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N H Terry

Publications and source records attributed to N H Terry.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence.

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.

Animals↗
PubMed1995

Specific staining of iododeoxyuridine and bromodeoxyuridine in tumors double labelled in vivo: a cell kinetic analysis.

The simultaneous and specific staining of iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) allows for more accurate estimates of potential doubling time (Tpot). Because CldUrd is not approved for human use, the procedure was adapted for the staining of IdUrd and bromodeoxy-uridine (BrdUrd). The fluorescein isothiocyanate-conjugated B44 antibody (B44-FITC) stained both IdUrd and BrdUrd in tumor nuclei labelled singly with one or the other pyrimidine analogue. However, when MCaK tumors in exponential growth in vivo were pulse labelled with both IdUrd and BrdUrd, the staining of BrdUrd was not seen, and the labelling pattern reflected specificity to IdUrd. These observations were confirmed using tumors pulse labelled with IdUrd and/or BrdUrd at 6 h and/or 0.3 h prior to tumor removal in all possible combinations. Simultaneous specific staining of BrdUrd by Br3 and of IdUrd by B44-FITC was documented by quantification of labelling indices (LIs) from double-labelled tumors. The specificity of B44-FITC for IdUrd in double-labelled tumors was due to a greater affinity of this antibody for IdUrd than for BrdUrd. This technique allowed for two independent estimates of LI and Tpot when tumors were double labelled for 3.0 and 5.5 h. Both IdUrd and BrdUrd are approved for clinical use, and this double-labelling technique should prove to be valuable for measuring the cell kinetics of solid tumors in vivo.

Animals↗
PubMed1995

Cellular kinetics in rectal cancer.

Measurements of dynamic tumour cell kinetic parameters, particularly the potential doubling time (Tpot) may have potential as predictive assays for treatment outcome after radiotherapy. This paper details the distributions of Tpot and other kinetic and DNA content parameters measured in rectal cancers. Biopsies were taken from 119 patients approximately 6 h after infusion of 200 mg m-2 bromodeoxyuridine (BrdUrd). The samples were analysed by bivariate DNA/BrdUrd flow cytometry. The primary purpose of the study was to measure the kinetic parameters of labelling index (LI), duration of S-phase (TS) and Tpot. Secondarily, tumour DNA ploidy (DNA index) and S-phase fractions (SPFs) were also estimated from the univariate DNA histograms. The 101 evaluable patients were classified according to clinical stage as T2 (n = 12), T3 (n = 53), T4 (n = 28) or recurrent tumours (n = 8). Of the evaluable tumours, 73 were DNA aneuploid. The median LI, TS, and Tpot of the aneuploid tumours were 21%, 20 h and 3.3 days respectively. The calculated LI, TS, and Tpot of diploid tumours were subject to uncertainties because of the contribution of normal cells. The LI and SPF of all tumours were, however, significantly (P < 0.001) correlated, having a correlation coefficient of only 0.76. The wide distributions of values for LI (quartiles 13.5%, 26.9%) and Tpot (quartiles 2.4, 5.6 days) that were found are necessary baseline information if these parameters are to be useful in individual treatment selection or as predictors of treatment outcome.

Adenocarcinoma↗
PubMed1995

[Clinical value of the potential doubling time (Tpot) measured by flow cytometry].

The potential doubling time (Tpot) is defined as the time necessary to double the number of proliferating tumor cells in the absence of spontaneous cell loss. Tpot is thought to be a better index of tumor proliferation than clinically observed tumour volume doubling time. The in vivo measurement of Tpot is a possible way to detect fast growing tumours which would be better controlled by accelerated radiotherapy. The published data demonstrate the feasibility and the safety of the technique. Large variations in Tpot values were observed in tumors with similar histology, indicating an accelerated proliferation rate in some tumors. There are technical difficulties related to intra-tumor heterogeneity, sample contamination by normal cells, and inter-laboratory variability, which question the biological interpretation of Tpot. Further studies are ongoing to establish whether the in vivo measurement of Tpot 1) provides information that is independent of the "classical" prognostic factors, and 2) allows the early recognition of the patients likely to benefit from accelerated treatment.

Cell Cycle↗
PubMed1994

Near-diploidy: a new prognostic factor for clinically localized prostate cancer treated with external beam radiation therapy.

