60 results on '"Dethlefsen LA"'
Search Results
2. Characterization of camptothecin-resistant Chinese hamster lung cells.
- Author
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Chang JY, Dethlefsen LA, Barley LR, Zhou BS, and Cheng YC
- Subjects
- Animals, Base Sequence, Cell Line drug effects, Cricetinae, Cricetulus, DNA isolation & purification, DNA Topoisomerases, Type I genetics, DNA Topoisomerases, Type I metabolism, DNA Topoisomerases, Type II metabolism, Dose-Response Relationship, Drug, Drug Resistance genetics, Etoposide analogs & derivatives, Etoposide pharmacology, Molecular Sequence Data, Podophyllotoxin pharmacology, RNA, Messenger analysis, Vincristine pharmacology, Camptothecin pharmacology, Lung drug effects
- Abstract
Three camptothecin-resistant sublines (V79r, IRS-1r and IRS-2r) of V79 cells and their irradiation-sensitive mutants, IRS-1 and IRS-2, were developed by stepwise, continuous exposure to camptothecin (CPT). The degree of resistance varied among these cells. Based on the biochemical characterizations of these resistant cell lines, the mechanisms which could be responsible for the resistance to CPT were proposed to be: (a) a decrease in the intracellular accumulation of CPT with or without alteration of DNA topoisomerase I, (b) a decrease in the amount of DNA topoisomerase I, or (c) a decrease in the sensitivity of DNA topoisomerase I to CPT. The resistant cells which exhibited down-regulation of DNA topoisomerase I were collaterally sensitive to etoposide (VP-16) and its analogue, 4'-demethy-4 beta-(4"-fluoroanilino)-4-desoxypodophyllotoxin, despite the fact that there were equal amounts of DNA topoisomerase II in the parental and in the resistant cell lines. Alternating the usage of CPT and VP-16 for the treatment of cancer is indicated.
- Published
- 1992
- Full Text
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3. Poly(ADP-ribose) metabolism in proliferating versus quiescent cells and its relationship to their radiation responses.
- Author
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Sweigert SE, Marston JM, and Dethlefsen LA
- Subjects
- Animals, Cell Division, In Vitro Techniques, Interphase, Mice, Radiation Tolerance, Tumor Cells, Cultured cytology, Cell Cycle, Cell Survival radiation effects, Nucleoside Diphosphate Sugars metabolism, Poly Adenosine Diphosphate Ribose metabolism
- Abstract
In the murine tumour cell lines 66 and 67 growing in vitro, quiescent (Q; unfed plateau-phase) cells are more sensitive to X-ray-induced cell killing than are proliferating (P) cells, while St4 cells (Q cells that have been re-fed and returned to 37 degrees C for 4h) are similar to P cells in radiosensitivity. We have been investigating parameters of poly(ADP-ribose) metabolism in order to determine whether such factors contribute to the variations in radiosensitivity of these growth states. These parameters were cellular NAD content, the activity of poly(ADP-ribose) transferase (ADPRT) in permeabilized cells and the activity of poly(ADP-ribose)-degrading enzymes. The results suggest that in line 66, but not 67, a reduced ability to regenerate NAD following irradiation was associated with the reduced survival of Q cells. However, neither the baseline activity of ADPRT nor the degree of stimulation of ADPRT by X-rays was found to correlate with survival, or with the induction and repair of DNA strand breaks. Stimulation of ADPRT by X-rays was dependent on dose and was greatest for a 2-min incubation with 3H-NAD. For a 2-min incubation the stimulation of ADPRT following a dose of 50 Gy was 7- and 10-fold in 66 and 67 P cells, respectively, versus 3-4-fold in Q cells. Detectable stimulation was observed in 66 P and Q cells for doses as low as 5 Gy. P and Q cells did not differ in the rate of degradation of the poly(ADP-ribose) polymers.
- Published
- 1990
- Full Text
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4. Nuclear thiols: technical limitations on the determination of endogenous nuclear glutathione and the potential importance of sulfhydryl proteins.
- Author
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Loh SN, Dethlefsen LA, Newton GL, Aguilera JA, and Fahey RC
- Subjects
- Animals, Bridged Bicyclo Compounds, Buthionine Sulfoximine, Cell Line, Chromatography, High Pressure Liquid, Cytoplasm analysis, Female, In Vitro Techniques, Indicators and Reagents, Methionine Sulfoximine analogs & derivatives, Cell Nucleus analysis, Glutathione analysis
- Abstract
Significant discrepancies were found between the values for glutathione levels determined by the Tietze enzymatic assay and those measured by labeling with monobromobimane followed by HPLC analysis when these methods were applied to proliferating and quiescent cells of the 66 murine mammary tumor line depleted of glutathione by buthionine sulfoximine or to nuclei prepared from these cells by permeabilization with Nonident detergent. The probable origin of the discrepancy was traced to the presence of acid-soluble sulfhydryl proteins in the extracts which are thought to lead to erroneous values in the Tietze assay method. Using the monobromobimane-HPLC method it was found that the low-molecular-weight thiol levels in nuclei prepared by detergent permeabilization equilibrate in less than 1 min with the permeabilizing medium, indicating that (i) endogenous nuclear glutathione levels cannot be determined reliably using conventional methods of cellular disruption and (ii) the endogenous nuclear glutathione level is likely to be the same as the cytoplasmic value. The levels of protein sulfhydryl associated with the nuclear preparations were found to be of the same magnitude as the cytoplasmic GSH level and must therefore be considered a potentially significant source of thiol capable of repairing DNA radicals.
- Published
- 1990
5. Comparison of two flow cytometric assays for cellular RNA--acridine orange and propidium iodide.
- Author
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Wallen CA, Higashikubo R, and Dethlefsen LA
- Subjects
- Animals, Cell Line, DNA analysis, Fluorescence, Interphase, Mammary Neoplasms, Experimental, Mice, Mitosis, Acridine Orange, Flow Cytometry methods, Phenanthridines, Propidium, RNA analysis
- Abstract
Two flow cytometric assays for cellular RNA, two-step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNa (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (greater than 97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.
- Published
- 1982
- Full Text
- View/download PDF
6. Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy.
- Author
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Bauer KD and Dethlefsen LA
- Subjects
- Acridine Orange, Animals, Cell Line, Cricetinae, Cytological Techniques, Female, Fluorescence, HeLa Cells analysis, Ovary analysis, Spectrophotometry, Ultraviolet, Staining and Labeling, RNA analysis
- Abstract
Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.
