Separation of triphenyl atropisomers of a pharmaceutical compound on a novel mixed mode stationary phase: a case study involving dynamic chromatography, dynamic NMR and molecular modeling.

Analysis of atropisomers is of considerable interest in the pharmaceutical industry. For complex chiral molecules with several chiral centers hindered axial rotation can lead to formation of interconverting diastereomers that should be separable on achiral stationary phases. However, achieving the actual separation may be difficult as the on-column separation speed must match or be faster then the rate of isomer interconversion. Often, this requirement can be satisfied by using low-temperature conditions and by improving selectivity via use of chiral stationary phases. In the current study, we present an alternative approach utilizing an Obelisc R column, a novel mixed mode stationary phase that provided acceptable separation of triphenyl atropisomers inside a conventional HPLC temperature range. The separation was investigated under various chromatographic conditions. The interconversion chromatograms exhibited classic peak-plateau-peak behavior indicating the simultaneous atropisomer separation and interconversion. The elution profiles were integrated in order to deconvolute the peak areas of the "pure" (non-exchanged) and interconverted species; these data were used to obtain kinetic information. Analysis of retention data rendered thermodynamic information on the mechanism of retention and selectivity. Chromatographic kinetic data were complemented with variable-temperature NMR and molecular modeling studies, which provided additional support and insights into the energetics of the interconversion process.

Preparation and novel reduction reactions of vinamidinium salts.

Substituted acetic acids and formamides react in the presence of phosphorus oxychloride to yield the vinamidinium hexafluorophosphate salts 5a-d, 6a-d, and 7 in moderate to good unoptimized recrystallized yields (40-67%) as easily handled nonhygroscopic solids. The 1,3-differentially substituted vinamidinium salts 8 was prepared by amine exchange in 81% yield as are the cyclic diazapinium salts 9 and 10 in > 76% yield. The symmetrical 2-chlorovinamidinium 11 was prepared by displacement of 3 in 71% yield. The 2-chlorovinamidinium salts are cleanly reduced to the parent vinamidinium salts 12-16 using HI or PPh3/pTSA in up to 99% assay yield.

A general preparation of pyridines and pyridones via the annulation of ketones and esters.

A general preparation of pyridines 4a-f from stabilized ketones 3a-c and aryl ketones 3d-f is described. The annulation of stabilized esters 3g,h gives access to the corresponding 2-pyridones 4g,h. The annulation reactions proceed in fair to excellent yields (46-87%) with vinamidinium hexafluorophosphate salts 2a-d containing electron-withdrawing groups at the beta-position. The mechanism of the reaction was investigated by NMR and proceeds through the formation of a dienaminone intermediate.

A practical synthesis of a COX-2-specific inhibitor.

A number of synthetic strategies to the Cox-2 specific inhibitor 1 have been described. These studies have led to the identification of a novel pyridine construction using annulation of ketone 2 using a vinamidinium species 29 and ammonia in 97% assay yield. Three approaches to the synthesis of ketone 2 are described that allow for its preparation in large quantities in >65% overall yield from methyl 6-methylnicotinate.

Factors associated with fatigue in a family medicine clinic in the United Arab Emirates.

BACKGROUND: Fatigue is a common symptom in Family Medicine and it has many associated factors. The Arabian Gulf provides a unique setting for studying these factors, in particular the UAE where rapid development has been a prominent feature. OBJECTIVES: The aim of the study was to sample a group of GP attenders and examine the factors which were associated with fatigue in the UAE. METHODS: A fatigue scale, psychological questionnaire, detailed history, physical examination and laboratory testing were administered to a sample of attenders at a Family Medicine clinic. RESULTS: Fatigue was more prevalent than in western studies (males 34.0%, females 38. 2%). It was strongly associated with anxiety, especially in younger adults, and it has been recognized that rapid social change is felt most acutely in young adults and adolescents. Depression in females was also a factor. Lack of exercise, obesity and illiteracy played a minor role in the severity of fatigue. CONCLUSIONS: Fatigue appears to be a cultural 'idiom of distress', a way of expressing anxiety or depression in a rapidly changing society.

An efficient preparation of vinamidinium hexafluorophosphate salts.

Substituted acetic acids or acetyl chlorides react with phosphorus oxychloride in DMF to yield the vinamidinium salts 3a-j in moderate to excellent recrystallized yields (28-90%). The cations are conveniently isolated as their hexafluorophosphate salts, which are easily handled nonhygroscopic solids. The nitro compound 3l is prepared in 91% yield by nitration of the parent vinamidinium 3k. The X-ray crystal structure is reported for the 2-phenyl isomer 3e and displays minimal overlap of the two pi-systems.

