Cytokinetic Studies Research Articles

Effects of IL-6 and TNF-α Cytokines on Cell Proliferation in Androgen Dependent/Independent Prostate Cancer Cell Lines

Prostate cancer is one of the most common types of cancer worldwide. Prostate cancer, caused by abnor-mal and uncontrolled growth of the cells that make up the prostate tissue, is second only to relalated deaths in men. Cytokines, which have a significant impact on the response of the immune system, play an active role in the development of prostate cancer. This study determined the effect of proinflammatory cytokines, IL-6 and TNF-a on the proliferation of prostate cancer cell lines. MTT test was used for the effect of cytokines applied at different doses and hours on cell viability. According to our results, IL-6 cytokine caused a high increase in proliferation in androgen-dependent LNCaP cells, while androgen-independent PC-3 cells showed different proliferative effects in time and dose-dependent manner. TNF-α cytokine had a negative effect on the proliferation of androgen-dependent LNCaP cells, while it increased the proliferation level of androgen-independent PC-3 cells. These results show that the effects of cell lines-on the proliferation of cells are different depending on their androgen sensitivity, which will be used in different cytokinetic studies to determine the inflammatory response to treat prostate cancer. It is espe-cially important to consider this condition during the development of prostate cancer treatment strategies.

Cytokinetic pattern in the thoracic spinal cord of chick embryos (incubation day 5-13) using PCNA staining and TUNEL method.

For the cytokinetic studies using spinal cords of chick embryos, chronological patterns of cell proliferation and programmed cell death (apoptosis) should be known. Information in the early stages of chick embryos is available while data on later stages are seldom available. To investigate the chronological patterns of cell proliferation and apoptosis in the thoracic spinal cord of normal chick embryos on incubation day 5, 6, 8, 10 and 13 (Hamburger and Hamilton stage 26-40), proliferating cell nuclear antigen (PCNA) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method were used. Cell proliferation was active at the germinal layer on days 5 and 6. It markedly declined on day 8 and became negligible on day 13. TUNEL-positive cells were mainly found in the germinal layer, the ventrolateral part of the mantle layer and the dorsal root ganglion. Compared to PCNA-positive cells, TUNEL-positive cells were sparse, especially after day 10, when only a few positive cells were scattered. These results will be used as a control data for the studies such as an experimental research for neural tube defects.

Therapeutic management of hematological malignancies in elderly patients. Biological and clinical considerations. Part 1. Myelodysplasias and the acute leukemias.

The different therapeutic options that may be employed in the treatment of elderly patients with acute leukemias and myelodysplastic states are considered following an analysis of certain biological features, that have been investigated by cytochemical, cytogenetic and cytokinetic techniques, immunophenotyping, and studies on G-6-PD isoenzymes. These studies imply that in the elderly the pattern of hematological malignancies and the lack of response to conventional treatment derive from intrinsic biological differences between these pathological states in older and younger patients. Treatment in elderly patients has ranged from palliative treatment to intensive chemotherapy, often with disappointing results in both cases. Palliative treatment does not induce remissions, and median survival is short. On the other hand, elderly patients do not tolerate well both induction and post-remission therapy due to the degree of toxicity and the effects of drug-induced pancytopenia. In this scenario, in vitro drug-sensitivity testing and karyotyping assume increasing importance, because they may predict which patients are likely to benefit from intensive therapy. In both acute leukemias and myelodysplasias, treatment ideally should be designed case by case, according to the hematological, clinical and biological features.

