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Secreted protein acidic and rich in cysteine (SPARC) has a suppressive effect on U87 glioma cell proliferation when assessed in vitro and in vivo using parental U87T2 and U87T2-derived SPARC-transfected clones. Since SPARCinteracts with extracellular matrix (ECM) proteins, we examined the effect of SPARC secretion on proliferation, morphology, and cell density of glioma cells grown in vitro, in the absence and presence of ECM proteins under standard (10% fetal bovine serum [FBSI) and reduced (0.1% FBS) serum stress conditions. Under standard conditions, MTT (3-(4,5-cimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) growth curves, morphology, and Western blot analyses demonstrated that SPARC had a suppressive and biphasic effect on growth that was not grossly modulated by the ECMs. The SPARC-induced changes in morphology observed at 24 h were not altered by the presence of ECMs. Under reduced-serum stress conditions, Western blot, morphological, and flow cytometric analyses indicated that the SPARC-induced suppressive growth effects were eliminated when the cells were grown on plastic. However, ECM-specific changes in growth were observed, some of which correlated with secreted SPARC levels. These results indicate that the differential effects of SPARC and ECMs on proliferation are dependent on culture conditions. Since the results obtained under standard conditions agree with our in vivo observations, we conclude that the ability of SPARC to suppress proliferation is regulated to a greater degree by the level of SPARC and that this suppressive effect is not influenced by the presence of any of the ECMs examined.

A multidimensional chromatographic 2-D liquid-phase separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of protein expression between Peninsularinone-treated and untreated HCT-116 human colon adenocarcinoma cells. The method involves fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous reversed-phase HPLC. A 2-D map of the protein content of each cell line based upon pI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or downregulated proteins displayed using ProteoVue and DeltaVue software. Using this method, > 1000 protein bands could be detected in 0.2 pH fractions over a pH range of 4-7. In addition, the liquid eluent from the separation was directed on-line into an electrospray TOF-MS to obtain an accurate molecular weight of the intact proteins. An accurate molecular weight together with the peptide map was used to obtain protein identification using database searching. The method has been shown to have high reproducibility for quantitative differential display analysis of interlysate comparisons, generation of accurate protein identifications, and ease of data interpretation. It has been used herein to identify proteins that change as a function of drug treatment. The relative simplicity of the current procedure and the potential for full automation will make this technique an essential tool in future proteomic studies.

The cellular paradigm presented here defines the cellular action profile of new anticancer agents that complements our discovery and development paradigm. The main elements of this profile include a concentration clonogenicity response relationship on proliferating and plateau phase cells, flow cytometry studies assessing progression delay and apoptosis, macromolecular synthesis inhibition, and DNA damage assessment by the comet assay; other specific assessments then derive from these findings such as topoisomerase assays. XK469 is a new anticancer agent derived from the herbicide Assure that is the inactive parent compound of a family of quinoxaline analogs found to have anticancer activity in vivo. We have applied the described cellular action profile paradigm to XK469 to define a novel action at the cellular level. XK469 is a G2M phase-specific, antiproliferative agent whose activity is related to the 7-position of the chlorine ion in the benzene ring and expressed through a unique cellular action profile resulting in the irreversible increase in cyclin B1 (possibly by specific inhibition of its ubiquitination) and leading, in the absence of apoptosis, to the final mitotic arrest of HCT-116 cells in prophase with subsequent loss of clonogenicity.

XK469 is an investigational anticancer agent that exhibits antiproliferative activity in tumor-bearing animal models. We examined the drug-action profile of this agent at the molecular level regarding alterations induced in gene expression and proteins in HCT-116 human colon adenocarcinoma cells. We used a unique cDNA microarray (GeneMapTM Cancerarray) comprising 1152 human tumor-related genes and 2-D gel electrophoresis, respectively, following a 24-hour exposure to a drug concentration that killed a two-log fraction of HCT-116 clonogenic cells. Functional gene cluster profile (FGCP) analysis of the 71 out of 1152 genes that displayed a >2-fold increase or decrease in expression (over untreated control) identified a drug-specific involvement of the MAPK signal transduction pathway. MAPK signaling together with the involvement of ubiquitin proteins from 2-D gel electrophoresis suggest a novel drug-action profile at the molecular level for the in vitro antiproliferative activity of XK469. Cytometry 47:72–79, 2002. © 2001 Wiley-Liss, Inc.