- Published
- 1980
- Full Text
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7. Cell loss from three established lines of the C3H mouse mammary tumor: a comparison of the 125I-UdR and the 3H-TdR-autoradiographic methods.
- Author
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Dethlefsen LA, Sorenson J, and Snively J
- Subjects
- Animals, Autoradiography, Cell Line, DNA, Neoplasm metabolism, Female, Idoxuridine metabolism, Iodine Radioisotopes, Male, Mammary Neoplasms, Experimental metabolism, Mathematics, Mice, Mice, Inbred C3H, Thymidine metabolism, Tritium, Cell Cycle, Cell Survival, Mammary Neoplasms, Experimental pathology
- Abstract
The 125I-UdR method for measuring cell loss from solid tumors has been reevaluated. The rate of tumor cell loss from three established lines (S 102F, S102S and Slow) of the C3H mouse mammary tumor was determined by the 125I-UdR method and the results were compared to the estimates for cell loss as determined by the combined approach of cellular 3H-TdR autoradiography and volumetric growth-rate determinations. This detailed comparison shows that the two methods complement each other but cannot substitute for one another because they give different quantitative information. The combined approach measures the flow of viable cells, as determined morphologically, from the proliferating compartment to the quiescent comparment, the quiescent compartment out of the tumor, etc., but does not evaluate the flow of degenerate cells or acellular (necrotic) debris. In contrast, the 125I-UdR method indicates the net flow of intact cells and/or dead cells as well as debris from the tumor as the 125I-labeled material is lost from the tumor, but gives limited internal information. thus, depending on the specific experiment, an investigator could choose one or the other of the methods to answer the question. Perhaps both would be desirable at times; however, in most cases, one could not substitute one method for the other. The data from the Slow tumors also indicate that in certain tumors, the quantitative information from the 125I-UdR method may be quite limited, i.e. the confidence limits within an experiment as well as the replication error between experiments may be high.
- Published
- 1977
- Full Text
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8. The effects of metabolic inhibitors on the synthesis of inducible tyrosine aminotransferase in cultured hepatoma cells.
- Author
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Dethlefsen LA
- Subjects
- Cell Line, Cycloheximide pharmacology, DNA biosynthesis, Depression, Chemical, Dexamethasone pharmacology, Enzyme Induction drug effects, Protein Biosynthesis, RNA biosynthesis, Dactinomycin pharmacology, Deoxyadenosines pharmacology, Tyrosine Transaminase biosynthesis
- Abstract
The effects of actinomycin-D and 3'-deoxyadenosine (cordycepin) on the steroid-mediated induction of tyrosine aminotransferase (TAT) synthesis have been reexamined in view of recent reports that the primary inhibitory action of these compounds may affect synthesis of proteins as well as RNA. The present results confirm that cordycepin blocks the steroid-mediated induction of TAT in rat hepatoma cells (HTC), but unlike actinomycin-D, cordycepin neither increases nor maintains the levels of TAT found in HTC cells preinduced with dexamethasone. Indeed, cordycepin added to preinduced cells, either in the presence or absence of steroid, causes a prompt decline in TAT activity. These data also confirm that both actinomycin-D and cordycepin have an early inhibitory effect on protein synthesis, but the cordycepin effect is observed sooner and the extent of inhibition is greater. When actinomycin-D and cordycepin are added simultaneously to preinduced cells with the steroid removed, the actinomycin-td produced maintenance of preinduced levels of TAT persists. Also, the inhibition of protein synthesis in cultures with both inhibitors approaches that for the cells treated with actinomycin-D alone instead of cordycepin alone. These data suggest that cordycepin inhibits TAT synthesis in preinduced cells by its inhibition of protein synthesis, and this inhibitory effect of cordycepin is blocked by actinomycin-D. It is possible that actinomycin-D does this by preventing the incorporation of cordycepin into RNA. However, regardless of the correctness of this speculation, the multiple effects of cordycepin indicate that this inhibitor cannot be used either to prove or rule out the post-transcriptional model for regulation of gene expression. Also, this requirement that protein synthesis must continue in order to maintain pre-induced levels of TAT is inconsistent with the assumption that the maintenance of these induced TAT levels by actinomycin-D is due to inhibition of TAT degradation.
- Published
- 1975
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9. Delayed enhanced effects of Adriamycin on the X-irradiation-induced gastrointestinal toxicity in mice.
- Author
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Dethlefsen LA, Riley RM, and Lehman CM
- Subjects
- Animals, Digestive System drug effects, Female, Male, Mice, Mice, Inbred C3H, Time Factors, Whole-Body Irradiation, Digestive System radiation effects, Doxorubicin toxicity
- Abstract
The delayed responses of C3H mice which had been pretreated with various single-dose and two-dose fractionated Adriamycin/X-irradiation protocols were evaluated by stressing the 120-day survivors with either whole-abdomen X-irradiation (LD50/7 assay) or whole-body X-irradiation (crypt colony survival). Pretreatment with Adriamycin alone was as toxic as Adriamycin plus X-irradiation for the animals stressed at 120 days (LD50/7 assay). There was no induced cellular radioresistance (D0) and no apparent increase in crypt size as indicated indirectly by the 10-clone dose at 120 days after completion of treatment. The increased lethality of the X-irradiation-stressed 120-day survivors was most likely a primary gastrointestinal response with little or no contribution from either bone marrow or kidney toxicity. The effect was apparently due to a persistent Adriamycin-induced antiproliferative response at the cellular level but the molecular mechanisms are unknown. Such data suggest caution to our clinical colleagues. Cancer patients treated with high doses of Adriamycin, independent of concomitant X-irradiation, will most likely be moderately to severely compromised in their ability to respond to a stress which requires cellular proliferation, and, based on the murine data, this effect is persistent if, indeed, not permanent.