Intestinal Growth in Altricial European Starling (<i>Sturnus vulgaris</i>) and Precocial Japanese Quail (<i>Coturnix coturnix japonica</i>)

This paper introduces the comparison of altricial and precocial birds as a model system for the study of patterns of intestinal growth. It reports on comparative morphometric and cytokinetic studies of intestinal growth in altricial European starling (Sturnus vulgaris) and precocial Japanese quail (Coturnix coturnix japonica). Morphometric data at five gut segments of neonates and adults of both species are provided to evaluate the growth of the intestine. A double labelling technique combining 3H-thymidine autoradiography with 5-bromo-2'-deoxy-uridine immunohistochemistry gives labelling index, length of S-phase, and tissue turnover time for hatchlings and adults of both species. The results of morphometry show a similar pattern of intestinal growth in both species, a strong dependency of intestinal growth on body size, and a clear topographic separation of areas of cell proliferation from the functional mucosal epithelium. The results of the cytokinetic experiments indicate a constant length of the S-phase independent of species, development, and age. The 3H-thymidine labelling index is significantly higher in neonate quail than in neonate starling. Between adult animals of both species, the labelling index goes not differ significantly except for the duodenum which has a higher index in starling. The turnover time necessary to replace the mucosal epithelium is 3 days in hatchlings of both species and ranges between 10 and 17 days in adults. A diurnal pattern of cell proliferation, as previously reported in adult quail, is not present in neonate quail. Thus, differences in intestinal growth among altricial and precocial birds are primarily based on size differences of proliferation areas whilst cellular proliferation rates have only minor impact on intestinal growth. The short turnover time and the high rates of proliferation suggest that the intestine is a flexible organ that may be adjusted in size and function to the actual needs of the growing bird.

Morphologic alterations and cytokinetic studies of tracheal autograft epithelium in rabbits

Although tracheobronchoplasty has been used widely in the field of thoracic surgery, few details of the morphologic changes in and cytokinetics of the graft epithelium have been reported. The aim of this study was to focus on these aspects in autografted rabbit tracheas. Resected cervical tracheas were anastomosed immediately after removal, retrieved on postoperative days 1 through 28, and examined morphologically. Mitotic and bromodeoxyuridine-labeling indices of the graft epithelium were analyzed. On postoperative days 1 to 4, the graft epithelium showed focal desquamation at the anastomoses. Ciliated cells disappeared during postoperative days 4 to 7 and then increased gradually. Nonciliated cells retained a somewhat columnar shape on postoperative days 4 to 7, except at denuded foci. Thereafter, the grafts were covered completely with pseudostratified mucociliary epithelium. On postoperative day 4, both indices were maximal and appeared higher at the anastomotic than midgraft sites. Most of the graft epithelium was preserved during acute ischemia and then started to regenerate. The increased regenerative activity near the anastomoses may be attributable to mechanical damage or different nutritional conditions.

Cytokinetic studies of oral cancer cells using bromodeoxyuridine labelling in relation to factors influencing prognosis

Cytokinetics of 27 untreated oral squamous cell carcinomas were evaluated by in vitro bromodeoxyuridine (BrdU) labelling. A mononuclear cell suspension was prepared for the labelling using collagenase and DNase treatment. The labelled cells were visualised by immunofluorescence staining with an anti-BrdU monoclonal antibody. The labelling index (LI%) was calculated by determining the percentage of BrdU labelled cells. The LIs of the carcinomas ranged from 3.8 to 19.2%. The LIs of poorly-differentiated (HG3) tumours were 2-3-fold higher compared with those of well-differentiated (HG1) and moderately-differentiated (HG2) carcinomas. Results also showed statistically significant (P < 0.0005) increases in LIs from nuclear grade 1 (NG1) to nuclear grade 3 (NG3) carcinomas. Higher LIs were observed in stage III/IV (10.82 +/- 4.62; 10.36 +/- 4.90) than those in stage I (6.87 +/- 2.09) and II (7.14 +/- 1.87) carcinomas. A significant (P < 0.0005) difference in LI values was found between the patients with positive and negative lymph nodes. Good correlation (r = 0.77) was exhibited between the LI values and mitotic counts (MC) of the specimens. These results on oral cancer cell proliferation seem to have prognostic implications.

The Use of Bromodeoxyuridine Cytokinetic Studies as a Prognostic Indicator of Cancer of the Head and Neck

Traditional measures of head and neck tumors often fail to predict patient outcome or clinical course, particularly in nonadvanced disease. This problem of unpredictable tumor behavior has been one focus of cell proliferation studies. Such studies, however, have been limited by difficult methodology. A newer method of quantifying tumor cell proliferation using bromodeoxyuridine is applicable for head and neck squamous cell carcinomas, as shown in the present study. The relative ease with which cell proliferation can be evaluated using this technique will allow large numbers of head and neck tumors to be studied, enabling correlations with tumor behavior to be made.