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.

Recombinant human granulocyte colony-stimulating factor, rhG-CSF, is widely applied to ameliorate neutropenia following chemotherapy. However, rhG-CSF can exert negative effects on megakaryocytopoiesis that might cause a delay of megakaryocyte recovery. Therefore, the present study was designed to test different rhG-CSF administration protocols with regard to their megakaryocytic inhibitory potential in a 5-fluorouracil (5-FU)-induced experimental model system. Splenectomized B6D2F1 mice received a single injection of 5-FU (150 mg/kg) on day 0 followed by 50 micrograms/kg/day rhG-CSF given daily for either zero, four, or eight days. Five days after 5-FU, bone marrow and blood hematopoiesis were reduced significantly when compared with controls, independent of whether or not animals received rhG-CSF. However, nine days after 5-FU, granulopoietic recovery from 5-FU-induced toxicity was faster for rhG-CSF-treated versus untreated mice as demonstrated by higher values for colony forming unit-granulocyte macrophage (CFU-GM) and granulocytes (CFU-GM: 7.2 +/- 0.4 versus 5 +/- 0.6 x 10(4)/femur, granulocytes: 4.3 +/- 2 versus 1.4 +/- 0.4 x 10(5)/ml, respectively). Furthermore, significant mobilization of CFU-megakaryocyte (CFU-Meg) and CFU-GM into the peripheral blood was induced by the eight-day administration of rhG-CSF following 5-FU (day 9: 911 +/- 102 CFU-Meg/ml, 2330 +/- 152 CFU-GM/ml). However, megakaryocytic cells in these same mice were considerably lower when compared with those of animals receiving no rhG-CSF (CFU-Meg: 2.7 +/- 0.2 x 10(3) versus 4.2 +/- 0.2 x 10(3)/femur; small acetylcholinesterase positive (SAChE+) cells: 4.9 +/- 0.3 x 10(3) versus 7.3 +/- 0.9 x 10(3)/femur; megakaryocytes: 2.5 +/- 0.2 x 10(3) versus 4.1 +/- 0.7 x 10(3)/femur; platelets: 2.67 +/- 0.5 x 10(9) versus 3.1 +/- 0.5 x 10(9)/ml, respectively). On the other hand, the shortening of the rhG-CSF treatment from eight to four days caused a rapid granulopoietic recovery comparable to animals receiving eight days of G-CSF with no significant delay in megakaryocytic recovery when compared with mice treated with 5-FU alone; however, with four days of rhG-CSF, the mobilization of CFU into the peripheral blood was significantly less effective. Taken together, the results showed that a shortening of rhG-CSF treatment after chemotherapy is capable of ameliorating neutropenia without negatively affecting megakaryocytopoietic recovery. If, however, maximum recruitment of CFU into the peripheral blood circulation by rhG-CSF for subsequent harvest and transplantation is needed, any shortening of rhG-CSF administration is not advisable.

Increasing evidence especially stemming from peripheral blood progenitor transplantation studies points to a possible biologic difference between mobilized blood and bone marrow progenitor cells. The objective of this study was to compare the in situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor (rhG-CSF)-recruited circulating granulopoietic (blood colony-forming unit-granulocyte-macrophage [CFU-GM(blood)]) and megakaryocytopoietic (blood CFU-megakaryocyte [CFU-Meg(blood)]) progenitors, with the nonmobilized fraction remaining in the bone marrow (CFU-GM(femur) and CFU-Meg(femur)). Splenectomized male B6D2F1 mice received 50 micrograms/kg/d rhG-CSF daily for 8 days to induce high levels of circulating progenitors, followed by either total body X-irradiation (TBI) or X-irradiation of the chest (CI) with 62.5, 125, 250, or 500 cGy. Progenitor cells were assayed 24 hours after irradiation. Circulating CFU-GM and CFU-Meg in the blood were decreased in a dose- dependent fashion by both TBI and CI, with TBI causing greater damage than CI. Average D0 values for TBI were 53 cGy for CFU-GM(blood) and 40 cGy for CFU-Meg(blood) D0 values for CI were 90 cGy for CFU-GM(blood) and 140 cGy for CFU-Meg(blood). As seen for blood progenitor cells, TBI caused a dose-dependent decrease of both CFU-GM(femur) (D0, 136 cGy) and CFU-Meg(femur) (D0, 148 cGy). However, radiation-induced bone marrow progenitor cell kill was significantly lower when compared with blood progenitors. Despite the fact that circulating blood elements only received a fraction of the total dose administered as Cl, the extent of blood progenitor kill caused by Cl was higher than the effects of identical TBI doses on bone marrow CFU. The results of this study showed that rhG-CSF-recruited CFU-Meg(blood) and CFU-GM(blood) were considerably more radiosensitive than femoral progenitors, thereby providing novel evidence for a biologic difference between rhG-CSF- recruited peripheral blood progenitors and the nonrecruited bone marrow CFU.

