gene. We also showed that transfection of a DNA cassette expressing small interference RNA (siRNA) targeted to NBS1 into 82-6 cells enhanced cell sensitivity to heat. These results suggest that NBS1 is involved in cellular responses to DNA damage which is induced by heat exposure as well as by radiation exposure in human skin fibroblast cells.     

In vitro holding and PLD repair

Summary The Stationary or Plateau-Phase of commonly used rodent cell lines like the V79 are often assumed to be quiescent (non-mitotic). An analysis of cell turnover in V79 plateau-phase cultures through BrUdR-incorporation combined with FUdR-block and light exposure (S-phase cytocide) revealed such cultures to be in a state of kinetic equilibrium. Even when the state of maximal permissible density was acquired, at least 50% of the population of cells were cycling within the time for one population doubling. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turnover-rate was reduced. Our assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing and confluent cultures. Elimination of cycling cells by S-phase cytocide removed these differences. Since a significant fraction of plateau-phase cells are not mitotically quiescent (Q), one must eliminate the proliferating (P) fraction if one wishes to examine the PLDR of the Q cells. For V79 cells, removal of the P cells eliminates the higher survival (usually interpreted as Q cell PLDR) of plateau-phase cells.     

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

To elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture. Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers. For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested. These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.     

The influence of caffeine on the mitomycin C-induced chromosome aberration frequency in normal human and xeroderma pigmentosum cells

The clastogenic effect of mitomycin C (MC) was determined in two normal fibroblast cell lines and two xeroderma pigmentosum (XP) cell lines, a variant and a group A excision-deficient line. The group A xeroderma cell line was substantially more sensitive to MC than either the XP variant or the normal human cells. On caffeine post-treatment potentiation of the MC-induced aberration frequency occurred in all the cell lines. The XP varian cell line exhibited a distinctly higher sensitivity to caffeine than the classical XP or the normal human cell lines.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.