Concomitant continuous infusion chemotherapy and radiation

Attempts to duplicate the cytotoxic effect of oxygen on radioresistant tissues spurred a search by radiation oncologists for other radiosensitizing techniques. This led to large-scale investigations using neutrons and other heavy particle radiations, hyperthermia, altered fractionation schedules, and the systemic use of the halogenated pyrimidines and the electron-affinic compounds. Unfortunately, the promise that the nitroimidazole compounds would selectively sensitize the radioresistant tumor cells and prove to be an effective systemic agent has not been borne out in clinical trials thus far. Existing pharmokinetic and cytokinetic studies have suggested that continuous infusion chemotherapy given concomitantly (CCIC) with irradiation (RT) acts synergistically, resulting in a significant increase in tumor cell killing. These observations have been supported by clinical research studies treating certain epithelial cancers that have resulted in considerably higher locoregional control rates and improved survival. Although initially used in treatment of only advanced or inoperable epithelial carcinomas, CCIC and RT are now being employed in the treatment of lower staged cancers as an organ-sparing procedure. Carcinoma of the anus treated by anteroposterior (AP) resection alone have reported 5-year survival rates of 30% to 60%. CCIC and RT using 5-fluorouracil (5-FU) and mitomycin C have achieved a local control rate of 90% to 100% and a 5-year survival rate of 80% to 86% with sphincter preservation in 90% of these cases. The 5-year survival rate in advanced urinary bladder carcinoma is 25% to 30% for either radiation or surgery and 42% when combined in a preoperative radiation schedule. Using 5-FU CCIC and RT, the local control rate of transitional cell carcinoma of the bladder has been 71% to 86% with a 5-year survival of 62%. 5-FU CCIC and cisplatin and RT used in the treatment of Stages III and IV carcinoma of the cervix yields a locoregional control of 74% compared with the radiation alone local control of 63% for Stage III and 30% for Stage IV disease. Advanced head and neck and paranasal sinus carcinomas treated by cisplatin CCIC and RT show improved tumor clearance even in the presence of bone destruction. A complete response rate of 87% has been reported with the use of cisplatin CCIC and hyperfractionated radiation. Hyperfractionated radiation also appears to improve the local control of advanced head and neck cancers over patients treated with single fractions of radiation with 66% surviving at 22 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro.

Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity. In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50-70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G2 phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G2 cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G2 phases. This was also observed with G1 cells from a G1-accumulated fraction in the first cycle. The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method). The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaptation of growth-cycle to these limitations.

Flow Cytometric DNA-Analysis of Plant Protoplasts Stained with DAPI

Abstract prepared from leaves of various higher plants were stained with the specific DNA fluorochrome DAPI and measured with a pulse cytophotometer. DNA distribution curves of protoplasts showed DNA histograms like assays of animal cells stained with the same dye solu­tion. The percentage of fractions of cell populations in G0/G1-, S- and G2 + M-phase of the cell cycle could be calculated. A simple method of protoplasts preparation and of flow cytometric estimation of DAPI stained plant protoplasts is described. This method allows cytogenetic and cytokinetic studies of cells from plant material and may be used for fluorescence activated cell sorting.

Cytokinetic studies and treatment results of estrogens followed by chemotherapy in locally advanced (LABC) and metastatic (MBC) human breast cancer

Cytokinetic studies and treatment results of estrogens followed by chemotherapy in locally advanced (LABC) and metastatic (MBC) human breast cancer

Cell cycle analysis using flow cytometry.