We here report the initiation of hematopoietic recovery in congenitally hypoplastic S1/S1d mice by the cytotoxic ablation of cells bearing the natural killer (NK) phenotype (NK 1.1+). The most striking finding was the early several-fold increase in the cycling fraction of stem and progenitor cells (with the exception of progenitors committed to megakaryocytopoiesis) in the anti-NK 1.1+ antibody-treated group. This increase resulted in an early, complete restoration of total marrow cellularity to the normal (+/+) littermate level. Our data suggest that NK 1.1+ cells exert functions critical to the negative control of hematopoietic cell proliferation in S1/S1d mice.

Summary Recently, we have reviewed studies regarding the growth-stimulating effect of CD4+ cells on haematopoietic cells in culture (Pantel & Nakeff, 1989a). In the present study we have tested the physiologic relevance of this interaction using a drug-perturbed mouse model. The long-term application of cyclophosphamide (CY, 30 mg/kg/d, five i.p. injections per week over 7 weeks) in B6D2F1 mice resulted in initial CY-induced cytotoxicity to CFU-S followed by the re-establishment to pretreatment values of the femoral content of CFU-S within 2–3 weeks of CY-treatment. An examination of CY-metabolism in these treated mice excluded a pharmacological explanation for the compensation of CY-cytotoxicity. However, a three-fold increase in the cycling fraction of CFU-S (determined by in vivo hydroxyurea suicide) was observed concomitant with a two-fold increase in the femoral content of L3T4+ cells (the murine equivalent to human CD4+ cells), as compared to the corresponding values in untreated mice. Ablating these L3T4+ cells in vivo by means of a cytotoxic monoclonal antibody (MoAb) to the L3T4 determinant resulted in a decrease in the cycling fraction of CFU-S from 56.8% to essentially zero and a decrease in the femoral content of CFU-S when comparing mice receiving either CY alone or CY plus MoAb, respectively. It would appear that the CY-induced increase in the proliferative activity of CFU-S requires the presence of L3T4+ cells. These data constitute the first in situ evidence for the physiologic relevance of immunocompetent L3T4+ cells as regulators involved in the recovery of stem cells from drug-induced myelosuppression.

Technological milestones in genomics have initiated a new approach in the development of novel anticancer drugs to specific genes. However, the heterogeneity of cancer involving multigene complexity calls upon a complementary approach to effectively develop novel anticancer drugs either to specific tumors or with broad range of anti-tumor activity. Among various techniques, fluorescence in situ hybridization (FISH) provides the opportunity to identify mRNA sequences at the subcellular level and has, therefore, become an important tool in gene expression studies. In our drug discovery and development program, we adopt a new hypothesis focusing on the whole cancer cell as a single target. A component of our unique developmental paradigm includes a drug-action profile paradigm defining the drug-specific antiproliferative effects of newly discovered investigational agents, at the molecular level using a genomic-proteomic interface. Such an approach using multicolor fluorescence hybridization on cDNA microarray and two-dimensional gel electrophoresis called, Painting with a Molecular Brush, has been successfully adopted to unravel the mechanism of action of a new anticancer agent, XK469.

Cryptophycin (CP)-52, a synthetic analog of CP-1, possesses potent and selective antiproliferative activity against human solid tumors both in vitro and In vivo. Based on an algorithm developed In this laboratory using HCT-116 human colon adenocarcinoma cells, CP-52 exhibited a time- and concentration-dependent antiproliferative effect In the In vitro clonogenic assay. Inhibition of both DNA and RNA synthesis was observed in the absence of any effect on protein synthesis following a 24-h exposure to CP-52, at a time when proliferating cells were arrested In the G 2 /M phase of the cell cycle. In summary, we interpret these data to Indicate that the selective inhibition of DNA synthesis may be a major causative factor responsible for the antiproliferative activity of CP-52 and subsequent G 2 /M arrest.

