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N H Terry

Publications and source records attributed to N H Terry.

At least 19 recordsLinked to original sources
PubMed1992

A quantitative method for evaluating bivariate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine.

A method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non-specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between-labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.

Animals↗
PubMed1991

Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells.

Various methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well-defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling. The relative movement (RM) of BrdUrd-labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, v (a function of the fraction of BrdUrd-labeled divided and the fraction of BrdUrd-labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time-point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.

Animals↗
PubMed1991

Measuring potential doubling times of murine tumors using flow cytometry.

Flow cytometric analysis of bromodeoxyuridine (BrdUrd) labeled cells has led to the description of various methods for determining population potential doubling times (Tpot) from samples obtained at a single time point. There have been several reports of results from human tumor samples using these various methods of analysis, but little documentation of the validity of these approaches in experimental tumor models. Recently, we reported results using in vitro cell cultures and determined the most reliable method for these. In this paper we report the results of this methodology when applied to two in vivo murine tumor models (MCaK and FSA). The analytic approach tested is summarized as follows: The relative movement (RMlu(t], a measure of the mean DNA fluorescence of BrdUrd-labeled cells that have not undergone division at the time of sampling, and a quantity v, a function of the fractions of BrdUrd-labeled divided and undivided cells, were measured for a series of time points following labeling. From each value of RMlu(t) an estimate of the length of S-phase (TS) was computed and from each v and TS a value for Tpot was found. These results were compared to the values of TS and Tpot obtained by fitting all the values simultaneously. Tumor MCaK values of TS and Tpot of 9.6 and 28.0 h were obtained from fitting all of the data. Tumor FSA gave values of 16.8 and 42.3 h for TS and Tpot, respectively. The results of this analysis show that single time point measurements can give reliable estimates of TS and Tpot.

Animals↗
PubMed1991

Molecular genetic evidence for a differentiation-proliferation coupling during DMSO-induced myeloid maturation of HL-60 cells: role of the transcription elongation block in the c-myc gene.

Proliferation-differentiation coupling was studied during dimethyl sulfoxide (DMSO)-induced myeloid maturation of HL-60 cells using transcription of the myeloperoxidase (MPO) and c-myc genes as indicators of differentiation and proliferation, respectively. Concomitant cell cycle kinetic analysis correlated the proliferation and transcription patterns. Transcription, cell cycle phases and rate of DNA synthesis were examined for up to 5 days of induction and, at 1-day intervals, analyzed during a 24-h reculture without the inducer. DMSO suppressed transcription of the c-myc and MPO genes with a t1/2 of 16 min and 7 h, respectively. The ability to recover transcription following reculture diminished with the progression of the induction and ultimately was lost; concomitantly, the cells irreversibly lost the capacity to divide. This indicated that the differentiation and proliferation processes are inseparable and that terminal differentiation accompanies irreversible proliferation arrest in HL-60 cells. We also studied the kinetics of the block to transcription elongation at the exon 1-intron 1 boundary of the c-myc gene. This block produces a 0.38 kb truncated transcript that is constitutively expressed in somatic cells (Re et al., Oncogene 5, 1247, 1990). During induction the level of the 0.38 kb RNA increased, while that of the complete c-myc mRNA decreased, indicating that this truncated RNA is generated instead of message through a monotonously initiated transcriptional process. Transcription initiation and synthesis of the 0.3 kb RNA persisted in terminally differentiated cells, suggesting a role for this RNA in non-proliferating cells.

Actins↗
PubMed1991

Measuring cell proliferation by relative movement. I. Introduction and in vitro studies.