This manuscript reviews the utility of flow cytometry for the study of cell proliferation. The applications of univariate DNA distribution analysis to cytokinetic studies of asynchronous and perturbed cell populations are discussed briefly. The newly developed technique for simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine is discussed in more detail. The cytochemistry required for this analysis is reviewed as are its applications to: determination of the fractions of cells in the G1-, S- and G2 + M-phases of the cell cycle; determination of the G1-, S- and G2 + M phase durations and dispersions and growth fraction for asynchronous cells; detection of ara-C resistant cells present at low frequency in an otherwise sensitive population; and analysis of the cytokinetic response of a solid murine tumour to treatment in vivo with a cell cycle specific agent.

Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies.

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol-fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine-conjugated wheat germ agglutinin (WGA), and a fluorescein-labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer-based multivariable pulse-height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S-phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.

Cytokinetic studies in acute leukemia during early induction therapy.

The proliferative activity of bone marrow blasts was determined in 19 patients with acute leukemia during early induction therapy using in vitro tritiated thymidine (3H-TdR) cytoautoradiography and propidium iodide DNA flow cytofluorometry. Following 1-3 causes of treatment, the aliquot of bone marrow blasts in 9 patients, who later achieved remission or marrow aplasia, was reduced to a greater extent that of the remaining 10 patients who failed to respond. In the first group of patients, the 3H-TdR labeling index was increased by 259-653% over the pretreatment value, whereas it was lower than 104% of the pretreatment value in all but one unresponsive patient. Leukemic blast recruitment is conceivable. It might have facilitated remission by increasing the effectiveness of the antileukemic agents administered later.

Differentiation and cytokinetic analyses of normal and neoplastic lymphoid cells in B and T cell malignancies of childhood

The labelling index (LI) of untreated B cell non-Hodgkin's lymphoma (B NHL) in children was significantly higher (P less than 0.002) than the LI of untreated T cell malignancies (median LI 27.7% and 10.3%, respectively). In B NHL abdominal tumour material showed a significantly higher in vitro incorporation of tritiated thymidine (3H-dT) than extra-abdominal tumour material (P less than 0.01). Sequential immunological, cytochemical and cytokinetic studies revealed tumour cells in up to three marker-defined subpopulations of lymphoid cells. In all 14 patients examined the LI of E-Ia+sIgM+ cells was higher than the LI of E+Ia+/-sIgM- cells; in all eight patients with Ia+ and Ia- tumour cells the former had a higher LI than the latter (P less than 0.002). In all T cell neoplasia (T ALL/NHL) investigated malignant cells were found in two of the three marker-defined subsets of lymphoid cells. In 10/13 patients the E-rosette positive blast cells had a higher LI than the E-rosette negative T blast cells. For the small group of patients with T cell malignancies no significant difference in the overall LI between OKT9+ stage I (early thymocyte) and OKT9- stage II (cortical thymocyte) neoplasias could be found. However, within the malignant clone of a T cell malignancy the expression of the OKT9 (transferrin receptor) but not the cortical thymocyte (HTA-1) antigen was related to the in vitro 3H-dT incorporation.

Neuroendocrinelike (small granule) epithelial cells of the lung.

