DNA Histogram Analysis Research Articles

フローサイトメトリーによる脳腫瘍細胞の生長解析と抗癌剤感受性試験への応用

Flow cytometry (FCM) has been used widely for clinical diagnosis and evaluation of chemotherapy. The author has conducted monolayer-culture of brain tumor cells and reported the changes of DNA-histograms of brain tumor cells for antineoplastic agents. In this paper, a new application of FCM was established to evaluate the choice of the best antineoplastic agent in chemotherapy for malignat brain tumor.After adjusting cell preparation and eva luating the analytic system of FCM, the sensitivity test to antineoplastic agents was developed through preliminary studies using the established brain tumor cell line. Antineoplastic agents were challenged to the cells in monolayer-culture, and the changes of DNA-histograms as well as cell viability were analyzed by FCM. Cell viability was measured by the FDA (Fluorescein Diacetate) vital staining method, and propidium iodide was used for the analysis of DNA-histograms. Judgment of the best agent was conducted, based on its effects on the changes of their cell kinetics.Cytocydal and cytostatic effects were evaluated quantita tively, and the results showed a well coincidence with the sensitivity of the cells to the appropriate antineoplastic agent.Secondly, the above system was applied to the clinical cases of 15 malignant b rain tumors. The best antineoplastic agent was selected for each case through this method and good therapeutic effects were demonstrated. The present results suggest that the above method is of much clinical value.

Inhibition of DNA Synthesis by a Small-Cell Lung Carcinoma-Derived Protein

A tumor-derived factor that inhibits cellular DNA synthesis was identified. The factor was extractable from a small-cell lung carcinoma cell line grown in either chemically defined medium or nu/nu mice and inhibited tritiated thymidine ([3H]dThd) incorporation by tumor cell lines of autologous, allogeneic, and xenogeneic origins. The viability of nonproliferating cells from normal tissue was not affected. Tumor extract inhibitory activity was trypsin labile but was resistant to other proteases, neuraminidase, lipase, DNase, RNase, glucosidase, extremes of pH-temperature, and reducing conditions. Inhibitory activity was reversibly bound to helix pomatia lectin but not to lentil, wheat germ, or concanavalin A lectins. Purification by size-exclusion high-performance liquid chromatography yielded a bioactive unimodal 12-kilodalton (kd) peak. The bioactive 12-kd moiety could be eluted from sodium dodecyl sulfate-polyacrylamide gels. Redosing of populations of the T-lymphoblastoid cell line CEM achieved an early (24 hr) sustained depression of pulse [3H]dThd incorporation and ultimately led to decreased population density of factor-treated populations. DNA histogram analysis demonstrated no change in cell cycle phase distribution after factor treatment. 5-Bromo-2'-deoxyuridine (BrdUrd) vs. propidium iodide with the two-parameter Fluorescence-Activated Cell Sorter analysis showed relative inhibition of non-S-phase BrdUrd uptake at 24 hours. A cell-free DNA polymerase assay demonstrated significant inhibition of non-alpha-polymerase-associated DNA synthesis in factor-treated cells. These studies suggest that this tumor-derived inhibitor of DNA synthesis represents a class of cellular products involved in the autoregulation of growth by regulation of DNA synthetic activity.

Flow cytometric analysis of tetanus toxin binding to neuroblastoma cells.

A neuroblastoma cell line was assessed for its capacity to bind tetanus toxin (TT) by using immunofluorescence and flow cytometry to analyze cells on a single cell basis. A clone of Neuro 2a, N2AB-1, was shown to bind variable amounts of TT per cell and this binding could be saturated by increasing doses of the toxin. Toxin binding was specific for neuronal cells, as the non-neuronal cell line, C6 glioma, bound negligible amounts of toxin. Variability of immunofluorescence staining was due in part to the increase in size of N2AB-1 cells as they progress through the cell cycle as measured by cell surface densities of toxin binding and DNA levels by propidium iodide (PI) staining. When N2AB-1 cells were treated with exogenous gangliosides for 24 h, cells were induced to sprout neurites and cell growth was inhibited. Analysis of DNA histograms indicated that ganglioside treatment caused more cells to appear in G0G1 of the cell cycle than that seen for untreated controls. Upon cytometric analysis of TT binding to ganglioside treated cells, it was apparent that treatment stimulated all cells to bind TT in larger amounts per cell than that seen with untreated N2AB-1 cells. These data suggest that TT binding and, therefore, toxin receptors are constant in density throughout the cell cycle of these neuroblastoma cells and that exogenous gangliosides can cause differentiation followed by increased toxin binding.

Differences between labeling index and DNA histograms in assessing S-phase cells from a homogeneous group of chronic phase CML patients.