This paper presents two new ways of analysing data which may be obtained from pulse labelling a population of cells with bromodeoxyuridine and analysing that population as a function of time with bivariate flow cytometry. The progression of cells is measured by the change in position in the cell cycle, as shown by a change in the mean DNA content of the labelled and unlabelled cells. The particular measures of the mean DNA content used are extensions of the relative movement of the labelled undivided cells, RMlu(t), which was introduced by Begg and co-workers to measure the DNA synthesis time, TS. In general, the relative movement is defined as the mean DNA fluorescence of a population of cells less the DNA fluorescence of the cells in G1 and divided by the difference in DNA fluorescence of the cells in G2 + M and G1. In this paper we examine the relative movements of all the labelled cells and all of the unlabelled cells, denoted RML(t) and RMU(t) respectively. It is found that RML(t) and RMU(t) exhibit clear cyclic behaviour and distinguishable characteristics which depend directly on the transit times (T) of the cell cycle phases, i.e. TG1, TS and TG2 + M. Furthermore, the peak heights of the RMU(t) curve are shown to depend strongly on the growth fraction of the population under consideration. A theoretical treatment of the curves so obtained is presented, and is shown to yield values in close agreement with those from other methods for measuring these transit times and a lower limit to values for the growth fraction of Chinese hamster ovary cells grown in vitro.

Animals↗
PubMed1990

Improved method for computing potential doubling time from flow cytometric data.

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.

Animals↗
PubMed1990

Effects of beta-all-trans retinoic acid on growth, proliferation, and cell death in a multicellular tumor spheroid model for squamous carcinomas.

The growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by beta-all-trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10-day-treated MTSs incorporated [3H]thymidine into trichloroacetic acid-precipitable material, and the [3H]thymidine labeling index, determined by autoradiography, was equivalent between control and RA-treated MTSs. Bivariate flow cytometric analysis of bromodeoxyuridine-labeled MTSs showed equivalent S phase progression of labeled cells over an 8-hour chase. MTS growth stasis was not related to RA-induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA-induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of acellular areas. MTS growth statis is thus related to an RA-induced cell loss in this MTS model for squamous carcinomas.

Autoradiography↗
PubMed1990

The accuracy of fine needle aspiration biopsy for flow cytometric determination of tumor DNA content.

The use of fine needle aspiration (FNA) to obtain a diagnosis of malignancy is established in the practice of oncology, but there is little information on its accuracy in sampling tumor DNA content. We therefore compared flow cytometric DNA data obtained from FNA-derived samples with that obtained after digestion of the same murine tumor from which the aspirates had been taken. Fifteen female C3Hf/Kam mice were implanted with MCA-29 tumor cells from the same source tumor. MCA-29 is a multiploid mammary adenocarcinoma with two aneuploid populations (DNA Index of A = 1.67, B = 1.89). The tumors were grown to a mean size of 8.6 mm. After sacrifice, three FNAs were performed on each tumor, following which the whole tumor (WT) was excised and homogenized. All FNA and WT samples were digested with 0.04% pepsin and the nuclei stained with propidium iodide in preparation for flow cytometry. DNA histograms of the aspirates were compared with the corresponding WT histograms. Any single FNA detected population A in all (100%) cases and detected the less prominent population B in 94.3% of instances. Any single FNA was able to detect the same populations that were present in the whole tumor in 95.4% of cases, while the set of three aspirates matched the corresponding WT in 100% of cases. We conclude that FNA DNA histograms are accurate for the assessment of ploidy, but that in order to ensure detection of all tumor populations present, multiple aspirates are needed.

Animals↗
PubMed1990

Comparative effect of the thiols dithiothreitol, cysteamine and WR-151326 on survival and on the induction of DNA damage in cultured Chinese hamster ovary cells exposed to gamma-radiation.