The presence of neuroendocrinelike epithelial cells in the lung of numerous species has been demonstrated by light and electron microscopy. Histochemical methods used to identify these cells have included staining with silver, amine-type fluorescence (APUD cell), periodic acid Schiff (PAS)-lead hematoxylin, and immunohistochemical localization of neuron-specific enolase. Cytoplasmic dense core vesicles (70-200 nm in diameter) have served as the major ultrastructural characteristic. Lung neuroendocrinelike cells have been shown to occur in fetal and adult mammals as solitary-type cells or as distinct organoids known as neuroepithelial bodies ( NEBs ). Although the frequency of both populations is considered low, solitary-type cells with dense-core granules can be found in as high as 5% of epithelial cells in the cricoid region of the guinea-pig larynx. The solitary cells can be found throughout the airways of mammals, whereas the NEBs are confined to the intrapulmonary airways. Unmyelinated fibers have been traced from the lamina propria and into the NEB, where they ramified between the component cells of the NEB. The function of lung neuroendocrinelike cells is not known, but morphological and cytochemical studies suggest that the NEBs are intrapulmonary chemoreceptors that can respond to changes in airway gas composition. Hypoxia or hypercapnia has been shown to decrease the amine cytofluorescence in these organoids and apparently to increase the exocytosis of dense core vesicles from the basal region of the cell. Immunohistochemical studies have suggested that some lung epithelial cells may contain a known neuropeptide(s), but further investigation is needed to confirm the presence of such compounds in lung neuroendocrinelike cells and their physiochemical properties. Apparent hyperplasia of lung neuroendocrinelike cells can occur readily in hamsters treated with diethylnitrosamine. It has been postulated that human lung tumors with endocrinelike properties, namely, bronchial carcinoids and lung small cell carcinomas, may originate from lung neuroendocrinelike cells. However, a more plausible explanation, based on cytokinetic studies of epithelial neuroendocrinelike cells in the lung and other organs, is that these cells originate from a nonneuroendocrine population. Interaction of such a progenitor cell population with selected carcinogens may lead to stimulation of the rate of normal differentiation or, alternately, to selection of an abnormal route of differentiation that possesses a neuroendocrine phenotype.

Differentiation block of prethymic lymphocytes during Moloney-virus-induced lymphoma development associated with a thymic epithelial defect.

Previous cytokinetic studies in Moloney-virus (M-MuLV)-induced lymphomas of BALB/c mice showed an intrathymic maturation block of prethymic lymphocytes derived from hematopoietic tissues. Thymus-cell cultures during the latent period of lymphoma development showed a proportion of nonlymphoid cells (NLC) from uninfected mice of 0.1% (3 days) to 0.002% (20 days), rising in infected mice to 1-2% after 5 weeks. Concomitantly, thymic epithelial cells exhibit progressive degenerative changes in vivo in infected mice with virus replication and in vitro a marked cellular polymorphism with nuclear atypia becomes overt. Immunofluorescence studies of thymopoietin II and serum thymus factor in epithelial cells indicate a marked decrease of these hormones in the epithelial cells from infected mice. These results suggest a functional defect of virus-infected thymic epithelial cells which causes a progressive accumulation of nondifferentiating T-cell precursors.

Response of recurrent acute lymphoblastic leukemia to glucocorticoids: Serial studies of receptor content, in vivo cytokinetic changes and clinical responses

The sensitivity of relapsed acute lymphoblastic leukemia (ALL) to treatment with steroids was assessed by measuring bone marrow blast, whole-cell glucocorticoid receptor (GR) levels and serial cytokinetic and clinical responses to steroids, both as single agents and in combination reinduction chemotherapy. GR levels ranged widely, from 1800 to 47,800 sites/cell (median 16,000), and did not differ significantly from levels measured in newly diagnosed patients ( p = 0.50). Nineteen of the 48 children studied had GR levels measured both at diagnosis and relapse, and in 14 the values at relapse were either decreased or increased by more than 25%. For nine children, the sensitivity to a three-day oral course of dexamethasone alone (6 mg/m 2 per day) was estimated from daily leukocyte counts and serial bone marrow cytokinetic studies. A clinical oncolytic effect of dexamethasone was associated with a reduction in the marrow cell proliferating compartment, as judged from labeling indices, mitotic indices and percent S + G 2 + M in five patients. We found, however, that GR levels either alone or combined with clinical or cytokinetic responses to dexamethasone as a single agent did not reliably identify patients who were likely to respond favorably to reinduction with steroid-containing combination chemotherapy. The 35 children who successfully attained subsequent remissions had GR levels similar to those who failed ( p = 0.35). While not statistically significant, two observations suggest an association between lower receptor content and drug resistance. Receptor levels from patients with ALL who relapsed while on chemotherapy were appreciably lower (median 15,700, n = 31) than GR levels from patients who relapsed off therapy (median 20,300, n = 17) ( p = 0.08). Moreover, three of seven patients with serial studies, whose GR levels at relapse were lower than at diagnosis, failed reinduction — compared to only one of twelve whose levels were either increased or remained unchanged ( p = 0.11). Although not having any obvious clinical utility, studies of GR at diagnosis and at relapse may aid in clarifying mechanisms of drug resistance in leukemic blasts.