- Published
- 1984
10. Matrix simulation of duodenal crypt cell kinetics. II. Cell kinetics following hydroxyurea.
- Author
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Roti Roti JL and Dethlefsen LA
- Subjects
- Animals, Cell Count, Cell Survival, Cells, Cultured, Duodenum cytology, Intestinal Mucosa cytology, Isotope Labeling, Kinetics, Mathematics, Mice, Mice, Inbred C3H, Mitosis drug effects, Thymidine, Tritium, Cell Division drug effects, Hydroxyurea pharmacology, Models, Biological
- Abstract
The perturbed cellular kinetics of the duodenal crypt following a single injection of hydroxyurea (HU) have been simulated using matrix algebra. Following the direct effects of HU (S-phase cytotoxicity and a G1/S block) the crypt cell kinetics undergo several alterations. Previously documented alterations include: (1) a temporary partial synchronization of the surviving cells, (2) a shortening of the cell-cycle transit time, and (3) recruitment of normally non-proliferating cells into active proliferation. These conclusions have been extended by constructing several different complex but theoretically possible recovery models and the validity of each of these models has been evaluated by simulating the following biological data: the number of cells in the S and M-phase of the cell cycle, total viable cells per crypt, and the per cent labeled mitosis and the number of labeled cells following 3H-TdR injections at 9 and 21 hr after HU treatment. The model which showed visually the best overall agreement with all sets of the data was chosen as "most probable' and leads to the following interpretations. Immediately after the end of the HU block (i.e. 5 hr after HU injection) the modal cell-cycle transit time is reduced to 8 hr. By 17 hr after HU, the modal transit time is increased to 10 hr. Repopulation of the proliferating compartment, i.e. restoration of the proliferating compartment back to the control value, occurs between 12 and 17 hr after HU injection and probably consists of both recycling of the proliferating cells (i.e. they do not progress up into the non-proliferating compartment) and recruitment of the non-proliferating cells into active proliferation. Also, the rate at which the non-proliferating cells move onto the villi is reduced temporarily. The overall recovery process results in a crypt which temporarily is larger than control and produces villi cells at a rate which is faster than the control. The time when the crypt size and villus cell production rate return to normal cannot be established using the available data.
- Published
- 1975
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11. Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA.
- Author
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Sandhu LC, Warters RL, and Dethlefsen LA
- Subjects
- Nucleic Acid Conformation, Spectrometry, Fluorescence, Benzimidazoles, DNA, Bacterial, DNA, Superhelical, Plasmids
- Abstract
The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.
- Published
- 1985
- Full Text
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12. 3H-5-iodo-2'-deoxyuridine toxicity. Problems in cell proliferation studies.
- Author
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Dethlefsen LA
- Subjects
- Animals, DNA biosynthesis, DNA, Neoplasm biosynthesis, Dose-Response Relationship, Drug, Duodenum metabolism, Ethanol administration & dosage, Ethanol pharmacology, Female, Idoxuridine administration & dosage, Idoxuridine metabolism, Intestinal Mucosa metabolism, Kinetics, Male, Mammary Neoplasms, Experimental metabolism, Mice, Mice, Inbred C3H, Thymidine metabolism, Tritium, Idoxuridine toxicity, Mitosis drug effects
- Published
- 1974
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13. Toxic effects of extended glutathione depletion by buthionine sulfoximine on murine mammary carcinoma cells.
- Author
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Dethlefsen LA, Biaglow JE, Peck VM, and Ridinger DN
- Subjects
- Animals, Buthionine Sulfoximine, Cell Line, In Vitro Techniques, Methionine Sulfoximine pharmacology, Methionine Sulfoximine toxicity, Mice, Time Factors, Glutathione metabolism, Methionine Sulfoximine analogs & derivatives
- Abstract
Extended depletion of glutathione to approximately equal to 5% of control in the murine mammary carcinoma cell line 66 was achieved with a concentration of 0.05 mM buthionine sulfoximine. At 24 hours, there was no evidence for cellular toxicity from the BSO treatment per se; however, by 48 hours, there was inhibition of protein and DNA synthesis and cell growth and cell kinetic data was suggestive of both a G1 and a G2 block. Glutathione depletion to this extent (i.e., 0.13 mM vs. 2.24 mM in control) did not modify the aerobic radiation response for cells in the physiological states of proliferation, quiescence, or stimulated quiescent cells. This degree of cellular toxicity may well be cell-type dependent, but the results do suggest that caution is in order if one should attempt long-term GSH depletion in vivo.
- Published
- 1986
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14. Heterogeneity of X-ray cytotoxicity in proliferating and quiescent murine mammary carcinoma cells.
- Author
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Wallen CA, Ridinger DN, and Dethlefsen LA
- Subjects
- Animals, Cell Division radiation effects, Cell Line, Cell Survival radiation effects, DNA Repair, DNA, Neoplasm analysis, Female, Glutathione analysis, Mammary Neoplasms, Experimental pathology, Mice, RNA, Neoplasm analysis, Mammary Neoplasms, Experimental radiotherapy, Radiation Tolerance
- Abstract
A highly enriched (greater than or equal to 97%) quiescent (Q) tumor cell population can be induced in both the 66 and 67 murine mammary carcinoma lines in vitro by nutrient deprivation (7-day, unfed plateau cultures), while exponential cultures (2-day cultures) of this line are composed of greater than 98% proliferating (P) cells. We have used these two cell lines to determine how the radiation sensitivity varies as a function of genetic heterogeneity (two cell lines derived from the same tumor) and proliferative status (physiological state). The 67 Q cells were significantly more sensitive than were the P cells to single doses of X-rays, with Dos of 52 and 90 rads and Dqs of 188 and 250 rads, respectively. Cells from transition cultures (cells that have essentially stopped proliferation but are not in the biochemical state of Q cells) have a radiation sensitivity similar to that of P cells. When exponentially growing 67 cells were induced into a Q state by reducing the serum concentration (0.5 versus 15%), they, too, were more sensitive to X-rays than were their proliferating counterparts. This sensitivity of the Q cells was decreased by placing them 30 min prior to irradiation in either fresh medium, a balanced salt solution, or a balanced salt solution with 24 mM glucose. However, the Q cells in these conditions were still an order of magnitude more sensitive than the P cells after a 523-rad dose. Therefore, the increased sensitivity of the well-oxygenated 67 Q cells appears to be primarily related to physiological alterations accompanying the transition from P to Q. The radiation sensitivity of 66 cells has also been measured in P and Q states. These cells are significantly more radioresistant than are the 67 cells and, again, the 66 Q cells were more sensitive than were the 66 P cells, with Dos of 90 and 109 rads and Dqs of 150 and 368 rads, respectively. Furthermore, the heterogeneous radiation response of the 66 and 67 cells continues to be expressed under various physiological states, albeit in qualitatively different ways; i.e., in 66 Q versus P cells, the shift in sensitivity is primarily due to a markedly reduced Dq while, in the 67 Q versus P cells, the lowered radiosensitivity is due to a marked reduction in both Do and Dq. At least in these cell lines, it is unlikely that Q cells will determine the response of the tumor to radiation.