The reliability of DNA histogram analysis in accurately estimating S-phase cells from human tumors was tested by comparing the results to those of simultaneously obtained tritiated thymidine labeling index (LI) studies. Patients with chronic myelocytic leukemia (CML) during chronic phase were selected for study because the Philadelphia chromosome (Ph) was the only cytogenetic abnormality in each case and, since it is a balanced translocation, the frequently encountered problem of aneuploidy in human neoplastic cells was avoided. Unfortunately, when 30 CML patients were studied simultaneously by DNA histogram analysis and LI studies, the correlation coefficient between the two results was only r = 0.611. A comparison of three different mathematical programs for DNA histogram analysis showed that none was completely satisfactory. We conclude that DNA histogram analysis does not provide the same data as autoradiographically processed labeling index studies even in patients with Ph-positive CML during the chronic phase when the situation is not complicated by additional aneuploidy.

Identification of the Flow Cytometry System for DNA Histogram Analysis Purposes

The maturity of a proliferating cell is expressed by the amount of DNA it contains. To determine the maturity distribution of a cell population DNA can be labeled with a fluorescent dye. By using a flow cytometer (FCM) a DNA histogram - frequency vs fluorescence intensity - can be made. A mathematical model was developed to obtain the input and transfer functions of the FCM from the observed, noise polluted output. A priori knowledge about some properties of the input and the nature of the FCM system was available. To quantify the model parameters a Gauss-Newton optimization algorithm was used in the Maximum Likelihood method, yielding error estimates as well. Results were satisfactory.

Lack of relationship between in vitro cell measurements and response to busulfan in chronic myelocytic leukemia.

Twenty-three patients with chronic-phase CML have been treated with intermittent courses of busulfan to determine if the duration of unmaintained remission becomes progressively shorter with successive courses of therapy and to determine whether there was a relationship between cluster and colony formation, suicide index of CFUc, labeling index, percentage of cells in S-phase (as determined by cytofluorographic DNA histogram analysis), in vitro sensitivity of the CFUc to busulfan, and response to busulfan therapy. Serial studies in individual patients and the group as a whole revealed no relationship between changes in the cellular parameters described above and response to busulfan. The white blood cell (WBC) doubling time with serial courses of busulfan in individual patients did not always progressively decrease as has been previously reported. In vitro studies of CFUc in chronic-phase CML appear to be of no value in predicting response to busulfan.

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations.

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.

High resolution method for the analysis of DNA histograms that is suitable for the detection of multiple aneuploid G1 peaks in clinical samples.

The DNA histogram obtained by flow cytometry can be considered as the product of an "ideal" measurement column vector and a measurement distortion matrix. In order to extract the ideal histogram from the real data, the measurement distortion matrix is commonly presumed to be a family of Gaussian coefficients that are centered on the diagonal. We have designed a feedback-controlled curve-fitting procedure that reconstructs the ideal histogram from the real data through successive iterations. The optimum coefficient of variation (cv) for the family of Gaussians in the measurement distortion matrix is determined from an analysis of the sums of squares of fits of the computed DNA histogram to the real data over an appropriate range of trial cv. Since this method assigns a Gaussian to each and every data channel, it permits the resolution of closely spaced multiple aneuploid G1 peaks in clinical samples. The effects of high frequency noise that may be present in the data can be attenuated by multiplying the real data histogram by a Gaussian matrix with cv close to but smaller than that of the measurement distortion matrix.

Sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) of glioma cells in vivo and in vitro.

A new water-soluble nitrosourea (ACNU) was tested for its antitumor activity against four glioma cell lines. Four factors were studied to determine its antitumor activity: inhibition of cell growth, morphologic observation, analysis of DNA histogram with flow microfluorometry, and sensitivity testing with microtest plate. Growth inhibition in response to ACNU was seen in two cell lines (EA285, U251-MG), whereas two cell lines (YE2-02, T98) were resistant to ACNU. The results of the present sensitivity test concur with those of other examinations that this test is useful in selecting a drug and determining the effective dose.

Flow Cytometric Analysis of the Response of the R3327-G Rat Prostatic Adenocarcinoma to Endocrine Manipulation

The technique of flow cytometric DNA histogram analysis (FCM) shows there to be two distinct cell populations (diploid vs aneuploid) in the poorly differentiated R3327-G rat prostatic adenocarcinoma. The following study compares tumor weight measurements with several FCM computer-based methods designed to determine rapidly the proliferative status of tumors. Hypophysectomy, bilateral adrenalectomy, orchiectomy, sham operations, or diethyl-stilbestrol treatments were initiated when the tumors were palpable (day 21) and continued until the tumors were excised (day 52). Hypophysectomy, orchiectomy, adrenalectomy, and diethylstilbestrol treatments all resulted in significant inhibition by tumor weight. Quantitation of the percentage of mid-S phase aneuploid cells by summation gave the best correlation with tumor weight. Tumors grown in hypophysectomized, orchiectomized, adrenalectomized, or diethylstilbestrol-treated animals showed a significant reduction in the proportion of mid-S phase cells as compared with controls. The calculation of the percentage of all aneuploid cells was significantly reduced in hypophysectomy, orchiectomy, and diethylstilbestrol-treated animals. However, tumors grown in adrenalectomized animals were not significantly different from controls by this method. Adrenalectomy was found to be the least effective form of therapy, and this was reflected in all of the parameters measured. These data show that FCM analysis may be useful in the quantitation of prostatic carcinoma response to therapy.