BACKGROUND: DNA ploidy is a significant prognostic factor in patients with prostate cancer. Using DNA/nuclear protein flow cytometry, a subpopulation of tumors with near-diploid DNA is identifiable. The prognostic significance of near-diploidy was examined. METHODS: Paraffin-embedded formalin fixed prostate tumor tissue from patients treated at M. D. Anderson Cancer Center with external beam radiation therapy was processed for DNA/nuclear protein flow cytometry. All patients had pretreatment and follow-up serum prostate specific antigen (PSA) levels. Seventy-six specimens were suitable for flow cytometric analysis. Tumors were classified as either diploid (n = 30), near-diploid (n = 24), or nondiploid (n = 22, tetraploid and aneuploid). Median follow-up time was 36 months. RESULTS: Diploid tumors were associated with a significantly better actuarial outcome at 4 years, compared with near-diploid tumors, using either biochemical relapse (rising PSA) or a composite end point of a rising PSA or clinical relapse (16% versus 52% relapse, P < 0.05, log-rank). Moreover, patients who had nondiploid tumors had the worst prognosis (77% relapse, composite end point). No significant difference was observed between diploid and near-diploid neoplasms regarding actuarial local control, freedom from metastasis, freedom from clinical relapse, or overall survival time. A Cox proportional hazards model, using the composite end point of a rising PSA or relapse, was performed with ploidy categorized as diploid, near-diploid, and nondiploid; pretreatment PSA, DNA ploidy, and tumor grade were found to be independent prognostic factors. When ploidy was categorized as diploid or near-diploid (nondiploid tumors excluded), pretreatment serum PSA and DNA ploidy were independent predictors of outcome. Ploidy remained an independent prognostic factor even when nondiploid tumors were excluded. CONCLUSIONS: These data show that patients who have near-diploid tumors have an intermediate prognosis between the more favorable diploid tumors and the less favorable nondiploid tumors.

Acid Phosphatase↗
PubMed1994

Simultaneous cytokinetic measurement of aneuploid tumors and associated diploid cells following continuous labelling with chlorodeoxyuridine.

Cells from a murine tumor, MCa-K, were continuously labelled with the thymidine analogue chlorodeoxyuridine (CldUrd) and analyzed by bivariate flow cytometry in order to measure the growth fraction (GF) and potential doubling time (Tpot) of both the DNA-aneuploid tumor cells and the associated DNA-diploid cells. MCa-K has a DNA index of 1.7, rendering two, partially overlapping, populations observable with labelled and unlabelled cells in each population. The data from these tumors may be divided into three regions of differing DNA content, with one region containing a pure DNA-diploid population, a second region with both cell types, and a third region including only DNA-aneuploid cells. Equations are presented to characterize the fractions of labelled cells in each region as a function of labelling time and cell type, thereby permitting estimation of the proliferative properties of the populations. These equations include the possibility that DNA-aneuploid cells cease cycling both in G1 and in S phase to account for the observed numbers of unlabelled cells with S phase contents. The estimated value of Tpot of the DNA-diploid cells is 126.0 h with a GF of 42%, while that of the DNA-aneuploid cells is 36.9 h with a GF of 69%. It is also estimated that between 2% and 6% of all DNA-aneuploid cells starting DNA synthesis cease cycling, leading to 25% of the cells having an S-phase DNA content being noncycling.

Aneuploidy↗
PubMed1994

The prognostic significance of DNA ploidy in clinically localized prostate cancer treated with radiation therapy.

PURPOSE: To determine the prognostic significance of deoxyribonucleic acid (DNA) ploidy in comparison to pretreatment prostate specific antigen (PSA) and other prognostic factors for patients with adenocarcinoma of the prostate treated with external beam radiotherapy. METHODS AND MATERIALS: Paraffin-embedded prostatic adenocarcinoma material was obtained from patients treated from 1987-1991. Sufficient histologic material for flow cytometric DNA content analysis was obtained from 86 patients and adequate histograms were obtained from 76 of these. The DNA histogram profiles were classified as diploid, tetraploid, or aneuploid. Median patient follow-up was 36 months. RESULTS: There were 54 patients with diploid tumors, and 22 with nondiploid tumors (11 tetraploid and 11 aneuploid). Since the disease outcome for tetraploid and aneuploid tumors was the same, these were pooled (nondiploid tumors). The distribution of diploidy and nondiploidy correlated with pretreatment PSA (p < 0.0005) and grade (p = 0.055), but not with stage, pretreatment prostatic acid phosphatase, transurethral resection, pretreatment serum testosterone, or age. In actuarial univariate analyses, DNA ploidy was a significant predictor of outcome for local failure, distant metastases, any clinical relapse, rising PSA, and rising PSA and/or relapse. Ploidy was not a significant predictor of overall survival, although there were only six deaths. Diploidy predicted for improved outcome, for example, 34.6% incidence of a rising PSA and/or relapse at 4 years compared to 76.9% with nondiploidy (p < 0.0001). An actuarial univariate analysis of other potential prognostic factors using the composite endpoint of rising PSA and/or relapse also revealed pretreatment PSA, grade, pretreatment prostatic acid phosphatase, stage, and serum testosterone to be significant predictors of outcome. In Cox proportional hazards analysis, pretreatment PSA, DNA ploidy, and grade were the only independent prognostic factors for disease outcome using the composite endpoint. CONCLUSION: DNA ploidy is an independent predictor of outcome in patients with Stages T1-T3 prostate cancer treated with definitive external beam radiotherapy.