- Published
- 1985
15. Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X-irradiation.
- Author
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Dethlefsen LA, Biaglow JE, Peck VM, and Ridinger DN
- Subjects
- Animals, Cell Survival, Glutathione metabolism, Mice, Sulfhydryl Compounds radiation effects, Time Factors, Mammary Neoplasms, Experimental metabolism, Sulfhydryl Compounds metabolism
- Abstract
The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.
- Published
- 1987
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16. Heat fractionation and thermotolerance: a review.
- Author
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Henle KJ and Dethlefsen LA
- Subjects
- Animals, Cell Cycle, Cell Division, Cells, Cultured, Chromatin metabolism, Hot Temperature, Humans, Hydrogen-Ion Concentration, Kinetics, Membrane Lipids metabolism, Neoplasms, Experimental therapy, Protein Biosynthesis, Skin pathology, Thermodynamics, Body Temperature Regulation, Hyperthermia, Induced methods, Neoplasms therapy
- Abstract
A rational approach to the design of clinical protocols combining fractionated hyperthermia plus X-irradiation or hyperthermia plus chemotherapy requires an understanding of the biology of fractionated heat alone. Mammalian cells growing in vitro can dramatically increase their tolerance to thermal damage (i.e., reduce the cellular inactivation rate) after prior heat conditioning. Although the mechanism(s) for this cellular thermotolerance is still unknown, it is apparent that the thermal history, the heat fractionation interval, and the recovery conditions all modify significantly the degree of thermotolerance subsequently exhibited. At the tissue level, the role of cellular thermotolerance is further complicated by host physiological mechanisms. Few data are available on heat fractionation in vivo, and the relative importance of physiological versus cellular effects remains to be defined.
- Published
- 1978
17. Analytical cytometric approaches to heterogeneous cell populations in solid tumors: a review.
- Author
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Dethlefsen LA, Bauer KD, and Riley RM
- Subjects
- Acridine Orange, Animals, Cell Line, Cell Survival, Chromatin analysis, DNA, Neoplasm analysis, Fluorescence, Humans, Kinetics, Models, Biological, Neoplasms, Experimental pathology, RNA, Neoplasm analysis, Staining and Labeling, Cell Cycle, Flow Cytometry, Neoplasms pathology
- Abstract
The problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics and proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cells heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.
- Published
- 1980
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18. DNA damage repair in quiescent murine mammary carcinoma cells in culture.
- Author
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Warters RL, Lyons BW, Ridinger DN, and Dethlefsen LA
- Subjects
- Animals, Cell Division, Cell Line, Centrifugation, Density Gradient, DNA isolation & purification, DNA radiation effects, Female, Interphase, Mice, Micropore Filters, Cell Cycle, DNA Repair, Mammary Neoplasms, Experimental metabolism
- Abstract
Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2-3) in which cells were proliferatively active and in day 3-5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7-9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.
- Published
- 1985
- Full Text
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19. The importance of intratumor temperature uniformity in the study of radiosensitizing effects of hyperthermia in vivo.
- Author
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Gibbs FA Jr, Peck JW, and Dethlefsen LA
- Subjects
- Animals, Extremities, Female, Male, Mammary Neoplasms, Experimental blood supply, Mice, Neoplasm Transplantation, Regional Blood Flow, Temperature, Transplantation, Homologous, X-Rays, Hot Temperature therapeutic use, Mammary Neoplasms, Experimental therapy, Radiation Tolerance
- Published
- 1981
20. A calmodulin antagonist has no effect on the repair of X-ray-induced damage in a murine mammary carcinoma cell line.
- Author
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Ridinger DN, Loh SN, Warters RL, and Dethlefsen LA
- Subjects
- Animals, Cell Line, In Vitro Techniques, Mice, Radiation Genetics, Calmodulin antagonists & inhibitors, DNA Repair drug effects, DNA, Neoplasm radiation effects, Mammary Neoplasms, Experimental genetics, Sulfonamides pharmacology
- Abstract
The effects of the calmodulin antagonist W13 were determined on potentially lethal damage repair, sublethal damage repair, and X-ray-induced DNA damage repair following X irradiation of 67 murine mammary carcinoma cells in the proliferative and quiescent states. Studies with W13 (20 micrograms/ml) on proliferating cells showed that the cells rounded up within 2 h but stayed attached to the dishes and there was a slight transient G2 block by 6 h. Also, the proportion of S-phase cells at 12 h was reduced to 65% of control with the concurrent [3H]thymidine incorporation reduced to 62% of control. There was no detectable effect from this pharmacological dose of W13 either on PLDR in proliferating cells at 400 and 800 rad or on quiescent cells at 200 and 400 rad. Likewise, there was no measurable effect on SLDR in either proliferating or quiescent cells at equally split doses of 800 and 600 rad, respectively. In addition, for control vs W13-treated proliferating cells, no difference was detected either in the induction of DNA damage by X irradiation or in the initial rate of repair (T 1/2 approximately equal to 7 min), as measured by the alkaline filter elution assay. In contrast to uv and bleomycin-induced damage, these data suggest that calmodulin may have no major role in either the molecular or cellular recovery from X-ray-induced damage in mammalian cells.
- Published
- 1986
21. Time-temperature relationships for heat-induced killing of mammalian cells.
- Author
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Henle KJ and Dethlefsen LA
- Subjects
- Animals, Cell Line, Cell Survival, Cricetinae, Cricetulus, Female, Hindlimb, Humans, Intestine, Small pathology, Kinetics, Methods, Mice, Models, Biological, Neoplasms, Experimental immunology, Neoplasms, Experimental pathology, Neoplasms, Experimental therapy, Ovary pathology, Skin Temperature, Time Factors, Hot Temperature therapeutic use
- Published
- 1980
- Full Text
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22. Matrix algebraic simulation of mitotic cell selection experiments.