Evidence for differences in the mechanisms by which antibodies against CD44 promote adhesion of erythroid and granulopoietic progenitors to marrow stromal cells.

Adhesive interactions between haemopoietic progenitor cells and stromal elements involve a number of different molecules, some of which may be progenitor- lineage- and stage-specific. CD44 is one such molecule, although little is known about the mechanism(s) by which it is involved. In this study, several anti-CD44 monoclonal antibodies (mAb) increased the adherence of clonogenic cells, without affecting the total number of types of progenitors recoverable from the adhesion cultures. All of these mAb recognized epitopes on the globular head of CD44. In contrast, two mAb that recognized other regions of CD44 reduced progenitor adhesion to stroma. The mechanism by which one of the anti-CD44 mAb (L178) enhanced progenitor adhesion did not involve CD44-crosslinking, and was independent of VLA-4-, VLA-5- or LFA-1-mediated interactions, Ca or Mg cations, or accessory cells. In addition, CD44 expression on both progenitors and stromal cells contributed to L178-enhanced progenitor adhesion. Baseline adherence of erythroid progenitors to stroma required tyrosine kinase activity, whereas that of granulopoietic progenitors did not. However, the increase in adhesion did require tyrosine kinase activation. Additional experiments suggested that enhanced adhesion of CFU-GM to stroma may also be adenylate cyclase-dependent. Taken together, the present studies indicate both similarities and differences in the mechanisms of CD44-mediated adhesion of erythroid and granulopoietic progenitors to stromal cells.

Antibodies to the beta 1-integrin chain, CD44, or ICAM-3 stimulate adhesion of blast colony-forming cells and may inhibit their growth.

Early hematopoietic progenitor cells adhere to bone marrow stromal cells (BMSCs) mainly through VLA-4/VCAM-1 interactions. However, many adhesion molecules are expressed by these cell types. Cell adhesion molecules not only mediate adhesion; some are also capable of triggering cellular signaling events. These signals can be induced by several anti-adhesion molecule antibodies. In this study, we investigated the effects of several of such stimulatory antibodies against alpha L (CD11a), alpha 4 (CD49d), and beta 1 (CD29) integrin chains, ICAM-3 (CD50), CD34, CD44, and CD45. All antibodies reacted strongly with CD34-positive bone marrow (BM) cells, but only those against beta 1 integrin (TS2/16, Lia1/2) or CD44 (NKI-P2) reacted with BMSCs. To test the ability of these antibodies to stimulate adhesive interactions, we analyzed their effect on stroma-adherent blast colony-forming cells (Bl-CFCs). We found that TS2/16 (anti-beta 1 integrin), NKI-P2 (anti-CD44), and 152-2D11 (anti-ICAM-3) enhanced adhesion of BM mononuclear cells to stroma (TS2/16:3.4-fold, NKI-P2: 3.8-fold, 152-2D11: 2.6-fold) when compared with isotype-control-treated cells. The increase in stroma-adherent cells was accompanied by an increase in Day 5-7 blast colonies of 3.8-, 2.6-, and 1.9-fold, respectively. One antibody against CD29:Lia1/2 strongly inhibited the formation of blast colonies, an effect that was at least partially caused by its growth-inhibitory activity. Of the other antibodies tested, none displayed growth-modulatory activity. We have found previously that Bl-CFCs depend strongly on VLA-4 and VCAM-1. However, in TS2/16- or 152-2D11-treated cultures, we observed not only these, but also VLA-5-dependent adhesive interactions. In contrast, VLA-5 did not appear to be involved in NKI-P2-treated cultures. Our data indicate that interactions mediated by beta 1-integrins are involved in the growth of Bl-CFCs. Furthermore, interactions mediated by beta 1-integrins, CD44, and ICAM-3 differentially modulate VLA-4- and VLA-5-dependent progenitor/BMSC interactions.

VLA-4-mediated interactions between normal human hematopoietic progenitors and stromal cells.