The cellular paradigm presented here defines the cellular action profile of new anticancer agents that complements our discovery and development paradigm. The main elements of this profile include a concentration clonogenicity response relationship on proliferating and plateau phase cells, flow cytometry studies assessing progression delay and apoptosis, macromolecular synthesis inhibition, and DNA damage assessment by the comet assay; other specific assessments then derive from these findings such as topoisomerase assays. XK469 is a new anticancer agent derived from the herbicide Assure that is the inactive parent compound of a family of quinoxaline analogs found to have anticancer activity in vivo. We have applied the described cellular action profile paradigm to XK469 to define a novel action at the cellular level. XK469 is a G2M phase-specific, antiproliferative agent whose activity is related to the 7-position of the chlorine ion in the benzene ring and expressed through a unique cellular action profile resulting in the irreversible increase in cyclin B1 (possibly by specific inhibition of its ubiquitination) and leading, in the absence of apoptosis, to the final mitotic arrest of HCT-116 cells in prophase with subsequent loss of clonogenicity.

XK469 (NSC 697887) is a novel antitumor agent with broad activity against a variety of tumors. Previous studies suggest that XK469 is a topoisomerase lip poison with functional activity similar to that of 4'- (9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The goal of our study was to investigate its mechanism of action further using a human HCT-116 (H116) colon tumor cell model. Concentration-survival curves with continuous exposure indicated that XK469 had low cytotoxic activity against H116 cells. Cell cycle analysis revealed that XK469 is a phase-specific cell cycle blocker that is associated with increased levels of cyclin BI, cyclin A and p53 but not CDKI (cdc2) or cyclin E. In contrast, treatment of H116 cells with m-AMSA caused a total degradation of both cyclin A and BI but enhanced expression of cyclin E and p53. Accumulation of cyclin BI in XK469-treated cells was correlated with the inhibition of cyclin BI ubiquitination, a metabolic process mandatory for proteasome-mediated protein turnover. However, no inhibition of cyclin BI ubiquitination was detected in cells treated with rn-AMSA or colchicine, a known mitotic inhibitor. Furthermore, unlike rn-AMSA, XK469 did not induce caspase activation or apoptotic cell death in H116 cells. Our results suggest that XK469 is a phase-specific cell cycle inhibitor with a unique mechanism of action that is correlated with the inhibition of cyclin BI ubiquitination and its accumulation at early M phase. (C) 2002 Wiley-Liss, Inc.

Flow cytometric analysis of nucleoids is useful to investigate nuclear matrix-DNA associations in cells. We have now applied this technique to examine whether differences in nucleoid structure can be detected between tumor and normal-like human breast epithelial cells. We have previously shown that MCF-7 tumor and MCF-10A normal-like human breast cells exhibit different levels of endogenous oxidative DNA damage as well as differences in response to hydrogen peroxide. We therefore examined whether flow cytometric analysis of nucleoids can be used to detect both endogenous DNA damage and DNA damage induced by hydrogen peroxide in these cells. Nucleoids were prepared by lysis of MCF-7 and MCF-10A cells with a high-salt buffer. The size of the DNA loops around the nuclear matrix core was detected by measuring the forward light-scatter signal in the presence of ethidium bromide. The relaxation and supercoiling of DNA in the presence of low and high concentrations of ethidium bromide, respectively, was similar in MCF-7 tumor and MCF-10A normal-like cells. After treatment of cells with 100 μM and higher of hydrogen peroxide, there was a statistically significant increase in the forward light scatter from nucleoids prepared in the presence of high concentrations of ethidium bromide, and this increase was similar in both cell lines. The changes in the forward light scatter signal with increasing hydrogen peroxide concentration did not mimic the patterns of increases in single strand breaks or formation of 5-hydroxymethyl-2′-deoxyuridine that we reported previously. This indicates that the flow cytometric technique probably detects other types of changes induced by hydrogen peroxide.