We compared the ability of three thiols--dithiothreitol (DTT), cysteamine and WR-151326--to protect aerated Chinese hamster ovary cells from the lethal and DNA-damaging effects of gamma-radiation. These results were compared with earlier measurements for WR-1065 and WR-255591. The time-course and the concentration dependence of protection against cell killing was determined after 10 Gy of gamma-rays. The aminothiols cysteamine and WR-151326 protected at much lower extracellular concentrations than the simple thiol DTT; however, there was no clear difference between the behaviour of cysteamine, WR-151326, WR-1065 and WR-255591 in this respect. Protection by DTT and cysteamine was complete within 1 min, whereas for WR-151326, WR-1065 and WR-255591 about 30 min was required before protection began to reach a plateau. Based on these data, complete radiation survival curves were generated for each thiol and protection factors calculated. Effects on the induction of DNA single-strand breaks (ssb) and double-strand breaks (dsb) by gamma-rays were measured using alkaline (pH 12.1) and neutral (pH 7.0 and 9.6) elution, respectively. All three thiols protected against ssb induction, although to a significantly lower extent than against cell killing measured under identical conditions. Each thiol also protected against dsb induction. After high radiation doses the protection factors for dsb induction were also less than the protection factors for cell survival; however, when dsb were assayed using the low-dose replicate plating neutral elution method, the relative effect of each thiol on cell survival and on dsb induction appeared to be equivalent. The hierarchy of protection against both ssb and dsb induction (based on the extracellular thiol concentration required to produce a given degree of protection) was similar to that for cell survival, i.e., WR-151326 congruent to cysteamine less than DTT.

Animals↗
PubMed1990

New methods for calculating kinetic properties of cells in vitro using pulse labelling with bromodeoxyuridine.

The transit times of Chinese hamster ovary cells through the phases of their cell cycle were measured using dual parameter flow cytometry to measure DNA content and the presence of monoclonal antibodies to bromodeoxyuridine. Up to four separate populations can be accurately measured: unlabelled cells in G2 + M; labelled cells that have not yet divided; labelled cells that have already divided; and the unlabelled cells that were originally in G1 plus the cells that were originally in G2 + M and have since divided. The fractions of cells in these populations can be easily followed in time and the usual kinetic properties can be estimated from these fractions, or combinations thereof, including the times through G1, S, G2 + M and the cycle time. We present equations for analysing this type of data and comment on which equations are most appropriate for measuring specific kinetic properties of the cells.

Animals↗
PubMed1989

The influence of bone marrow depletion on intestinal radiation damage.

These experiments were designed to test the hypothesis that bone marrow damage contributes to lethality when the endpoint used is LD50 for gastrointestinal damage. Specific pathogen-free mice were irradiated to the total body, total abdomen, or to the total body followed by rescue with syngeneic bone marrow cells. The relationship between animal survival and jejunal crypt survival was also examined under these three experimental conditions. The LD50/10 after total abdominal irradiation (15.6 Gy) was higher than that for total body irradiation (11.4 Gy). Rescue with syngeneic bone marrow cells after total body irradiation also increased the LD50 10 days to 14.6 Gy. The proportion of animals surviving after total body irradiation depended on the number of bone marrow cells injected as a rescue inoculum. Hence gastrointestinal death after total body irradiation is influenced by bone marrow depletion. Crypt survival, however, was similar following all three experimental procedures. These data, therefore, demonstrate a dissociation between a clonogenic and lethality assay of intestinal damage. Furthermore, a comparison of crypt survival at the LD50 under the different conditions showed that a factor of 10 times more crypts were needed to rescue a mouse from gut lethality when the total body was irradiated than when only the total abdomen was treated. Hence, the concept of the intestinal "tissue rescuing unit" as a precise and constant number of crypts is inappropriate and will vary with the experimental conditions.

Abdomen↗
PubMed1989

Time course of loss of residual radiation damage in murine skin assessed by retreatment.