- Author
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Tomasovic SP, Roti Roti JL, and Dethlefsen LA
- Subjects
- Animals, Benzimidazoles pharmacology, Cell Cycle drug effects, Cell Cycle radiation effects, Cell Line, Computers, Cricetinae, Female, Mathematics, Models, Biological, Ovary, Pyridines pharmacology, X-Rays, Cell Separation, Mitosis drug effects, Mitosis radiation effects
- Abstract
Mitotic cell selection experiments are frequently utilized in investigations on the effects of various metabolic inhibitors and/or X-irradiation on cell-cycle progression of mammalian cells in tissue culture. This study describes a method for matrix algebraic simulation of these experiments which involves the sequential multiplication of cell-population matrices, growth matrices, and mitotic cell-selection matrices. Simulations are shown for progression in control cultures and cultures perturbed either by X-rays or 2-mercapto-1(beta-4-pyridethyl) benzimidazole. Application of this model will enhance the planning of these investigations and allow more rigorous testing of alternate hypotheses concerning the mechanisms of action of perturbing agents.
- Published
- 1980
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23. Localized hyperthermia and X-irradiation of murine jejunum in situ: a new method.
- Author
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Peck JW, Gibbs FA Jr, and Dethlefsen LA
- Subjects
- Animals, Cell Survival radiation effects, Female, Fiber Optic Technology, Male, Mice, Mice, Inbred C3H, Radiotherapy Dosage, Thermometers, Xenon Radioisotopes, Hyperthermia, Induced methods, Jejunum pathology, Jejunum radiation effects, Jejunum surgery, Radiotherapy methods, X-Ray Therapy methods
- Abstract
In C3H mice, preparative surgery apposed a 15 mm length of jejunum to the peritoneal surface of the ventral abdominal wall. After the mice healed, manipulation of the abdominal wall made possible selective hyperthermic treatment and X-irradiation of the immobilized jejunal segment (IJS) while it remained within the peritoneal cavity and in continuity with the rest of the intestines. For hyperthermia, the ventral abdominal wall and attached IJS was flattened between two aluminium plates immersed in a standard water bath. The temperature at the mesenteric attachment of the IJS was within 0.5 degrees C of a 44 degrees C bath 100 s after immersion and within 0.3 degrees C at steady-state. Xenon-133 washout studies showed that the treatment technique did not compromise blood flow to the IJS. The preparative surgery left the IJS normally responsive to X-irradiation as determined by crypt microcolony assay. Hyperthermia treatment by itself at 44 degrees C for up to 20 min caused no loss of crypts or villi; however, adjuvant 5-15 min 44 degrees C hyperthermia displaced the X-ray crypt-survival curves towards lower doses without changing their slopes. Nevertheless, formal statistical analyses admitted both additive and multiplicative interpretations of the effects of X-irradiation and adjuvant hyperthermia on intestinal crypt survival. After adjuvant hyperthermia, the surviving crypts were preferentially clustered near the mesenteric attachment to a greater extent than was expected from the asymmetry of the heat dose.
- Published
- 1986
- Full Text
- View/download PDF
24. Duodenal crypt survival and crypt cellular recovery kinetics following combined treatment with adriamycin and X irradiation.
- Author
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Dethlefsen LA and Riley RM
- Subjects
- Animals, Autoradiography, Cell Survival drug effects, Cell Survival radiation effects, Dose-Response Relationship, Radiation, Duodenum drug effects, Duodenum pathology, Female, Male, Mice, Mice, Inbred C3H, Neoplasms drug therapy, Time Factors, X-Rays, DNA Repair, Doxorubicin therapeutic use, Duodenum radiation effects, Neoplasms radiotherapy
- Published
- 1980
25. Sensitization to hyperthermia (45 degrees C) of normal and thermotolerant CHO cells by anisotonic media.
- Author
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Henle KJ and Dethlefsen LA
- Subjects
- Animals, Cell Line, Cell Survival drug effects, Cricetinae, Cricetulus, Female, Ovary, Radiation Tolerance, Time Factors, Cell Survival radiation effects, Hot Temperature, Saline Solution, Hypertonic pharmacology, Sodium Chloride pharmacology
- Published
- 1979
- Full Text
- View/download PDF
26. Molecular cloning of the 3'-proximal third of Sendai virus genome.
- Author
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Dowling PC, Giorgi C, Roux L, Dethlefsen LA, Galantowicz ME, Blumberg BM, and Kolakofsky D
- Subjects
- Animals, Cattle, DNA genetics, DNA, Recombinant metabolism, DNA-Directed RNA Polymerases metabolism, Nucleic Acid Hybridization, Parainfluenza Virus 1, Human enzymology, Plasmids, RNA, Messenger genetics, Thymus Gland, Virion enzymology, Cloning, Molecular, Genes, Viral, Parainfluenza Virus 1, Human genetics
- Abstract
Portions of the Sendai virus genome were randomly cloned by using virion 50S RNA and calf thymus DNA pentanucleotides as primers. The recombinant clones were probed first with radiolabeled products of an in vitro virion RNA polymerase reaction to locate early message clones and then with a probe from the viral genome 3' end to locate the most 3'-proximal clones. Clones were then ordered from the 3' end of the genome and used to construct a genetic map of the 3'-proximal third of the genome by hybrid-selection of mRNAs. We report that the gene order for this region is 3'-NP - P + C - M-5' and that the genetic loci of the viral P and C proteins cannot be separated by these techniques.
- Published
- 1983
- Full Text
- View/download PDF
27. Adriamycin effects and the interactions of adriamycin with X irradiation on murine mammary tumors.
- Author
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Dethlefsen LA and Riley RM
- Subjects
- Animals, DNA biosynthesis, Flow Cytometry, Injections, Intraperitoneal, Mammary Neoplasms, Experimental metabolism, Mammary Neoplasms, Experimental radiotherapy, Mice, Mitosis, Mitotic Index, Neoplasm Transplantation, Doxorubicin administration & dosage, Mammary Neoplasms, Experimental pathology
- Published
- 1982
28. Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.
- Author
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Dethlefsen LA, Riley RM, and Roti Roti JL
- Subjects
- Animals, Autoradiography, Cell Cycle drug effects, DNA, Neoplasm biosynthesis, Doxorubicin pharmacology, Female, Mammary Neoplasms, Experimental pathology, Mice, Mice, Inbred C3H, Mitotic Index drug effects, Cytological Techniques, Doxorubicin therapeutic use, Mammary Neoplasms, Experimental drug therapy, Photometry
- Abstract
The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.