Squamous Metaplasia in Organ Cultures of Vitamin A-Deficient Hamster Trachea: Cytokinetic and Ultrastructural Alterations&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Cytokinetic and morphologic studies were performed to elucidate the origin of epidermoid metaplasia in organ cultures of tracheas derived from vitamin A-deficient Syrian golden hamsters. Focal areas of squamous metaplasia were observed 2 days after explantation, and extensive epidermoid metaplasia and cornification were present in approximately 89% of the explants at day 10. During the first 2 days, both basal and mucous cells proliferated actively. By day 3, the total mucous cell [3H]thymidine labeling index (LI) had declined and remained at very low levels during the remaining culture period. The basal cell LI also declined from its higher level, but it remained at relatively higher levels than those of the mucous cells between days 3 and 9 after culture. In the lesions, labeled cells were generally confined to the basal layer, and the LI was about thirtyfold greater than the total basal cell LI. As the lesions progressed, the surface layer containing the mucous and ciliated cells was exfoliated as a result of population pressure from the underlying actively proliferating basal cells and subsequent epidermoid differentiation of the daughter cells. These data support the hypothesis that epidermoid metaplasia of tracheobronchial epithelium caused by vitamin A deficiency originates from the generative or basal cells.

Clinical and cell kinetic studies of the effects of the epipodophyllotoxin VP16-213 during therapy of refractory acute nonlymphocytic leukemia.

After induction failure or relapse on primary therapy, acute nonlymphocytic leukemia (ANLL) has historically been highly resistant to reinduction chemotherapy. In order to evaluate a new drug combination for this condition, 24 children and young adults with ANLL failing to attain first remission [5] or in relapse [19] were treated with VP16-213 plus 5-azacytidine (5AZ). Each patient had previously received combination chemotherapy with several antileukemic agents, including anthracyclines and cytosine arabinoside. These patients received 5-day courses of VP16-213 (200 mg/m2) daily x 3 days, followed immediately by 5AZ (300 mg/m2) daily x 2 days, repeated every 1–2 days until the bone marrow became hypoplastic. After 2–4 courses, 19 patients demonstrated an objective anti-leukemic effect by achieving marrow hipoplasia without evidence of blasts. Eleven of these (11/24=46%) achieved complete remission after a median time of 45 days from the initiation of therapy, making this combination of VP16-213 and 5AZ the most active regiment we have tested for ANLL in relapse. In order to determine the effects of VP16-213 on leukemic blast proliferation in vivo, serial bone marrow aspirates were studied in eight of these children. VP16-213 200 mg/m2 IV was given daily x 3 days, and the mitotic index, labeling index and fractions of cells in G0/G1, S and G2+M by flow cytometric measurement of DNA content were determined. Bone marrow samples were obtained before, 4 h and 20–24 h after the third dose in all patients, and 20–24 h after the third dose in six of the patients. The cytokinetic studies 4 h after the first dose of VP16-213 consistently showed a G2 phase arrest of the bone marrow myeloblasts, indicating uptake of the drug and production of the same cell kinetic lesion which has been shown for cells treated with VP16-213 in vitro. In each patient studied 24 h after the third drug dose, there was a decrease in the fraction of cells in S phase compared to the pretreatment study (median decrease 56%, range 24–67%). The blast count per ml of marrow decreased by a median of 84% (range 72–91%) over this same interval. The observed decrease of fraction of cells in S phase after three doses of the drug suggests selective kill of cells in S compared to G0/G1. We conclude that the activity of VP16-213 against ANLL blasts can be demonstrated in vivo by the production of G2 phase arrest and reduction of marrow blast count with findings consistent with increased cytotoxicity for cells in S and G2 phase of the cell cycle. In addition, this intensive combination of VP16-213 and 5AZ has shown clear evidence of effectiveness in patients with refractory ANLL, and thus may be useful during initial therapy.