Adenocarcinoma↗
PubMed1994

Hormonal protection from procarbazine-induced testicular damage is selective for survival and recovery of stem spermatogonia.

Procarbazine produces long-term sterility in the male by killing stem spermatogonia. The degree and selectivity of protection of stem spermatogonia in rats from procarbazine by pretreatment with steroid hormones were investigated. Male LBNF1 rats were treated for 6 weeks with Silastic implants containing testosterone plus 17 beta-estradiol. The hormone-treated rats and sham-treated controls were given a single injection of graded doses of procarbazine and the hormone implants were removed the next day. Spermatogonial stem cell survival and function, assessed by the repopulation indices and sperm head counts 10 weeks later, showed that stem spermatogonia were protected by testosterone plus 17 beta-estradiol treatment from the toxic effects of procarbazine with a dose-modifying protection factor of about 2.5. In contrast, there was no hormonal protection from the procarbazine-induced killing of differentiating spermatogonia, preleptotene spermatocytes, and spermatocytes in meiotic prophase or from the delay in maturation of round spermatids, assessed 9 days after procarbazine injection by histological or flow cytometric methods. In addition, there was no hormonal protection from the procarbazine-induced decline in body weights and lymphocyte counts, indicating that the gastrointestinal, neurological, and hematological systems were not protected. The specificity of protection indicates that the hormonal protection of the stem spermatogonia is not the result of a systemic or overall testicular decrease in drug delivery, decrease in bioactivation, nor increase in drug detoxification, except possibly within the stem cells themselves. We conclude that the degree of hormonal protection and its specificity would be appropriate for clinical application provided that the mechanism of protection is elucidated and appears applicable to humans.

Animals↗
PubMed1994

Proliferation kinetics of recruited cells in a mouse mammary carcinoma.

Solid tumors contain populations of proliferating (P) and quiescent (Q) cells. Shifting between these populations occurs continuously and cells are recruited from quiescence to proliferate (Q-->P) as a result of exogenously applied or endogenous cell depleting stimuli. Direct measurements of the proliferation kinetics of these Q-->P cells in solid tumors are difficult to make because of the much larger percentage of P-cells. In order to specifically analyze the kinetics of the Q-->P cells, double thymidine analogue labeling was used. This was accomplished by first labeling in vivo all of the P-cells in MCaK tumors using continuous exposure to chlorodeoxyuridine (CldUrd) administered by a minipump over 21 h. About 75% of the aneuploid cells are P-cells based on CldUrd labeling. At different times after the pumps were removed, the tumors were pulse-labeled with iododeoxyuridine (IdUrd) and harvested 6 h later. A 3-color flow cytometry assay was used to simultaneously and independently analyze CldUrd and IdUrd incorporation, as well as DNA content. The Q-->P cells were identified as having only been labeled with IdUrd. The length of their S-phase was calculated from the movement of the Q-->P cells during the 6 h after IdUrd labeling. The results showed the length of S-phase for the recruited cells to be slightly, but significantly, longer than the length of S-phase for the total cells (11 h versus 9 h, respectively). Thus, the recruited cells appear to have slightly slower kinetics than the proliferating cells in the absence of a perturbing stimulus such as radiotherapy or chemotherapy.

Animals↗
PubMed1994

Relationship of tumor DNA-ploidy to serum prostate-specific antigen doubling time after radiotherapy for prostate cancer.