- Published
- 1979
- Full Text
- View/download PDF
29. Morphometric changes as a function of the proliferative status of murine mammary carcinoma cells.
- Author
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Yasui LS and Dethlefsen LA
- Subjects
- Animals, Cell Division, Cell Line, Cell Nucleus ultrastructure, Chromatin ultrastructure, Mammary Neoplasms, Experimental ultrastructure, Mice, Microscopy, Electron, Mitochondria ultrastructure, Mammary Neoplasms, Experimental pathology
- Abstract
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.
- Published
- 1987
30. Matrix simulation of duodenal crypt cell kinetics. I. The steady state.
- Author
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Roti Roti JL and Dethlefsen LA
- Subjects
- Animals, Cell Count, Cells, Cultured, Duodenum cytology, Hydroxyurea pharmacology, Isotope Labeling, Kinetics, Mathematics, Mice, Mice, Inbred C3H, Mitosis drug effects, Thymidine, Tritium, Cell Division drug effects, Models, Biological
- Abstract
Steady state crypt cell kinetics have been simulated using matrix algebra. The model crypt cell population is distributed through two proliferation compartments (P1 and P2) and a quiescent state (Q). Under steady state conditions half the daughter cells produced on completion of P1 enter G1 of P2 and half enter G1 of P1. Both P2 daughter cells enter Q. Cells in Q are non-dividing but retain the potential to divide. On completion of Q, cells lose the potential to divide and move up onto the villi. The model has been developed by simultaneously simulating the following biological data: (1) the per cent labeled mitosis (PML) curve, (2) the number of labeled cells per crypt as a function of time following an injection of 3H-thymidine, and (3) the total number of cells per crypt.
- Published
- 1975
- Full Text
- View/download PDF
31. Alteration of murine mammary tumor metastasis and growth by cytomegalovirus infection.
- Author
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Olsen GA, Glasgow LA, and Dethlefsen LA
- Subjects
- Animals, Cytomegalovirus, Female, Lung Neoplasms secondary, Mammary Neoplasms, Experimental microbiology, Mammary Neoplasms, Experimental pathology, Mice, Neoplasm Metastasis microbiology, Neoplasm Transplantation, Time Factors, Transplantation, Homologous, Cytomegalovirus Infections immunology, Mammary Neoplasms, Experimental immunology, Neoplasm Metastasis immunology
- Abstract
Host resistance to the development of metastatic lesions is complex and involves both lymphocyte and macrophage functions. Studies in both humans and animals have suggested that cytomegalovirus infection may alter these components of the defense mechanism of the host. In the present study, an experimental model was developed to determine whether cytomegalovirus infection would affect host resistance to the establishment of metastatic tumor nodules in the lungs of C3H mice after i.v. inoculation of a single-cell suspension of mammary tumor cells. The number of tumor nodules in the lungs, the lungs-heart/body weight ratio, and the mean day of death were determined in control animals inoculated i.v. with 10(6) mammary tumor cells and compared with groups of animals also receiving a sublethal i.p. inoculum of murine cytomegalovirus (MCMV) (10(5) plaque-forming units) either 3 days before, on the day of, or 10 or 13 days after tumor cell inoculation. The results suggest a biphasic effect of virus infection on tumor development in the lung. A preexisting or concurrent MCMV infection suppressed tumor growth and prolonged life, while a MCMV infection later in tumorigenesis enhanced tumor growth and shortened survival. These data suggest that MCMV modulates host resistance to the development of metastatic tumor nodules and that this experimental model may be utilized to investigate further the relationship between virus-induced alterations of host defense mechanisms and tumor growth.
- Published
- 1980
32. Can animal and in vitro studies give new, relevant answers to questions concerning mammorgraphic screening for human breast cancer?
- Author
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Dethlefsen LA, Brown JM, Carrano AV, and Nandi S
- Subjects
- Animals, Breast Neoplasms etiology, Cell Transformation, Neoplastic, DNA Repair radiation effects, Disease Models, Animal, Female, Humans, In Vitro Techniques, Mammary Neoplasms, Experimental etiology, Risk, X-Rays, Breast Neoplasms diagnostic imaging, Mammography adverse effects, Neoplasms, Radiation-Induced etiology
- Published
- 1978
33. Effects of hyperthermia and X irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells.
- Author
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Livingston GK and Dethlefsen LA
- Subjects
- Animals, Cells, Cultured, Cricetinae, Cricetulus, Dose-Response Relationship, Radiation, Female, Hot Temperature, Interphase radiation effects, Ovary cytology, Radiotherapy, Time Factors, X-Rays, Chromatids radiation effects, Ovary radiation effects
- Published
- 1979
34. Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells.
- Author
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Wallen CA, Higashikubo R, and Dethlefsen LA
- Subjects
- Animals, Cell Line, Cell Separation, Clone Cells, DNA analysis, Female, Flow Cytometry, Interphase, Kinetics, Mice, RNA analysis, Cell Division, Mammary Neoplasms, Experimental pathology
- Abstract
The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.
- Published
- 1984
- Full Text
- View/download PDF
35. DNase I sensitivity of nuclear DNA measured by flow cytometry.
- Author
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Roti Roti JL, Wright WD, Higashikubo R, and Dethlefsen LA
- Subjects
- Animals, Cell Line, Cell Nucleus analysis, Chromatin analysis, Ethidium, Flow Cytometry, HeLa Cells analysis, Humans, Mammary Neoplasms, Experimental analysis, Mice, DNA analysis, Deoxyribonuclease I
- Abstract
The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1985
- Full Text
- View/download PDF
36. The effect of L-buthionine sulfoximine on the aerobic radiation response of A549 human lung carcinoma cells.
- Author
-
Biaglow JE, Varnes ME, Tuttle SW, Oleinick NL, Glazier K, Clark EP, Epp ER, and Dethlefsen LA
- Subjects
- Buthionine Sulfoximine, Cell Line, Cell Survival drug effects, Cell Survival radiation effects, Glutathione metabolism, Humans, In Vitro Techniques, Methionine Sulfoximine pharmacology, Oxygen physiology, Time Factors, Methionine Sulfoximine analogs & derivatives, Radiation-Sensitizing Agents pharmacology
- Abstract
Our data show that A549 cells are increasingly radiosensitive with prolonged exposure to L-BSO. The resulting glutathione and protein thiol depleted cells show both loss of shoulder and slope modification. Furthermore, there is an increase in single strand DNA breaks and irrepairable cross-linking. The aerobic radiation damage in the thiol depleted state appears to be different from that obtained with hypoxic cells. Any postulated role for GSH in reducing or preventing peroxidative radiation damage must also include protection against single strand DNA breaks as well as involvement in repairing DNA-protein cross-links. The latter effect may be related to decreased protein thiol content as reflected in a decreased enzyme capacity to repair DNA damage.