The alpha 4 beta 1 integrin very late activation antigen-4 (VLA-4) has been implicated to play a role in the adhesive interactions between hematopoietic progenitor cells (HPC) and bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin (FN). Here, we summarize some of the recent advances made in the elucidation of the role of these particular adhesive interactions for the regulation of normal hematopoiesis. HPC bind to stroma mainly through VLA-4/VCAM-1 interactions. There is evidence which suggests that more primitive HPC constitutively express VLA-4 in a high-affinity state. In vitro studies in the mouse have shown that monoclonal antibodies (mAb) against VLA-4 partly block the development of lymphocytes, myelopoietic cells, and erythropoiesis, whereas in the human system outgrowth of TdT+ B cells is severely retarded by such mAb. In vivo studies revealed that VLA-4 is involved in erythropoietic development, and is particularly important for homing and lodgement of HPC in the bone marrow. Hematopoiesis in mice with deficient expression of alpha 4 integrin or VLA-4's ligand VCAM-1 appears to develop normally. However, chimeras developed from wild-type blastocysts and beta 1 -/- embryonic stem cells do not contain beta 1 -/- hematopoietic cells, although these are present as blood islands in the yolk sac. These beta 1 -/- hematopoietic cells are capable of forming colonies, indicating that beta 1-integrin is not involved in hematopoietic differentiation, but is primarily important for migration of hematopoietic cells into the fetal hematopoietic organs. In addition to the role of VLA-4 in migration, it may also have other regulatory functions. It has been demonstrated that ligation of VLA-4 induces phosphorylation of the protein tyrosine kinase (PTK) pp125FAK as well as other proteins which may be involved in the regulation of ligand affinity. Indeed, it has been shown that tyrosine kinase-dependent stimulation of CD34+ hematopoietic cell lines with c-kit ligand (KL), IL-3 or GM-CSF transiently activates the ability of VLA-4 to bind to VCAM-1 or FN. These events are most probably involved in the induction of quiescence in HPC which adhere to stromal cells. This claim was recently substantiated: when HPC were treated with Fab fragments of an anti-VLA-4 mAb, entry into S-phase of the cell cycle was prevented. Taken together, the present data point to a role for VLA-4 in HPC migration, cell cycle regulation, erythropoiesis and B-lymphopoiesis. Moreover, these insights may explain how defects in adhesive behavior of leukemic HPC through VLA-4 contribute to their dysregulated growth and provide a rationale for therapeutically correcting those defects.

Schistosoma mansoni infection in mice augments the capacity for interleukin 3 (IL-3) and IL-9 production and concurrently enlarges progenitor pools for mast cells and granulocytes-macrophages.

Mast cells and granulocytes-macrophages (GM) are components of the host defense system against worm infections, including schistosomiasis. Here we report the kinetics of changes in the number of colony-forming cells (CFC) for mast cells and GM during the course of a primary experimental infection of mice with Schistosoma mansoni cercariae over a period of 24 weeks postinfection (p.i.). Concurrently, we measured known myelopoietic and/or mast cell-stimulating cytokines (i.e., interleukin 3 [IL-3] and IL-9) in pokeweed mitogen-activated spleen cell-conditioned medium. Our results show that during the acute phase of the hepatic granulomatous reaction, the numbers of both mast-CFC and GM-CFC were significantly elevated in bone marrow. However, while femoral GM-CFC numbers had returned to normal control values at week 16 p.i., femoral and splenic mast-CFC numbers remained significantly elevated until week 20 p.i., which corresponds to the chronic fibrotic phase of hepatic granulomatous inflammation. Increased GM-CFC numbers correlated with elevated IL-3 levels, while increased mast-CFC numbers paralleled the increased IL-9 concentrations in spleen cell-conditioned medium. By the reverse transcription-PCR method, enhanced expression of IL-3 and IL-9 transcripts was found in RNA samples obtained from livers and spleens of infected mice. Our data demonstrate that during the course of infection of mice with S. mansoni, the coordinate need for mast cells and GM is at least partly regulated at the stage of progenitor cell commitment in the bone marrow and spleen. It appears that IL-3 and IL-9 help to promote at this stage the ultimate generation of mature effector cells.

Kinetics of interleukin-6 production after experimental infection of mice with Schistosoma mansoni.