OBJECTIVES: DNA-ploidy is a strong prognostic factor for prostate cancer patients treated with definitive external beam radiotherapy. Using DNA/nuclear protein flow cytometry, three prognostic groups based on DNA-ploidy were identified: from good to poor, these are diploid, near-diploid, and nondiploid tumors. Since recent evidence indicates that the rate at which prostate-specific antigen (PSA) increases in the presence of biochemical failure is predictive of the time to clinical relapse, we examined the relationship between DNA-ploidy and PSA doubling time (PSA-DT). METHODS: Formalin-fixed paraffin-embedded tissues from 76 patients treated at M.D. Anderson Cancer Center with definitive radiotherapy alone were analyzed for ploidy using DNA/nuclear protein flow cytometry. Of these, 24 of the 27 patients with a rising PSA profile had three or more post-treatment PSA values from which the PSA-DTs were calculated. PSA-DTs were estimated using nonlinear regression techniques. RESULTS: The average PSA-DT for the 24 patients in this cohort was 11.3 +/- 10.5 months (+/- SD) with a median of 8.4 months. Diploidy (n = 3) was associated with a PSA-DT of 27.0 +/- 22.8 months, near-diploidy (n = 7) with a PSA-DT of 12.2 +/- 5.7 months, and non-diploidy (n = 14) with a PSA-DT of 7.5 +/- 5.7 months (p = 0.004, Spearman rank test). Stage, grade, and pretreatment PSA, as well as the endpoints of local control, freedom from metastases, and freedom from any relapse, did not correlate significantly with PSA-DT values. However, when patients were subdivided by PSA-DT into those with values 10 months or less (n = 14) and those more than 10 months (n = 10), there was a correlation with 3-year actuarial freedom from relapse: 28% and 74%, respectively (p < 0.01, log-rank). This subdivision of PSA-DT also correlated with DNA-ploidy (p = 0.03, chi-square) and stage (p = 0.04). CONCLUSIONS: The results show that there is a significant correlation of DNA-ploidy with PSA-DT. Diploidy was associated with the longest PSA-DTs, near-diploidy with intermediate PSA-DTs, and nondiploidy with short PSA-DTs. Patients with short PSA-DTs also had significantly higher actuarial rates of disease relapse at 3 years. These data confirm that PSA-DT is a strong predictor of tumor behavior and that patients who have nondiploid tumors probably require more aggressive, combined modality, treatment.

Actuarial Analysis↗
PubMed1994

Double labelling to obtain S phase subpopulations: application to determine cell kinetics of diploid cells in an aneuploid tumour.

We studied the cell kinetics of the murine mammary carcinoma MCa-K using iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) given at different times as independently detectable labels of S phase cells. The presence of IdUrd and CldUrd, and the amount of DNA were measured by three-colour flow cytometry making it possible to define three subpopulations within S phase and to measure the progression through the cell cycle during the time following labelling. In DNA histograms of these subpopulations, the diploid and aneuploid cells (which had a DNA index of 1.7) are essentially completely separated. From appropriate combinations of cells labelled with IdUrd only, CldUrd only, or both, it was possible to construct separate DNA distributions for the labelled diploid and aneuploid cells at the times of administration of each label. The kinetics of the diploid and aneuploid cells could be calculated for individual tumours from these two time points without having to make corrections for the presence of the second population. The diploid and aneuploid populations had indistinguishable S and G2 + M phase durations, T(S) and T(G2 + M), of about 9 and 2 h; however, the potential doubling time values for the aneuploid and diploid populations were 30.2 and 101.2 h respectively.

Aneuploidy↗
PubMed1994

Relationships between DNA damage and the survival of murine bone marrow cells irradiated in situ.

The relationships between DNA damage and the survival of murine bone marrow cells irradiated in situ were examined. Cell survival was assayed by the ability of bone marrow cells from irradiated mice to form colonies in vitro (CFU-C). DNA double-strand breaks (DSBs) were measured by neutral (nondenaturing) filter elution and pulsed-field gel electrophoresis (PFGE). Double-strand breaks were measured in the proliferating bone marrow cells, identified by injecting the mice with [3H]dThd at various times before gamma irradiation, as a model of the behavior of the radiosensitive target cells. To assess how the DNA lesions measured using these techniques correlated with cell killing, the effect of the radioprotective agent WR-2721 on the induction of DSBs in proliferating bone marrow cells was compared with its effect on CFU-C survival. WR-2721 protected against the killing of both granulocyte-macrophage and erythroid burst-forming CFU-C by a factor of about 2. In contrast, little (1.2-fold) protection was observed in the PFGE assay at radiation doses between 5 and 20 Gy. Similarly, at the lowest dose studied (5 Gy) there was little protection against DSBs as measured by neutral elution; only after doses of between 10 and 30 Gy was significant protection observed. Thus the previously reported predictive relationship between DSBs and cell survival in vitro does not appear to extend directly to murine bone marrow cells irradiated in vivo.