“You’ve come a long way, baby!” is an adage that characterizes the substantial advances made in the applications of flow and image cytometry since their inception, some few decades ago. Prior to the last ten years, much of the progress had been of a technical nature, one that was focused on improving instrumentation. At the present time, and probably even more so in the near future, the majority of effort has been, and will be, directed to biomedical applications aimed at measuring physiologically-relevant cell functions in both normal and cancerous tissue. A great deal of discussion has centered on how best to utilize these data to alter the course of effective medical treatment in both a prospective and interactive fashion. There are several important but difficult issues that need to be addressed in future applications.

The aim of this study was to systematically characterize possible rhG-CSF effects on the murine megakaryocyte-platelet system (untreated and recovering from chemotherapy or extramedullary irradiation). In untreated, splenectomized male B6D2F1 mice, rhG-CSF treatment (50 micrograms/kg/d for up to 8 d) markedly decreased femoral megakaryocytopoiesis. CFU-Meg, small acetylcholinesterase-positive (SAChE) cells, and megakaryocytes were significantly reduced to 35-70%; platelets, however, were not affected. Peripheral CFU-Meg and CFU-GM increased up to 200-fold. Following a single injection of 5-FU (150 mg/kg) on day 0, rhG-CSF (50 micrograms/kg/d) on days 1-8 suppressed the megakaryocytopoietic recovery as indicated by significantly lower platelet numbers on day 9. Granulopoietic recovery was accelerated by rhG-CSF. When rhG-CSF treatment was started on day 5, no beneficial effect on granulopoietic recovery was observed, but again platelet levels were significantly lower on day 9, indicating that within the first 4 d of rhG-CSF application, recruitment or lineage competition was not a critical event. To test for the effects of extramedullary irradiation on circulating progenitors, mice pretreated with 50 micrograms/kg/d of rhG-CSF for 8 d received irradiation to the chest with 500 cGy resulting in a substantial kill of circulating CFU-Meg and CFU-GM of up to 99%. However, this striking decrease of blood progenitors did not significantly affect their total body contents. This study indicates that rhG-CSF treatment can impair bone marrow megakaryocytopoiesis, which might be an important consideration for those clinical situations that carry a high potential for treatment-induced thrombocytopenia.

Thirteen previously untreated patients with metastatic melanoma entered into a phase II chemo-immunotherapy trial were monitored immunologically during treatment. Treatment consisted of dacarbazine (DTIC) 750 mg/m2 and cisplatin 100 mg/m2 on day 1 followed by interleukin-2 (IL-2) 4 x 10(6) U/m2 by daily intravenous bolus on days 12-16 and 19-23. Cycles were repeated every 28 days. On days 1 (pretreatment), 12, 16, and 23 of each cycle, lymphokine-activated killer (LAK) cell and natural killer cell activity as well as total lymphocyte count and CD3, CD4, CD8, and CD56 lymphocyte subsets were analyzed. Despite pretreatment with full-dose cytotoxic chemotherapy, all patients were able to respond immunologically to IL-2. Spontaneous LAK cell activity was generated by the end of each course of IL-2 administration and persisted for at least 5 days thereafter. Lymphocytosis was maximum at 5 days after IL-2 administration and included increased numbers of all measured lymphocyte subsets. IL-2 administration caused a relative increase in CD56+ cells and a relative decrease in CD3+ cells. There was a direct correlation between the increase in LAK cell activity and the increase in CD56+ lymphocytes. Antitumor responses occurred in five patients but these responses did not correlate with any of the measured changes in LAK activity or lymphocyte subsets. DTIC and cisplatin administered in this schedule does not abrogate the immunological effects of IL-2.

We have examined the role of natural killer (NK) cells in situ in the recovery of marrow hematopoiesis in B6D2F1 mice receiving various doses of total-body irradiation (TBI) as a well-characterized model for treatment-induced myelosuppression. Applying an in situ cytotoxic approach for ablating NK 1.1 cells, we have demonstrated that NK 1.1 cells differentially inhibit the recovery of hematopoietic stem cells (CFU-S) and their progenitor cells committed to granulocyte-macrophage differentiation from a sublethal dose of TBI (9 Gy) while not affecting the recovery of progenitor cells committed to either erythroid or megakaryocyte differentiation from TBI. However, recoveries of CFU-S and progenitor cells were unaffected by the ablation of NK cells prior to a moderate dose of TBI (2 Gy). These findings provide in situ evidence that NK cells are potential inhibitors of hematopoietic recovery from treatment-induced myelosuppression.