It has been reported that interleukin-6 (IL-6) is expressed in cells of acute inflammatory granulomas experimentally induced in mice by eggs of Schistosoma mansoni. Moreover, in vitro IL-6 was shown to enhance the cytotoxic activity of human platelets against larvae of S. mansoni. To elucidate further a proposed biological significance of this cytokine during the course of schistosomiasis, we studied the kinetics of IL-6 production and concomitantly performed a histopathological analysis of the livers in BALB/c mice subcutaneously infected with S. mansoni cercariae. Over a period of 24 weeks postinfection (p.i.) we monitored serum IL-6 levels, IL-6 production in vitro by pokeweed mitogen (PWM)-stimulated spleen cells as well as IL-6 mRNA expression in livers, spleens and kidneys. We found significantly elevated IL-6 levels in PWM-stimulated spleen cell-conditioned media (SCM) at weeks 6 to 20 p.i., peaking at week 10 p.i. In contrast, serum IL-6 concentrations started to rise not before week 8 but remained significantly elevated above normal control values until week 24 p.i. The time pattern of enhanced IL-6 mRNA expression detected in spleens and livers, but not in kidneys, as well as the rises of IL-6 in SCM and with a delay of 2 weeks in serum samples correlated with the onset of the egg-induced inflammatory reactions as well as the incidence and the number of the granulomas observed histopathologically in the livers of infected mice. Our data emphasize both a local and a systemic role of IL-6 in the host immune response following infection of mice with S. mansoni.

Regulated plasma levels of colony-stimulating factors, interleukin-6 and interleukin-10 in patients with acute leukaemia and non-hodgkin's lymphoma undergoing cytoreductive chemotherapy.

Endogenous plasma levels of granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF),IL-6 and IL-10 were measured in a total of 70 patients undergoing cytoreductive chemotherapy for treatment of acute leukaemia or non-Hodgkin's lymphomas. the diagnoses were acute myeloid leukaemia (AML; n = 30), acute lymphoblastic leukaemia (ALL;n=6), non-Hodgkin's lymphomas (NHL; n=11) and other malignant haematological disorders including myelodysplastic syndromes (n=23). After chemotherapy, plasma G-CSF was elevated (mean 5.6 ng/ml; range 1.2-10 ng/ml), and was inversely correlated with white blood cell counts (WBC) (r=-0.7, p<0.001). Occurrence of fever (T>38.0 degrees C) during severe myelosuppression (WBC<1x10(9)/1) was associated with an additional increase of G-CSF levels (P<0. (P<0.001). Plasma IL-6 correlated significantly with fever (range <1 to 1100 pg/ml, mean 130 pg/ml; r=0.5, P<0.001) but revealed only a weak association with WBC or platelet counts. In patients treated with recombinant G-CSF (n = 9), an association between IL-6 and fever was still observed after chemotherapy. During the nonfebrile status (total n = 242; AML n = 124), IL-6 levels remained <9 pg/ml in 90% of cases, whereas G-CSF increased with leucopenia (r = -0.72;P<0.001). In contrast, endogenous GM-CSF remained normal and IL-10 showed only a slight increase (21% of samples; maximum 22 pg/ml) in severe leucopenia. In particular, IL-10 levels did not correlate with G-CSF or IL-6 levels. We conclude that systemic release of G-CSF and IL-6 is obviously nit abrogated by cytoreductive chemotherapy in acute leukaemia and NHL may add to the therapeutic efficacy of recombinant cytokines. Also, plasma levels of G-, GM-CSF or IL-6 appear to be regulated by separate mechanisms.

Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.

We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.

Adhesion of human hematopoietic progenitor cells to bone-marrow-derived stromal cells is enhanced by antibodies to CD44.

It has been suggested that CD44 mediates adhesive interactions between hematopoietic progenitor cells and the stromal microenvironment. Ligands of CD44 include several extracellular matrix components, such as hyaluronic acid and fibronectin. Antibodies against CD44 have been shown to induce homotypic T cell aggregation, and to stimulate T and natural killer cell activity. We hypothesized that CD44 could similarly amplify interactions between blast-colony-forming cells and bone marrow stromal cells (BMSCs). Indeed, we have previously found that the anti-CD44 antibody NKI-P2 enhanced VLA-4-dependent interactions. Here, we studied an additional panel of nineteen anti-CD44 antibodies from the 5th Workshop on Leukocyte Differentiation antigens, to find out whether amplification was associated with a particular CD44 epitope. None of these antibodies showed inhibitory activity, whereas nine significantly increased the number of blast colonies more than 2-fold. Seven of these recognized epitope 1, and two epitope 2. More than 4-fold enhancement was only observed with epitope 1 antibodies: 4.C3 (4.4-fold), 212.3 (6.3-fold), L178 (9.1-fold), and NIH44-1 (9.2-fold). Our data suggest that primarily epitope 1 is associated with enhancement of colony formation. Furthermore, the findings support a role for CD44 as an amplifier in progenitor-BMSC interactions.

VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells.

The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CEC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against beta 1-, alpha 4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the alpha 5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as BI-CFC, remains to be determined.

Role of cell type-specific promoters in the developmental regulation of T1, an interleukin 1 receptor homologue.

Murine T1, an orphan receptor related to interleukin 1 receptors, exhibits a bimodal expression in mouse development. The molecular analysis of cultured cell lines now reveals the contribution of alternate promoters of the T1 gene to its differential expression. In nonhemopoietic cell types, where T1 synthesis in vivo is restricted to organogenesis and neoplasia, a recently characterized AP-1-dependent promoter directs a proliferation-associated expression of the gene. In hemopoietic cells, which express the T1 receptor throughout ontogenesis in vivo, T1 gene activity is driven by a novel serum factor-independent, constitutive promoter. The tissue-specific use of constitutive versus growth factor-dependent alternate promoters thus directs the permanent activity of the T1 gene in hemopoietic tissue versus the developmentally restricted expression of the gene in nonhemopoietic tissues in vivo.

IL-4 renders mast cells functionally responsive to endothelin-1.

It has previously been shown that mouse bone marrow-derived mast cells (BMMC) synthesize and secrete endothelin-1 (ET-1) and express ETA-type endothelin receptors (ETA-R). The study presented here was designed to elucidate the influence of different cytokine conditions for cellular differentiation and maturation on the ability of primary mouse BMMC to respond to exogenous ET-1. BMMC were grown for 2 wk in IL-3 alone and then cultured for 2 to 3 wk with kit ligand (KL) and/or IL-3 in the presence or absence of IL-4. ET-1 induced a very rapid (< or = 1 min) and dose-dependent release of histamine and serotonin from BMMC cultured in the presence of both IL-3 and IL-4. The effect of ET-1 was quantitatively comparable with IgE/Ag-induced mediator release and comprised up to 20% and 16% of total cellular histamine and serotonin, respectively. In BMMC grown with KL or KL plus IL-3, a substantial effect of ET-1 on amine release was only observed when IL-4 had been included in the culture medium. These IL-4 effects could not be observed if BMMC grown in IL-3 and/or KL were preincubated for 1 or 24 h with IL-4 before activation with ET-1, suggesting that a differentiation process rather than a functional priming effect had been initiated by IL-4. In BMMC, the histamine and serotonin release induced by ET-1 (10(-6) M) was inhibited by an ETA-R-specific antagonist (cyclic [D-Asp-Pro-D-Val-Leu-D-Trp]) in a dose-dependent manner, with complete inhibition at an antagonist concentration of 10(-8) M. ET-1 stimulated leukotriene C4 biosynthesis up to 4.5-fold in BMMC cultured in the presence of IL-4. No such ET-1 effect was observed in BMMC cultured in media containing IL-3, KL, or a combination of both cytokines. Peritoneal cells (containing 2 to 3% serosal mast cells) obtained from BALB/c mice released 87 +/- 2% of histamine within 1 min after challenge with ET-1. Our results demonstrate that ET-1 can directly act as a histamine and serotonin secretagogue and as a stimulator of leukotriene C4 production in mast cells. IL-4 appears to be critically involved in the differentiation of immature mast cell precursors to an ET-1-reactive phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity.

We describe the establishment of two permanent Simian virus 40-transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B-cell line BL70.

Growth factors and cell kinetics: a mathematical model applied to Il-3 deprivation on leukemic cell lines.

We assume the existence of a specific G1 protein which is an initiator of DNA replication. This initiator is supposed to be synthesized according to Michaelis-Menten kinetics. In order to start DNA replication, it is assumed that this G1 specific protein must be produced in a required amount. Intra-cellular growth inhibitors and extra-cellular growth factors control the production of the initiator. This model allows to calculate the average G1 phase time as a function of the various chemical concentrations of nutrients, enzymes, growth inhibitors and growth factors. This model is compared to cell kinetics experiments on a leukemic cell line responding to Interleukin 3 deprivation. The curves giving the experimental average G1 phase times with respect to Interleukin-3 concentrations are fitted by the mathematical model with a quite good agreement.

Measurement of efflux from G1-phase in a growth factor dependent cell line.

In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the G1-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.