An improved sum-of-normals technique for cell cycle distribution analysis of flow cytometric DNA histograms.

An improved method for determining the fraction of cells in the G1, S and G2 + M phases of the life cycle, from DNA distributions of S phase rich cultures, is presented. Picolinic acid synchronized cell cultures were used to demonstrate the usefulness of the technique for the cell cycle distribution analysis. The algorithm presented quantifies DNA histograms from FCM analyses using an extension of the previously reported 'sum of discrete normal curves' technique. It alleviates problems encountered with earlier methods by extending the contribution of the S phase cells to the distribution under the G1 and G2 + M peaks; at the same time it allows more of the descriptive parameters to be determined from the individual histograms. The fractions of cells in the G1, S, and G2 + M phases of the life cycle obtained by this method compare favourably with autoradiographic analyses and other analytical results.

Flow cytometric analysis of the response of the R3327-G rat prostatic adenocarcinoma to endocrine manipulation.

The technique of flow cytometric DNA histogram analysis (FCM) shows there to be two distinct cell populations (diploid vs aneuploid) in the poorly differentiated R3327-G rat prostatic adenocarcinoma. The following study compares tumor weight measurements with several FCM computer-based methods designed to determine rapidly the proliferative status of tumors. Hypophysectomy, bilateral adrenalectomy, orchiectomy, sham operations, or diethylstilbestrol treatments were initiated when the tumors were palpable (day 21) and continued until the tumors were excised (day 52). Hypophysectomy, orchiectomy, adrenalectomy, and diethylstilbestrol treatments all resulted in significant inhibition by tumor weight. Quantitation of the percentage of mid-S phase aneuploid cells by summation gave the best correlation with tumor weight. Tumors grown in hypophysectomized, orchiectomized, adrenalectomized, or diethylstilbestrol-treated animals showed a significant reduction in the proportion of mid-S phase cells as compared with controls. The calculation of the percentage of all aneuploid cells was significantly reduced in hypophysectomy, orchiectomy, and diethylstilbestrol-treated animals. However, tumors grown in adrenalectomized animals were not significantly different from controls by this method. Adrenalectomy was found to be the least effective form of therapy, and this was reflected in all of the parameters measured. These data show that FCM analysis may be useful in the quantitation of prostatic carcinoma response to therapy.

A rabbit bone marrow model system for evaluation of cytotoxicity: characterization of normal bone marrow cell cycle parameters by flow cytometry.

Characterization of a rabbit model system for the study of cell cycle effects of myelotoxic agents in normal bone marrow is described. Cell cycle phase distributions are obtained by computer analysis of flow cytometric single cell DNA histograms. Comparison of marrow aspirates with marrow samples from sacrificed animals indicates that dilution of aspirates with peripheral blood is not significant. Aspiration of marrow from one bone does not affect the cell cycle distribution of unsampled bones. Hence, sequential aspirates of different bones in a single animal may be used as representative samples for further study of effects of myelotoxins on marrow proliferation and differentiation.

Quantitative analysis of cell cycle progression of synchronous cells by flow cytometry

Chinese hamster ovary (CHO) cells were synchronized by the mitotic selection procedure and their subsequent progression through the cell cycle was monitored by flow cytometry. It is shown that our previously proposed method for DNA histogram analysis is applicable even to highly synchronous cell populations lacking significant G1 and G2/M components, provided an appropriate control is used as a reference standard. We have also obtained estimates of cell cycle parameters from the DNA histograms in this system, including phase durations, relative cellular DNA synthesis rates as a function of position in S, and synchrony indices. In particular, the rate of DNA synthesis appears to be very low in early S phase in this cell line, giving rise to an unexpected early S phase peak in the histogram of asynchronous, exponentially growing cells.

Multi-user system for analysis of data from flow cytometry

A new program is described for the analysis of DNA histograms from flow cytometry. The fundamental model representing the cell population is similar to one described previously. It assumes the population is grouped into compartments, each consisting of cells having approximately the same DNA content. After staining the cells with an appropriate fluorochrome, the fluorescence distribution of cells within each compartment is assumed to be Gaussian. In the present algorithm, the parameters of the model can either be computed directly by the program from the data, or can be specified as input by the user. When synchronous cell populations lacking distinct G1 and G2/M phases are analyzed, the parameter values must first be obtained using an appropriate control. Percentages of cells in the various compartments are computed using a gradient search method described by Bevington.