Amifostine↗
PubMed1993

Calculating potential doubling time using monoclonal antibodies specific for two halogenated thymidine analogues.

PURPOSE: A new flow cytometric technique that allows for two-incorporated thymidine analogues to be measured simultaneously and independently has been used to improve the accuracy of in vivo cell kinetic estimates, i.e., the length of S-phase (TS) and potential doubling time (Tpot). METHODS AND MATERIALS: The analogues chlorodeoxyuridine and iododeoxyuridine were injected at different times into mice bearing the mouse mammary tumor MCaK. At different times after labeling, the tumors were harvested and prepared for three color flow cytometric analysis of DNA, chlorodeoxyuridine, and iododeoxyuridine. Control experiments showed that similar estimates of Tpot were obtained from each label when administered singly, or as staggered pulses. Comparisons were made between TS and Tpot calculated from a single label (single point), from the averaged result of the two labels from the same tumor (two point-ave), and from the simultaneous nonlinear fitting of the measured parameters from the two labels, from the same tumor (two point-fit). These estimates of TS and Tpot were then compared to reference values obtained by fitting the pooled measured parameters from all the tumors, that were labeled for different periods of time. RESULTS: While all of the methods resulted in similar mean estimates of TS and Tpot that were close to the reference values, the fewest assumptions, and the least variability in the results, were obtained using the two point-fit data. CONCLUSION: The estimation of Tpot using two thymidine analogues is more accurate than that obtained from a single label.

Animals↗
PubMed1993

Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo.

A technique was developed for the staining of nuclei for DNA using propidium iodide, and incorporated chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd) using two monoclonal antibodies that showed negligible cross-reactivity. The mouse mammary solid tumor MCaK was labeled in vivo by intraperitoneal injection of the nucleosides. Tumor cell nuclei were stained after isolation from ethanol-fixed solid tumor tissue and acid denaturation. The Br3 antibody, which specifically recognizes CldUrd, was applied first, followed by indirect staining with goat anti-mouse phycoerythrin. The direct fluorescein isothiocyanate conjugate of the B44 antibody, which specifically recognizes IdUrd, was then applied. In the direct conjugate form this antibody reacted only minimally with CldUrd. The nuclei were then stained with propidium iodide. With this dye combination the coefficients of variations of the DNA histograms were consistently in the 2-4% range. Two other dye combinations were compared. The propidium iodide/phycoerythrin/fluorescein isothiocyanate dye combination was the simplest because of the compatibility with single laser flow cytometry.

Animals↗
PubMed1993

Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.

Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4 degrees C for 24-48 h before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

Animals↗
PubMed1993

Influence of intracellular thiol and polyamine levels on radioprotection by aminothiols.

We examined the effect of manipulating the levels of two endogenous radioprotectors, glutathione (GSH) and polyamines, on the ability of exogenous aminothiols to protect Chinese hamster ovary cells from the lethal effects of gamma-radiation. Treatment with 0.5 mmol dm-3 buthionine sulfoximine (BSO) for 24 h depleted GSH levels to < 1% of control and significantly sensitized the cells to irradiation in air. Undepleted control cells were protected by WR-1065 (4 mmol dm-3; 30-min preirradiation treatment at 37 degrees C) by 2.09-fold (range 1.98-2.21) at the 10% survival level, whereas BSO-treated cells were protected by a factor of 1.98 (range 1.95-2.14) at this survival level. Thus, GSH depletion had no significant effect on the radioprotective capacity of WR-1065. Treating cells with 1 mmol dm-3 alpha-difluoromethyl ornithine (DFMO) for 48 h depleted the polyamines putrescine and spermidine to very low levels, while spermine was not significantly depleted. DFMO also sensitized cells to aerobic irradiation. WR-1065 protected DFMO-treated cells by 2.29-fold (range 2.08-2.53), whereas undepleted control cells were protected by 2.09-fold (range 1.98-2.21) at the 10% survival level. Thus, WR-1065 appeared to offset the radiosensitizing effect of the DFMO treatment. Cysteamine, on the other hand, protected control and DFMO-treated cells to the same extent. We also examined the effect of combinations of exogenous thiols on radiosensitivity. Cells were treated with WR-1065 (4 mmol dm-3) for 30 min and then with increasing concentrations of dithiothreitol for 5 min prior to irradiation. The protective effects of these two thiols were simply additive.

Animals↗