The effects of colcemid on hematopoiesis in the mouse.

Colcemid was found to induce a dose and schedule dependent marrow magakaryocytosis and peripheral thrombocytosis. The response could be divided into early and late components. The early component appears to have been due to a direct stimulatory effect, probably by enhancement of endoreduplication in metaphase arrested megakaryocyte precursors. The ealy stimulatory response was blunted on toxic drug schedules. In contrast, the late component of the thrombopoietic response was demonstrated best on the most toxic drug schedules. It coincided temporally with the reactive restoration of the mononuclear marrow and blood cell elements, respectively. Thus, the late component appears to be a nonspecific rebound phenomenon. On comparing the thrombopoietic properties of Colcemid with those of the vinca alkaloids in experimental systems, the former appears to have a more favorable therapeutic index. The data suggest that colchicine and its derivatives may be useful agents in the treatment of clinical thrombocytopenic states.

Dual parameter flow cytometry studies in human lymphomas.

Dual parameter flow cytometry studies using Coulter volume and cell DNA content were carried out in monodisperse cell suspensions of 64 samples of human lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and benign lymphoid proliferations. Differences in mean Coulter volume among the lymphomas were due both to the intrinsic differences in mean G1 cell Coulter volume and to the presence of increased fractions of larger S and G2 cells, especially among the large B cell lymphomas. However, the relative contribution of large non-G1 cells to the overall population Coulter volume distribution was a relatively minor one; the presence of cells in S did not increase mean Coulter volume by more than 10%, even in samples with high S fractions. There was a good correlation between mean G1 cell Coulter volume and the log of the fraction of cells in S among the B cell lymphomas (r = 0.55). Evidence is presented that within individual samples, large cells proliferate more rapidly than small cells. This was seen in every case, both in the normal samples and in the lymphomas, and in the T cell lymphomas as well as in the B cell lymphomas. Aneuploidy was detected by flow cytometry in 11 cases; in 7 cases the aneuploid cell component could be analyzed separately from the diploid cell component on the basis of cell Coulter volume differences. The aneuploid components of diploid-aneuploid mixtures had higher S fractions than the diploid components in six of seven cases (0.16 +/- 0.04 [SE] vs. 0.08 +/- 0.02). These findings are considered in relation to the histopathological classification of the lymphomas, and in relation to the concept of clonal selection and clonal evolution of tumors.

The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases.

Dual parameter flow cytometry studies (cell DNA content and electronic cell volume) were performed in 220 cases of non-Hodgkin's lymphoma. All cases were characterized as B or T cell malignancies, based on immunologic surface marker characteristics. Aneuploidy by flow cytometry was more common among the B cell lymphomas than among the T cell lymphomas, and was most common among the large B cell lymphomas and B cell lymphomas of intermediate size. Ploidy index distributions showed a prominent hyperdiploid peak, as well as tumor cell populations with near-tetraploid DNA contents. In serial studies, a decrease in ploidy index was observed in association with clinical and histologic transformation in one case. The highest S fractions were observed among the large and intermediate B cell lymphomas and among the aggressive T cell lymphomas. In clinical samples consisting of mixtures of diploid and aneuploid populations, the data on the aneuploid components could often be separated from other components of the mixture in multiparameter studies on the basis of the larger electronic cell volumes of the aneuploid cells. In each case, the aneuploid large cell component almost invariably had a higher S fraction than the residual component(s) of the mixture. Overall, the data are consistent with a model of clonal selection and clonal evolution in the lymphomas in which early cytogenetic abnormalities that involve little or no change in total cell DNA content are followed by cell tetraploidization that is associated with cytogenetic instability and chromosome loss over the course of time.

The radioautographic transfer function and its implications for radioautographic methodology.

A function was developed to relate the radioautographic detectability of tritiated thymidine to its incorporation into nuclear DNA under various experimental and specimen processing conditions. The implications of this function for the interpretation of cell kinetic studies were outlined, and its implications for radioautographic methodology were discussed. The standardization of radioautographic methods for facilitation of comparisons of data obtained from different sources under dissimilar experimental conditions was discussed.

Rapid grain and cell counting for cell kinetic studies.

A commercially available bacterial colony counter has been adapted for the counting of radioautographic grains over individual cells in smears and for the counting of cells in histologic sections. For radioautographic grains, the correlation coefficients between counts obtained visually by 2 observers and between counts obtained visually and with the use of the instrument were similar (r=0.999 and r=0.998, respectively). The instrument counts were obtained more rapidly than the visual counts and were associated with less observer fatigue. Even though the performance of the instrument in counting cells in mouse bone marrow sections was less accurate than that in counting radioautographic grains, a good estimation of marrow cell number was obtained (r=0.968). Data on bone marrow cellularity were obtained far more rapidly than those with semiquantitative methods.