The amount of radiation damage remaining in mouse foot skin has been assessed by retreatment from 10 days to 6 months after a range of first doses. The acute skin reaction was used as the endpoint. Mice hind feet were first irradiated with a range of single doses (15-37.5 Gy) covering zero to near full effect. Feet were retreated with a full range of single doses together with groups of non-previously treated age-matched control mice. No age-related changes in radiation sensitivity were observed. Dose-response curves were constructed for all retreatment times for each priming dose, and isoeffect doses were calculated for both peak and average skin reactions. If 2-6 months were allowed to elapse before retreatment, the skin could be reirradiated as if it were previously untreated. However, if only 1 month was allowed to pass before retreatment, damage was 'remembered' after all first doses. The amount of damage 'remembered' in terms of dose was 11 Gy after a first dose of 37.5 Gy, and was less after the lower first doses.

Animals↗
PubMed1988

Residual radiation damage in murine lung assessed by pneumonitis.

The amount of radiation damage remaining in mouse lung has been assessed by retreatment from 1 to 6 months after a range of first doses. Pneumonitis at 196 days after retreatment was used as the endpoint. Lungs were first irradiated with a range of single doses (6-10 Gy). Ten Gy was the highest dose that, on its own, produced no changes in breathing rate or deaths due to pneumonitis. One to 6 months later lungs were retreated with a full range of single doses. Isoeffect doses were calculated for lethality for all retreatment times after each priming dose. The amount of residual damage remaining in the lung has been calculated as both a proportion of first doses and as the effect equivalent of remembered dose. Following a 10 Gy first dose, there was evidence of remembered irradiation injury at all retreatment intervals. After a 6 Gy priming dose, the lungs could be retreated to tolerance. The amount of residual damage was proportional to the size of first dose and was highest at 1 month (27% after 6 Gy and 70% after 10 Gy) and lowest after 3 months (0% after 6 Gy and 46% after 10 Gy). This partial recovery of lung function between 1 and 3 months was followed by an increase in amount of damage "remembered"; that is, a reduction in the retreatment dose that could be delivered. The proportion of residual damage after 10 Gy was never less than 25%. The data suggest an early target cell depletion and regeneration in the lung (within 3 months), the extent of which is dependent on the size of initial injury.

Animals↗
PubMed1988

Tissue repair and repopulation in the tumor bed effect.

These experiments were designed to study the kinetics and magnitude of cell repair and repopulation in tissues whose damage results in the tumor bed effect. The right hind thighs of mice were irradiated with single doses or two equal gamma-ray fractions. Interfraction intervals ranging from 30 min to 24 h (to measure the kinetics of repair from sublethal damage) and 6 and 12 weeks (to determine the extent of repopulation) were used. One day after the second radiation dose 5 X 10(5) FSA tumor cells were inoculated into the center of the irradiated field. Radiation dose-response curves were obtained by calculating the time required for tumors to reach 12 mm diameter. No recovery occurred within 6 h of the radiation delivery as measured by this assay. Some recovery, 3.2-4.6 Gy above a single radiation dose, occurred when the interval between two fractions was 24 h. With increasing interfraction intervals of 6 and 12 weeks further dose sparing occurred in the amount of 5.0-6.9 and 7.5-8.3 Gy, respectively. The data suggest that repopulation is the major contributor to the radiation dose-sparing recovery of stromal tissue and that some proliferative response may occur as early as 1 day after the first irradiation.

Animals↗
PubMed1985

Misonidazole toxicity and pharmacokinetics in mice: dependence on strain and size.

The toxic side-effects of misonidazole (MISO) have been studied in two strains of mice over a wide weight range. The sensitivity of the mice, determined as the LD50 within 7 days of administration, varied by almost a factor of two. Differences were seen in the same strain, with heavy mice being most sensitive, and from strain to strain, with CBA mice being more susceptible than WHT albino mice. The toxicity of the MISO was closely related to a change in the peak blood levels, but showed no correlation with biological half-life. The tissue volume available for drug distribution appears to be reduced in large, heavy mice at high drug doses, and cannot be explained simply on the basis of drug exclusion from adipose tissue. For radiobiological studies with MISO doses of more than 0.5 mg/g it is recommended that it should be administered in a dosage that will give a known blood level of the drug, rather than simply by giving a constant dose based on body weight.

Animals↗