The R3327 class of rat prostate tumors consists of both the androgen-dependent R3327 adenocarcinoma and androgen-independent sublines, the R3327At spindle cell tumor, and the R3327A, a squamous cell carcinoma. We have developed in vitro clonogenic cell assays to measure and compare systematically the proliferative potential of these tumors following various monodispersion techniques. Linear relationships between the number of monodispersed tumor cells cultured at low cell density and the number of colonies formed 10 days later establish these assays as the first quantitative cellular approach to those proliferative subpopulations ultimately responsible for the growth of these tumors. We have chosen the name colony forming cell-prostatic adenocarcinoma (CFC-PA) to refer to the members of the proliferative subpopulation of the R3327 tumor. An ultrastructurd comparison of R3327 adenocarcinoma tissue sections with the cells produced during culture provides evidence that the cells of the proliferative subpopulation may be derived from the acinar epithelium of the tumor.

We have employed flow cytometry (FCM) and cell sorting to quantitate and study megakaryocytes in mouse and rat femoral marrow following their 20- to 30-fold concentration by centrifugal elutriation (CE). This enrichment of megakaryocytes permitted the first determination of their DNA-related fluorescence by FCM analysis following DNA staining. Fluorescence distributions of CE-enriched cell fractions following supravital staining with Hoechst 33342 were similar to those following chromomycin A3 staining of ethanol-fixed cells. Microscopic examination of cells sorted onto glass slides on the basis of their DNA-related fluorescence following supravital staining together with specific acetylcholinesterase staining for megakaryocytes indicated that megakaryocytes generally increased in cell size with increasing DNA content. This technologic application represents a significant advance in the study of megakaryocytopoiesis, since the kinetics of either the normal or perturbed population can now be studied rapidly and quantitatively.

Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the "shape" of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.

The density profile of mouse bone marrow megakaryocytes, as determined by discontinuous albumin density gradient centrifugation, was characterized by a single density population (1.088 to 1.119 g/ml) with a peak density of 1.10 g/ml and a maximum enrichment of 15.5. This single population comprised both mature and immature megakaryocytes. The density profile of small, acetylcholinesterase-positive cells, a class of potential megakaryocyte precursors, was almost identical to that of morphologically recognizable megakaryocytes.

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.

T lymphocytes from normal human controls and sickle cell disease (SCD) patients were isolated from peripheral blood and cultured for 72 hours following addition of phytohemagglutinin. The ratio for the fraction of cells in DNA synthesis (S phase) over the fraction in G2 phase (S/G2) was significantly higher in SCD patients in comparison to the controls (mean +/- SD) (4.01 +/- 0.78 vs. 2.78 +/- 0.76, P less than 0.02). Following in vivo zinc supplementation to two subjects, the S/G2 ratio was normalized. We conclude that the distribution of T lymphocytes in cell cycle is altered in SCD patients and that this effect may be zinc-dependent.

SummaryA plasma culture system has been used successfully to grow and quanti-tate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinester-ase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 104 nucleated cells plated.

Cell colonies derived from the clonogenic tumor cell [colony-forming cell, prostate adenocarcinoma (CFC-PA)] assayed in vitro from the R3327 rat prostate adenocarcinoma demonstrate prostate acid phosphatase activity when assayed histochemically and convert testosterone to stanolone. The number of CFC-PA/10(4) cells plated in steroid-free cultures was increased following the addition of testosterone or stanolone and decreased following the addition of 17 beta-estradiol. The decreased rate of growth of the R3327 tumor in castrated male inbred Copenhagen rats when compared to the growth measured in normal (intact) male and female inbred Copenhagen rats was reflected in a large decrease in the number of CFC-PA/10(4) cells plated from tumors grown in castrated male rats when compared to the values obtained from tumors that were grown in normal male and female rats. Furthermore, the replacement of fetal calf serum with normal male or castrated male rat serum resulted in little change in CFC-PA/10(4) cells plated in cultures established from tumors grown in castrated rats, although significant increases in CFC-PA were observed in cultures established from tumors grown in normal male or female rats.

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye : DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.