- Published
- 1986
- Full Text
- View/download PDF
37. The effects of adriamycin on murine duodenal crypt cell proliferation.
- Author
-
Dethlefsen LA and Riley RM
- Subjects
- Animals, Cell Cycle drug effects, Cell Survival drug effects, DNA biosynthesis, Duodenum metabolism, Female, Kinetics, Male, Mice, Mice, Inbred C3H, Cell Division drug effects, Doxorubicin adverse effects, Duodenum drug effects
- Published
- 1979
- Full Text
- View/download PDF
38. Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures.
- Author
-
Bauer KD and Dethlefsen LA
- Subjects
- Acridine Orange, Clone Cells, Cytological Techniques, DNA metabolism, Fluorescence, HeLa Cells, Humans, Kinetics, RNA metabolism, Staining and Labeling, Interphase, Mitosis
- Abstract
Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.
- Published
- 1981
- Full Text
- View/download PDF
39. In vivo effects of 5-fluorouracil and ftorafur[1-(tetrahydrofuran-2-yl)-5-fluorouracil] on murine mammary tumors and small intestine.
- Author
-
Pallavicini MG, Cohen AM, Dethlefsen LA, and Gray JW
- Subjects
- Animals, Body Weight drug effects, Cell Division drug effects, DNA biosynthesis, Female, Fluorouracil toxicity, Male, Mice, Mice, Inbred C3H, Tegafur toxicity, Fluorouracil analogs & derivatives, Fluorouracil pharmacology, Intestine, Small drug effects, Mammary Neoplasms, Experimental pathology, Tegafur pharmacology
- Abstract
The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G1/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate 32P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.
- Published
- 1979
- Full Text
- View/download PDF
40. The effect of pH on potentially lethal damage recovery in A549 cells.
- Author
-
Varnes ME, Dethlefsen LA, and Biaglow JE
- Subjects
- Cell Line, Humans, Hydrogen-Ion Concentration, In Vitro Techniques, DNA Repair radiation effects, DNA, Neoplasm radiation effects, Lung Neoplasms genetics
- Abstract
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.
- Published
- 1986
41. Cellular recovery kinetic studies relevant to combined-modality research and therapy.
- Author
-
Dethlefsen LA
- Subjects
- Animals, Doxorubicin pharmacology, Doxorubicin toxicity, Female, Intestines drug effects, Intestines radiation effects, Kinetics, Pregnancy, Radiation Injuries, Experimental, Research Design, Antineoplastic Agents pharmacology, Cell Survival drug effects, Cell Survival radiation effects, Neoplasms, Experimental therapy
- Published
- 1979
- Full Text
- View/download PDF
42. The combined effects of hydroxyurea and X irradiation on murine duodenal mucosa and host survival.
- Author
-
Dethlefsen LA, Ohlsen JD, and Riley RM
- Subjects
- Animals, Cell Survival drug effects, Cell Survival radiation effects, DNA metabolism, DNA radiation effects, Duodenum drug effects, Female, Hydroxyurea pharmacology, Injections, Intraperitoneal, Interphase drug effects, Interphase radiation effects, Intestinal Mucosa drug effects, Lethal Dose 50, Male, Mice, Mice, Inbred C3H, Radiation Dosage, Radiation-Protective Agents administration & dosage, Time Factors, X-Rays, Duodenum radiation effects, Hydroxyurea administration & dosage, Intestinal Mucosa radiation effects
- Published
- 1980
43. Fractionation of combined heat and radiation in asynchronous CHO cells. II. The role of cell-cycle redistribution.
- Author
-
Tomasovic SP, Henle KJ, and Dethlefsen LA
- Subjects
- Animals, Cells, Cultured, Cricetinae, Cricetulus, DNA analysis, Female, Ovary cytology, Radiation Tolerance, Time Factors, X-Rays, Cell Cycle radiation effects, Cell Survival radiation effects, Hot Temperature
- Published
- 1979
44. The physiological state as a modifier of radiation-induced cytotoxicity in heterogeneous murine tumor cells growing in vitro.
- Author
-
Cheng WY, Ridinger DN, Lehman CM, Puryear RL, and Dethlefsen LA
- Subjects
- Animals, Cell Separation, Energy Metabolism radiation effects, Glucose metabolism, Hydrogen-Ion Concentration, Mice, Time Factors, Cell Cycle radiation effects, Cell Survival radiation effects, Tumor Cells, Cultured radiation effects
- Abstract
The oxic radiation response (cytotoxicity) of two heterogeneous murine tumor-cell lines cultured in vitro was studied as a function of the cell's physiological state at the time of X-irradiation. The proliferating (P) 66 and 67 cells displayed equal radiosensitivities; however, the quiescent (Q) cells were considerably more radiosensitive than the P cells, and the 66Q cells were even more radiosensitive than the 67Q cells. Also, the 66Q cells continued to proliferate slowly with about 85 per cent in the G1 phase and 10 per cent in the S phase, while the 67 Q cells displayed a more complete G1 arrest (92-95 per cent). A detailed analysis of the metabolic status vs cell-cycle age (i.e. G1 vs S phase) indicated that the cell-cycle age was the predominant factor influencing radiation-induced cytotoxicity in 67 cells. The data also showed that in the plateau phase Q-cell cultures, pH and cell contact were not influencing factors and that the increased radiosensitivity of the Q cells could not be explained on the basis of energy deprivation. Moreover, the 66Q, but not the 67Q cells displayed an increased sensitivity in addition to that caused by the predominant cell-cycle age shift. This extra increase in radiosensitivity is of unknown metabolic origin, but could be related to cellular membrane fragility in the stressed 66Q cells since this extra component of Q-cell radiosensitivity was reduced both by refeeding (metabolic activation) 4 h before X-irradiation and by delayed plating while incubating the cells in Q medium at 37 degrees C after X-irradiation.