Lethal and sublethal effects of hydroxyurea in relation to drug concentration and duration of drug exposure in sarcoma 180 in vitro.

The lethal and sublethal effects of hydroxyurea were studied in Sarcoma 180 in vitro in relation to drug concentration and drug exposure duration using cloning methods, radioautography, and flow microfluorometry. It was shown that postperturbation changes in radioautographic labeling intensity reflected real changes in the rate of DNA synthesis in individual cells. The data suggest that both the lethal and sublethal effects of hydroxyurea are dependent on the rate of DNA synthesis. These findings have important implications for the interpretation of DNA content distributions under perturbation conditions and for the development of drug treatment regimens that are based on cell kinetics.

The T-lymphocyte as a diploid reference standard for flow cytometry.

T-lymphocytes prepared from the peripheral blood of normal individuals exhibit uniform postmitotic DNA contents that are constant from individual to individual when fixed in ethanol and stained with mithramycin, or when fixed in formalin and stained with acriflavine-Feulgen and analyzed by flow microfluorometry. T-lymphocytes fixed by either method are stable on storage for several months. These cells are suitable for use as an external diploid reference standard for flow cytometric detection of cells with abnormalities in postmitotic content exceeding 5% of diploid reference. Cells with abnormalities in postmitotic DNA content of less than 5% can be detected in mixtures containing both normal and malignant cells by multiparameter analysis.

Schedule-dependent synergism of combinations of hydroxyurea with adriamycin and 1-beta-D-arabinofuranosylcytosine with adriamycin.

The lethal effects of combinations of adriamycin (ADR) and either hydroxyurea (HU) or 1-beta-d-arabinofuranosylcytosine (ara-C) were studied in relation to drug-scheduling interval in Sarcoma 180 cells grown in vitro. Drug lethality was determined by cell cloning in soft agar. Serial kinetic changes induced by a priming dose of HU or ara-C were monitored by flow cytometry and related to schedule-dependent cell killing by ADR. All drug exposures were for 1 hr. When ADR was given together with either HU or ara-C, cell log kill was additive. However, when ADR was given after exposure to either HU or ara-C, cell killing was increased up to 200- to 500-fold, respectively. Maximum schedule-dependent synergism was observed at a drug-scheduling interval of 2 hr; schedule-dependent synergism decreased as the interval between drugs was increased beyond 2 hr. Schedule-dependent synergism was not observed when the same drug combinations were given in reversed order. Drug-induced changes in the DNA histogram were not seen until 5 hr after HU exposure and 8 hr after ara-C exposure. Thus, the schedule-dependent synergism between ADR and either HU or ara-C cannot be explained by cell cycle blockade with synchronization of cells in S phase.

Kinetic effects of sangivamycin in sarcoma 180 in vitro.

The lethal and sublethal effects of sangivamycin (SGM) were studied in sarcoma 180 in vitro in relation to drug concentration and duration of drug exposure. SGM lethality was found to be dependent on both drug concentration and duration of drug exposure. Pronounced effects on cell survival were observed only when SGM exposure was prolonged; with prolonged drug exposure, small increments in SGM concentration resulted in large increases in cell killing. Log-phase cells were more susceptible to the lethal effects of SGM than were early-plateau-phase cells. Measurements of incorporation of [3H]thymidine and [3H]uridine into the acid-insoluble cell fraction demonstrated inhibition of both DNA and RNA synthesis by SGM which was also dependent on drug concentration and duration of drug exposure, reflecting the lethality characteristics of SGM. As SGM concentration was increased, DNA synthesis was inhibited more rapidly than was RNA synthesis. Flow cytometry demonstrated a concentration- and time-dependent accumulation of cells in the late S and G2-M region of the DNA histogram. Our findings indicate that maximum lethality is obtained by prolongation of SGM exposure, and they suggest that pharmacokinetic studies may be important for determining regimens which provide such exposure in humans.

Schedule dependence of vincristine lethality in Sarcoma 180 cells following partial synchronization with hydroxyurea.