The stem cell-megakaryocyte-platelet cell system is responsible for the production of blood platelets which are essential to the initiation of hemostasis and maintenance of the integrity of the blood vasculature. Platelet production is dependent upon the steady supply of megakaryocytes, which are themselves incapable of cellular proliferation, being almost exclusively in a state of cytoplasmic maturation (14). The production of megakaryocytes is thus dependent upon progenitor cells that are: (a) morphologically unidentifiable by conventional cytology; (b) committed to megakaryocyte differentiation; and (c) capable of cellular proliferation—including endoreduplication of DNA to produce polyploid cells at various ploidy levels—in addition to cell mitosis. Ultimately, of course, these progenitors must be derived through differentiation or “commitment” of pluripotent hematopoietic stem cells by a process we do not presently understand.

A technique for separating mouse bone marrow megakaryocytes by unit gravity velocity sedimentation is described using a modified sedimentation chamber. The velocity sedimentation profile for megakaryocytes comprises three peaks corresponding to sedimentation velocities of 30, 60, and 100 mm/hr. Morphologic examination of megakaryocytes in the 30 mm/hr peak reveals them to be primarily immature with the latter two peaks corresponding to larger, more mature megakaryocytes.

We have systematically analyzed the proliferative properties of the clonogenic cells of the R3327 transplantable rat prostate adenocarcinoma (colony forming units, prostatic adenocarcinoma, CFU-PA). Pronase was determined to be more effective than either trypsin or collagenase in obtaining the largest number of viable cells from a solid R3327 tumor. Digestion with 2.5 mg/ml yielded a maximum of 5.6 X 10(4) CFU-PA/g of tumor tissue, with higher concentrations resulting in s substantially lower fraction of CFU-PA while producing larger overall cell yields. Bacto-agar at 0.35% supported the growth of the largest number of CFU-PA and was sharply concentration dependent with concentrations greater than 0.4% completely suppressing CFU-PA growth. The addition of conditioned medium (CM) from R3327 liquid cell cultures to agar cultures resulted in a specific twofold increase in CFU-PA/10(4) cells, whereas CM from R3327A cells was less effective and CM from rat skin fibroblasts least stimulatory. The addition of washed rat red blood cells (RBC) either within the agar culture itself or in an overlayer was highly stimulatory, resulting in as much as a fivefold increase in CFU-PA to 6 to 8 x 10(4)/g.

It is clear from extensive in vitro data that different subsets of lymphocytes stimulate and inhibit the growth of hematopoietic stem and progenitor cells. In order to clarify the complexity of the network between regulatory lymphocytes and hematopoietic target cells, we have examined the stimulatory and inhibitory effects derived from different lymphoid subsets. The regulatory influence of lymphocytes is transmitted mainly through the release of cytokines including the interleukins, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-beta and the interferons, all of which have non-specific effects on a variety of hematopoietic cells. Since these cytokines amplify the effects of other, more lineage-specific cytokines (e.g., erythropoietin, thrombopoietin and granulocyte or macrophage colony-stimulating factor) on the proliferation and differentiation of progenitor cells, the present review supports the conclusion that lymphoid subsets play a critical role in ensuring an optimal hematopoietic response to specific demands.

A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C- arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.

The definition of the cytokinetics of megakaryocytic differentiation has rested mainly on morphologic classifications that lack a quantitative data base. The approach that we have developed comprises the systematic combination of new techniques for quantitative megakaryocyte separation and correlated, multiparameter analysis of multiply labeled single cells by flow cytometry. We have coined this approach a multivectorial analysis of megakaryocytic differentiation. Modifications of combined centrifugal elutriation (CE) and density gradient centrifugation permit the rapid collection of large numbers of intact, essentially homogeneous, marrow megakaryocytes. These cells, labeled simultaneously with multiple markers of DNA content and surface membrane immunofluorescence have been subjected to correlated, dualparameter analysis by flow cytometry and cell sorting, in an attempt to relate polyploidization (as a unique megakaryocytic property) to cytoplasmic progress towards end-state platelet formation. This approach provides a reasonable data base for an objective appraisal of the interactive roles of nuclear endoreduplication and cytoplasmic development during differentiation. Furthermore, the potential usefulness of matrix models and other innovations inherent in their exploitation hold the promise of describing regulatory mechanisms that are poorly understood, both in normal megakaryocytes and those produced in disease.