- Published
- 1989
- Full Text
- View/download PDF
45. Kinetic models of C3H mouse mammary tumor growth: implications regarding tumor cell loss.
- Author
-
Roti JL, Bohling V, and Dethlefsen LA
- Subjects
- Animals, Cell Cycle, Cell Division, Kinetics, Mice, Mice, Inbred C3H, Mitotic Index, Thymidine, Tritium, Mammary Neoplasms, Experimental pathology, Models, Biological
- Abstract
Three models of tumor cell loss are described. The effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of 3H-TdR injections. The relative volumetric growth rate has the same mathematical form for each model. The PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. The predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. The most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of 3H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.
- Published
- 1978
- Full Text
- View/download PDF
46. Murine mammary tumour cells in vitro. I. The development of a quiescent state.
- Author
-
Wallen CA, Higashikubo R, and Dethlefsen LA
- Subjects
- Animals, Cell Count, Cell Line, Cell Separation, DNA analysis, Female, Flow Cytometry, Interphase, Kinetics, Mice, Mitotic Index, RNA analysis, Cell Division, Mammary Neoplasms, Experimental pathology
- Abstract
Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G1 DNA content increasing from 50% in the P state to greater than 97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of greater than or equal to 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since less than 2.5% of the cells incorporated [3H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was greater than 95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents.
- Published
- 1984
- Full Text
- View/download PDF
47. The effects of adriamycin and X-irradiation on the murine duodenum.
- Author
-
Dethlefsen LA and Riley RM
- Subjects
- Animals, Cell Survival drug effects, Cell Survival radiation effects, Dose-Response Relationship, Radiation, Duodenum radiation effects, Female, Kinetics, Male, Mice, Mice, Inbred C3H, X-Rays, Doxorubicin adverse effects, Duodenum drug effects, Radiation-Sensitizing Agents
- Published
- 1979
- Full Text
- View/download PDF
48. Effects of double and multiple doses of hydroxyurea on mouse duodenum and mammary tumors.
- Author
-
Dethlefsen LA, Sorensen SP, and Riley RM
- Subjects
- Animals, Body Weight, Cell Division, DNA biosynthesis, DNA, Neoplasm biosynthesis, Duodenum metabolism, Hydroxyurea pharmacology, Kinetics, Mice, Mice, Inbred C3H, Thymidine metabolism, Time Factors, Duodenum drug effects, Hydroxyurea administration & dosage, Mammary Neoplasms, Experimental drug therapy
- Abstract
Previous data on the effects of a single dose of hydroxyurea on C3H mouse duodenum and mammary tumors from a fast growing line (S102F) were used to predict times that may be optimal (i.e., minimize killing of the duodenal S-phase cells while enhancing the killing of tumor S-phase cells) for the administration of subsequent doses of hydroxyurea. These predicted protocols were tested by giving tumor-bearing mice injections of 2 doses at 24 hr intervals. A preliminary in vivo tumor treatment experiment was also done wherein multiple doses (up to 10) were given either at 12, 20, or 24 hr intervals with the mouse survival, body weights, and tumor volumes being recorded daily. The data show that partial cell synchronization was achieved in both tissues and the initial knetics of the surviving cells was essentially the same after a single dose, 2 doses, or 4 doses of hydoxyurea. Also, the different intervals between the 2 doses did not affect the timing of the initital peaks of DNA synthesis in partially synchronized cells; however, the height of the peaks was affected The results demonstrate that kinetic data can be useful for predicting optimal intervals for 2-dose regimes and probably multiple-dose regimes involving a single cell-cycle phase-speeific drug when applied to a mouse tumor model. However, the recovery phenomena in the respective tissues are extremely complicated and more animal tumor data need to be collected before one can make adquate use of cell-synchronizing agents and perturbed cellular kinetic data for routine clinical chemotherapy or combined modality therapy.
- Published
- 1975
49. Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells.
- Author
-
Dethlefsen LA, Lehman CM, Biaglow JE, and Peck VM
- Subjects
- Animals, Buthionine Sulfoximine, Cell Division drug effects, Cell Line, DNA, Neoplasm biosynthesis, Depression, Chemical, Dimethyl Fumarate, Drug Therapy, Combination, Glutathione metabolism, In Vitro Techniques, Methionine Sulfoximine therapeutic use, Mice, Fumarates therapeutic use, Glutathione physiology, Mammary Neoplasms, Experimental therapy, Methionine Sulfoximine analogs & derivatives
- Abstract
Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.
- Published
- 1988
50. Repair of DNA single- and double-strand breaks in proliferating and quiescent murine tumor cells.
- Author
-
Sweigert SE, Eguchi-Kasai K, Warters RL, and Dethlefsen LA
- Subjects
- Animals, Cell Division radiation effects, DNA, Neoplasm metabolism, DNA, Single-Stranded radiation effects, Hydrogen-Ion Concentration, Mice, Tumor Cells, Cultured cytology, Tumor Cells, Cultured metabolism, DNA Repair radiation effects, DNA, Neoplasm radiation effects, Tumor Cells, Cultured radiation effects
- Abstract
We evaluated the relationship between the repair of DNA single- and double-strand breaks and cellular radiosensitivity in proliferating vs. quiescent cells of the mouse mammary tumor lines 66 and 67 in vitro, using the technique of filter elution at pH 12.2, pH 7.2 and pH 9.6. In these lines, quiescent (Q; unfed plateau-phase) cells are more radiosensitive than are proliferating (P) cells. At doses of 4-6 Gy, both 66 and 67 Q cells repair single-strand breaks (ssb) with kinetics similar to those of P cells. However, repair of ssb was slightly retarded in Q cells at a higher dose (10 Gy) than at the lower doses. In contrast, repair of ssb in P cells was dose-independent, at least for doses up to 10 Gy. The rate of repair of DNA double-strand breaks (dsb), measured at pH 7.2, was dose-independent in P and Q cells of both lines. The repair kinetics were biphasic, with an initial half-time less than 15 min, and the early phase was similar in all cell groups. The half-time for repair in the slow phase ranged from about 2 to greater than 20 h. The fraction of damage repaired by the slow phase was relatively high in all cell groups (40-70 per cent). In line 66, P cells repaired a higher percentage of dsb by 2 h postirradiation than did Q cells. The opposite was observed in line 67: Q cells repaired more dsb in 2 h than did P cells. The survival of 66 St4 cells (Q cultures which have been refed with complete medium and incubated 4 h) was significantly greater than that of 66 Q; nevertheless St4 cells repaired both ssb and dsb at rates similar to those of Q cells. Therefore, survival does not necessarily correlate with the rates of either ssb or dsb repair among these cell lines in different growth states.
- Published
- 1989
- Full Text
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