Radiosensitivity and recovery of mouse L cells during the cell cycle.

Mouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age response functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D0 and extrapolation number n were found to vary during the cell cycle. The D0 has a maximum value of 176 +/- 1 rad in the middle of G1 phase and minimum of 71 +/- 1 rad at the S/G2 transition, while the extrapolation number is rather constant from the beginning of G1 phase (1.9 +/- 0.1) to the middle of S phase (2.3 +/- 0.1) and reaches a steep maximum of 9.3 +/- 1.1 at S/G2 transition. The values of n in the various phases of cell cycle are compared with the respective values of the recovery factor gamma determined after fractionated irradiation. --Cell survival after a single dose of 616 rad has minima for irradiation at G1/S transition and in early G2 phase; the survival in early G2 being about 40 times smaller than in early G1 phase. Implications for a cell cycle specific therapy are discussed.

DNA histogram analysis of human hemopoietic cells

The proliferative activity of human neoplasms may be an important determinant for therapeutic management. The advent of automated flow- through systems measuring cellular DNA content by means of fluorescence has considerably facilitated the analysis of cellular kinetics. Using a pulse cytophotometer ICP-11 (Phywe Co., Gottingen, Germany), three different fluorescent staining techniques for DNA histogram measurement on human hemopoietic cells were tested: mithramycin, ethidium bromide, and a combination of ethidium bromide and mithramycin. Employing the tritiated thymidine labeling index as reference standard for comparison with the DNA histogram-derived S-phase fractions, linear correlations were obtained using ethidium bromide alone and ethidium bromide in combination with mithramycin as staining techniques. The fluorescence intensity was increased fourfold to fivefold by the use of the two-dye combination, resulting in a substantial decrease in the coefficient of variation of DNA histograms to 1.5%-2%. This augmented histogram resolution is an important codition for detecting small-degree numeric chromosomal aberrations and discrete drug perturbation effects.

Zur Analyse von DNA-Histogrammen durch Computer

Introduction Quantitative DNA cytophotometry, the study of morphology of chromosomes and cell kinetics are important approaches toward characterization of genetic information. Each approach has its own problems and limits. Difficulties with interpretation of DNA histograms arise from a lack of a proper terminology, and from attempting to interpret them in terms of the existing terminologies for chromosomal analysis and cell kinetics. Aim of this research was to develop a computerized mathematical analysis of DNA histograms. Materials and methods As a basis for the comparison of normal with those of tumor cell populations the DNA histograms of heart muscle cells were selected for the normal cell population from 14 normal hearts of adults, 16 hypertrophic hearts, 14 non-polyploidized hearts of children, and 14 hypertrophic hearts of children with congenital malformations. Normal diploid cell populations from 7 effusions were also included. The 24 populations of malignant cells consisted of primary tumors and metastatic effusions. DNA cytophotometry was performed on single nuclei in Feulgen stained preparations by means of the UMSP/XD 50 ZEISS. The approximation of the DNA histograms by linear combination of normal distributions was done according to spline-function and calculated by means of the IBM-375. Results The nuclear classes 2C, 4C, and 8C show no differences between the normal, left and right heart with respect to mean values (x), standard deviations (s x), variances (s x) and coefficients of variance (s x/x). However, coefficients of variance are smaller in hypertrophic (2.15 to 8.37%) than in normal hearts of adults (8.90 to 10.85%), and larger in hypertrophic hearts of children (8.01 to 15.88%) than in non-polyploidized normal hearts of children (4.06 to 7.09%) The mean values of the DNA classes 2C, 4C, 8C, and 16C vary within ± 18.6% with a probability of 95.5%. Benign effusions contain only 2C and 4C nuclei with a variance of 4.00% and 8.75%, respectively. In DNA histograms of malignant cells, only one third has a first peak outside of 2C ± 18.6%. In approximately one fifth of the histograms the position of the second or third peaks deviates significantly from normal polyploid values. Since a large proportion of poly-ploid nuclei is limited to only a few normal tissues, pronounced polyploidy is suggestive of malignancy in all other tissues. If in the cases containing only two DNA classes, 2C and 4C, the populations are malignant, the proportion of 4C is more than 8% while in the corresponding benign populations the proportion of 4C attains only 2.97 ± 2,5%. In some cases a few highly polyploidized nuclei not taken into a account by our computer program are suggestive of malignancy. In only one DNA histogram out of the 24 analysed, all of these criteria are negative. In six cases the computer analyses reveal two stemlines of tumor cells with corresponding polyploid values.