The effects of exposure to 0.1, 0.5, or 2 microM vincristine for 4 hr were studied in Sarcoma 180 cells at various times after synchronization with 5 mM hydroxyurea for 1 hr. Maximum sensitivity to the lethal effects of vincristine was observed at 10 to 14 hr after hydroxyurea exposure at the higher vincristine concentrations, compared to a period of a maximum sensitivity to a second dose of hydroxyurea at 8 to 12 hr. Serial flow cytometry studies indicated that the apparent decrease in sensitivity to vincristine at 14 to 18 hr was due to the division of cells in the leading segment of the synchronized wave and their entry into the relatively resistant G1 phase prior to vincristine exposure. Synchronized cells that had not divided at the time of vincristine exposure were blocked transiently in G2. Serial metaphase index studies suggested that the G2 cells closest to the end of the cell cycle at the time of vincristine exposure were likely to exhibit the greatest degree of mitotic disorganization when they overcame the G2 block and entered metaphase. The present studies suggest that sensitivity to vincristine increases progressively as cells approach mitosis. The molecular mechanisms underlying this phenomenon are considered in relation to the increase in cell tubulin content during the course of cell cycle progression.

Effects of vincristine on cell survival, cell cycle progression, and mitotic accumulation in asynchronously growing Sarcoma 180 cells.

The effects of vincristine (VCR) on cell survival, cell cycle progression, DNA synthesis, and metaphase accumulation were studied in relation to drug concentration and drug exposure duration in Sarcoma 180 cells in vitro. VCR was found to affect cells in interphase, producing a transient G2 block at all drug concentrations and drug exposure durations studied. VCR did not affect DNA synthesis directly. Increases in the metaphase index were delayed and always peaked at approximately 8 hr after drug removal, regardless of the duration of drug exposure. Increases in the metaphase index of sufficient magnitude to be commensurate with VCR lethality were observed only with prolonged drug exposure. VCR produced both nuclear fragmentation and polyploidy. The proportion of cells undergoing polyploidy increased progressively with increasing drug exposure duration. Interference with cytokinesis during prolonged VCR exposure may represent a lethal effect of VCR that is separate from its short-term effects. This could serve as the basis for the clinical study of the antitumor effects of prolonged VCR infusions.

Model for the genetic evolution of human solid tumors.

A conceptual model is proposed for the genetic evolution of many human solid tumors that is based on the observations that cancer cells may spontaneously double their chromosome number; that cells with excessive chromosome numbers may be cytogenetically unstable, both losing chromosomes randomly during subsequent cell divisions, and often developing structural abnormalities in the chromosomes that are retained; and that some structural chromosome abnormalities may activate growth-promoting genes. The sequence of tetraploidization with chromosome loss can occur repeatedly in a given tumor. The available evidence supporting the model is reviewed. A computer simulation system that embodies these concepts is described and the model is used to generate distributions of chromosome number/cell under various simulated conditions and in a variety of simulated biological settings. A simulation of the time course of changes in chromosome number per cell that accompany the spontaneous neoplastic transformation of mouse fibroblasts in vitro is described. The best fit to the data was obtained when provision was made for the activation of at least two growth-promoting genes. The conditions for generating discrete aneuploid peaks in cytogenetic and flow cytometric studies were explored; our modeling studies suggest that the activation of a growth promoting gene is required in order to produce a discrete aneuploid peak. Our modeling studies suggest that the overrepresentation of individual oncogene-bearing chromosomes in aneuploid cell lines may require the activation of gene dose-dependent growth-promoting genes and is not likely to occur in cell lines in which at least two copies of each normal chromosome are required for cell survival. Overall, the results obtained using the model are consistent with a wide variety of flow cytometric and cytogenetic studies in human solid tumors.

Karyotypic evolution of a human undifferentiated large cell carcinoma of the lung in tissue culture.

Serial cytogenetic studies were performed on a cell line derived from a pleural effusion from a patient with undifferentiated large cell carcinoma of the lung. The initial sample had a broad range of chromosome numbers per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid sideline. A sequence consisting of a doubling of chromosome number per cell followed by chromosome loss was observed repeatedly during 40 culture passages. The presence of metaphase spreads showing evidence of endoreduplication suggested this as a likely mechanism for the doubling of chromosome number per cell. Eleven marker chromosomes were observed in the cells of the primary sample; these markers persisted through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16 were consistently overrepresented; each of these chromosomes was involved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22 were consistently underrepresented. Every chromosome, either in its normal form and/or as part of a marker, was represented on the average by at least one copy per diploid cell. Eighteen new marker chromosomes were observed during the course of cell cultivation; one of these evolved into a clonal marker over the course of six cell passages. Of the new marker chromosomes that were formed during the observation period, the majority were found in hypotetraploid cells.

Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas.

Human solid tumors develop multiple genetic abnormalities that accumulate progressively in individual cells during the course of tumor evolution. We sought to determine whether there are specific sequences of occurrence of these progressive evolutionary changes in human breast cancers by performing correlated cell-by-cell measurements of cell DNA content, p53 protein, Her-2/neu protein, and ras protein by multiparameter flow cytometry in 56 primary tumor samples obtained at surgery. In addition, p53 allelic loss and Her-2/neu gene amplification were determined by fluorescence in situ hybridization in cells from the same samples. We reasoned that if there is a specific order in which genetic changes occur, the same early changes would be found consistently in the cells with the fewest abnormalities. We reasoned further that late-developing abnormalities would not occur alone in individual cells but would almost always be found together with the early changes inherited by the same cells. By these criteria, abnormalities involving p53 generally occurred early in the course of development of invasive breast cancers, whereas ras protein overexpression was found to be a late-occurring phenomenon. Within individual tumors, cellular p53 overexpression was often observed alone in individual cells, whereas ras protein overexpression was rarely observed in the absence of p53 overexpression and/or Her-2/neu overexpression in the same cells. Furthermore, the intracellular level of each abnormally expressed protein was found to increase progressively as new abnormalities were acquired. Infiltrating ductal carcinomas exhibited characteristic phenotypic patterns in which p53 allelic loss and/or p53 protein overexpression, Her-2/neu amplification and/or overexpression, aneuploidy, and ras overexpression accumulated within individual cells. However, this pattern was not a prominent feature of lobular breast cancers. All six lobular breast cancers studied were diploid. p53 allelic loss and/or early p53 overexpression, and late ras cooverexpression in the same cells were less common in lobular breast cancers than in infiltrating ductal carcinomas. Although Her-21neu overexpression was a common finding in lobular breast cancers, Her-2/neu amplification was not observed in these tumors.

Intracellular coexpression of epidermal growth factor receptor, Her-2/neu, and p21ras in human breast cancers: evidence for the existence of distinctive patterns of genetic evolution that are common to tumors from different patients.

Multiparameter flow cytometry studies were performed on cells from the primary tumors of 94 patients with breast cancer. Correlated cellular measurements of cell DNA content, Her-2/neu, epidermal growth factor receptor (EGFR), and p21ras levels were performed on each of 5,000 to 100,000 cells from each tumor. When criteria for positivity were matched with those in common use for immunohistochemical studies, 28 of 94 (30%) breast cancers were classified as positive for Her-2/neu overexpression. When similar criteria were applied to the EGFR measurements, 23 of 94 (24%) cases were classified as positive for EGFR overexpression. Similarly, 23 of 94 (24%) cases were classified as positive for p21ras overexpression. By conventional flow cytometric criteria for DNA ploidy, 24 cases were diploid, 28 were tetraploid, and 42 were aneuploid. When the measurements were treated as separate sets of data, the only statistically significant correlations noted were the high frequency of diploid tumors, which did not overexpress any of the three oncogenes studied (P < 0.05), and an association between Her-2/neu overexpression and aneuploidy (P < 0.03). When the data were treated as correlated intracellular measurements, 90 of the 94 tumors studied contained a population of cells in which the intracellular levels of Her-2/neu expression were directly correlated with the levels of EGFR expression in the same cells. The ratio of Her-2/neu molecules to EGFR molecules in the same cells exceeded 1 in the majority of tetraploid and aneuploid cases and was close to or less than 1 in the majority of diploid cases. In nearly all tumors, p21ras overexpression was observed only in cells that overexpressed Her-2/neu, EGFR, or both, and p21ras levels per cell were more closely correlated with levels of EGFR per cell in the same cells than with Her-2/neu levels per cell. The data are consistent with a model in which heterodimerization of Her-2/neu and EGFR in individual cells is achieved by one of several genetic evolutionary pathways, all of which commonly lead to p21ras overexpression. The two major genetic evolutionary pathways identified in this study are an aneuploid, Her-2/neu overexpression-driven pathway seen in 59 of 94 tumors, and a diploid, EGFR overexpression-driven pathway seen in 19 of 94 tumors. All tumors with Her-2/neu:EGFR ratios greater than 2 contained an infiltrating ductal carcinoma component, whereas all infiltrating pure lobular carcinomas had Her-2/ neu:EGFR ratios that were less than 2. All of the genetic evolutionary pathways identified in this study were represented among the 11 tumors from patients who experienced early tumor recurrences.

Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

Electrolytic degradation of DNA fluorochromes during flow cytometric measurement of electronic cell volume.

Changes in flow cytometric measurement of DNA content can result from electrolytic chemical degradation of mithramycin, ethidium bromide, and propidium iodide during simultaneous measurement of electronic cell volume. Bench electrolysis also degrades these fluorochromes without changing the quantum yields, even when they are complexed to DNA. In the flow cytometer, electrolytic production of chlorine at the anode is the probable cause of this degradation, since exposure of these fluorochromes to chlorine gas produces the same effect. It is therefore advisable to measure the DNA content distribution alone before simultaneously measuring the DNA content and the electronic cell volume. If unavoidable effects on the DNA distribution are present, narrow forward-angle light scatter should be used as the cell size indicator during dual parameter measurements. Modifying instrument design by reversing electrode polarity might eliminate this problem.

Genetic evolutionary staging of early non-small cell lung cancer: the P53 --> HER-2/NEU --> ras sequence.

INTRODUCTION: The sequence of genetic evolutionary abnormalities that have occurred in a given lung cancer tumor before tumor sampling can be inferred from patterns of intracellular co-occurrence of these abnormalities in tumor cell subpopulations at the time of sampling. The same evolutionary sequences that are present within each lung cancer were evident in intertumor comparisons. METHODS: Correlated cell by cell measurements of cell DNA content, p53, Her-2/neu, and ras proteins were obtained by multiparameter flow cytometry on 46 surgically resected stage I-III primary non-small cell lung cancers. Early evolutionary changes were identified by the fact that they could appear alone in individual cells. Later appearing abnormalities were identified by the fact that they were accompanied by early abnormalities in the same cells. Patients were followed prospectively. Evolutionary patterns observed in individual tumors were correlated with subsequent clinical outcome of patients undergoing surgical resection. RESULTS: Three common patterns were identified: (I) a diploid DNA pathway consisting of the sequence p53 overexpression --> Her-2/neu overexpression --> ras overexpression, (II) an aneuploid DNA pathway with the same p53 --> Her-2/neu --> ras sequence, and (III) a pathway in which none of the intracellular protein measurements made here were abnormal. Fourteen tumors recurred after 11.5 months' median study time. Nine of 12 recurrences in pathways I and II occurred in patients whose tumors were far advanced along these molecular genetic pathways. CONCLUSIONS: Multiparameter cell-based genetic evolutionary studies may be a promising approach for identifying patients with stage I-III non-small cell lung cancer at high risk for recurrence.

A dynamic interpretation of multiparameter studies in the lymphomas.

A general approach to the analysis of cellular life cycle traverse patterns is described. This approach is exemplified in human lymphomas, using Coulter volume and DNA content measurements obtained by flow cytometry. Among the B-cell tumors the large-cell lymphomas generally show larger fractions of DNA-synthesizing cells and a higher degree of proliferative activity than the small B-cell lymphomas. Paired cell-by-cell measurements of Coulter volume and DNA content can be used to distinguish large aneuploid tumor cells from smaller diploid cells in mixed cell populations. The paired measurements can also be used to explore the kinetic properties of subpopulations within individual tumor samples. It can be shown that within individual lymphoma samples there are subpopulations of large cells that have greater proliferative activity than the small cells.

Pseudo-Gaucher cells in the bone marrow of a patient with Hodgkin's disease.

The authors studied an 18-year-old woman with stage IIIB nodular sclerosis Hodgkin's disease whose bone marrow contained abnormal storage cells that resembled Gaucher cells by light microscopic examination ("pseudo-Gaucher" cells). Electron microscopic examination revealed that these cells differed from true Gaucher cells and resembled storage cells previously described in chronic myelogenous leukemia. The patient's peripheral blood leukocyte beta-glucosidase and serum acid phosphatase levels were elevated, ruling out the diagnosis of inherited Gaucher's disease. After treatment with six monthly cycles of systemic chemotherapy (nitrogen mustard, vincristine, procarbazine, bleomycin, doxorubicin, and prednisone), all signs of Hodgkin's disease and pseudo-Gaucher cells disappeared. Repeat leukocyte beta-glucosidase and serum acid phosphatase levels were unchanged. The present case is unique with its documentation of classical enzyme patterns for beta-glucosidase and acid phosphatase and electron microscopic features. The authors postulate that pseudo-Gaucher cells result from excessive cell breakdown with an overload of available beta-glucosidase.