John L. Lewis

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression.

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27Kip1 to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27Kip1 expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27Kip1 on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27Kip1 have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27Kip1, cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.

Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids

Summary. Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR‐ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor‐deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X‐irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU‐GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide‐stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR‐ABL influences the expansion of the malignant clone in CML.

Progenitor cells divide symmetrically to generate new colony-forming cells and clonal heterogeneity

Summary. Self‐renewal is the most fundamental property of haemopoietic stem and progenitor cells. However, because of the need to produce differentiated cells, not all cell divisions involve self‐renewal. We have used a colony replating assay to follow the fates of individual haemopoietic progenitor cell clones. For this, human myeloid colony‐forming cells (CFCs) were cultured by standard methodology. Onset of proliferation and growth rates were established by a video recording method. Individual colonies were replated several times to document the rate of clonal extinction, and the numbers of secondary, tertiary and quaternary CFCs. The clonogenic population exhibited similar kinetics in terms of onset of proliferation and growth rate. Clonal extinction was progressive so that only 30 ± 7% (mean ± standard error of the mean; n = 4) of the original primary colonies formed quaternary colonies after the third replating step. However, individual primary CFCs that produced colonies throughout the experiment generated, on average, 40 ± 8 secondary and tertiary CFCs overall. The values obtained in standard culture conditions were modified when granulocyte colony‐stimulating factor (G‐CSF) or G‐CSF plus interleukin 3 were used to stimulate colony growth, showing that the kinetics of colony formation respond to extrinsic regulation. Examination of the replating potential of individual secondary colonies in the clones demonstrated that they generated different numbers of tertiary colonies. The data best fit a stochastic model of haemopoietic cell development where event probabilities can be modified by extracellular factors.

Phosphatidylinositol‐3 kinase inhibitors reproduce the selective antiproliferative effects of imatinib on chronic myeloid leukaemia progenitor cells

We investigated the role of the phosphatidylinositol‐3 kinase (PI‐3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210Bcr‐Abl. The effect of imatinib on the expression of PI‐3K pathway proteins was investigated by kinase assays and Western blotting; PI‐3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self‐renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210Bcr‐Abl with imatinib indirectly suppressed the activity of PI‐3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI‐3K pathway in p210Bcr‐Abl‐mediated signalling in primary CML progenitor cells. The PI‐3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0·001) and reducing CML cell proliferation (P = 0·0003). This differential effect was attributable to dysregulated signalling by granulocyte colony‐stimulating factor in CML. The responses of individual patients cells to wortmannin correlated with their responses to imatinib (P = 0·004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon α (IFNα) (P = 0·016). Imatinib‐resistant K562 cells were sensitive to LY294002. Inhibition of the PI‐3K pathway may be common to imatinib and IFNα and reflect dysregulated cytokine signalling. As imatinib‐resistant cells remained sensitive to wortmannin and LY294002, targeting the PI‐3K pathway may provide an alternative therapy for imatinib‐resistant patients.

A two-color BCR–ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia

The t(9;22) translocation resulting in the fusion of BCRand ABLgenes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two‐color BCR–ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR–ABLfusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR–ABL fusion gene (≤368 kb); the “gap” between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two‐color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR–ABL and ABL–BCRin CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte‐macrophage‐colony‐forming cell (CFU‐GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0.19 ± 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 ± 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR–ABL‐positive and ‐negative interphase cells and may be of use in the clinical monitoring of CML. Genes Chromosomes Cancer 23:109–115, 1998.

A role for the Fas/Fas ligand apoptotic pathway in regulating myeloid progenitor cell kinetics

Bone marrow from wild-type mice and mice with mutated Fas (lpr) or mutated Fas ligand (gld) was used to investigate the role of the Fas/FasL system in the regulation of myeloid progenitor cell kinetics.Granulocyte-macrophage colony-forming cells (CFU-GM) were measured by a standard colony assay and the proliferative activity of CFU-GM was measured by replating primary colonies and observing secondary colony formation. Fas expression was restored to lpr mouse bone marrow cells by retrovirus-mediated gene transfer and gld mouse marrow cells were treated with soluble FasL. Wild-type marrow cells were treated with YVAD (a caspase inhibitor) or anti-Fas monoclonal antibodies. There were greater frequencies of myeloid progenitor cells (CFU-GM) in lpr and gld mouse marrow compared to wild-type (WT) marrow (p = 0.0008). The proliferative capacity of CFU-GM was also significantly greater for lpr and gld CFU-GM compared to WT CFU-GM (p = 0.0003 and 0.0001, respectively). Retrovirus-mediated restoration of Fas into lpr marrow, and provision of soluble FasL (sFasL) to gld CFU-GM reduced CFU-GM proliferation to WT levels. Treatment of WT CFU-GM with YVAD or anti-FasL monoclonal antibody increased CFU-GM proliferation to the levels found in lpr and gld CFU-GM. YVAD significantly increased and anti-Fas significantly reduced the proliferative capacity of human CFU-GM (p = 0.015 and 0.04, respectively).Fas, FasL, and caspase activation may play an important role in regulating myeloid progenitor cell kinetics.

Abnormal kinetics of colony formation by erythroid burst-forming units (BFU-E) in chronic myeloid leukaemia.

We have investigated the kinetics of colony formation by progenitor cells in chronic myeloid leukaemia (CML) using erythroid burst‐forming units (BFU‐E) as a model system. For this, we scored the numbers of subcolonies produced by individual BFU‐E in cultures of normal marrow and blood cells and in cultures of CML blood cells. The formation of an erythroid burst consisting of a single subcolony was taken as evidence for immediate terminal differentiation; the formation of multiple subcolonies was taken as evidence for commitment to terminal differentiation only after several cell generations. Therefore the probability of differentiation can be obtained by scoring the numbers of subcolonies in individual erythroid bursts. We found that the probability of differentiation is decreased (P = 0.0004) and the number of subcolonies increased (P = 0.01) in CML BFU‐E compared with normal BFU‐E. The cellularity of the BFU‐E was also increased in CML. Using the probabilities of differentiation and renewal obtained from the BFU‐E cultures the results fitted the predictions of a stochastic branching model. These results indicate that (a) commitment to terminal erythroid differentiation occurs over several cell generations in populations of BFU‐E, (b) the probability of commitment to terminal differentiation (PD) within a particular population of BFU‐E, remains a constant independent of the number of cell generations involved, (c) PD is lower during burst formation by CML BFU‐E than by normal BFU‐E, and (d) commitment to terminal differentiation occurs over more cell generations in CML burst formation than in normal burst formation. Therefore a reduced probability of differentiation may be a primary defect and could explain the expansion of the erythroid progenitor cell compartment in CML.

Evaluation of ‘discordant maturation’ in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (CFU‐GM)

The ‘discordant maturation hypothesis’ proposes that the most mature proliferating cells in chronic‐phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph‐positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (PΔ assay for PΔ cells) which allows investigation of the kinetics of granulocyte‐macrophage progenitor (CFU‐GM) production. The frequencies of these primitive (PΔ) cells were similar in CML blood (14.5/105 mononuclear cells), CML marrow (17.3/105) and normal marrow (11.6/105) The average frequency in normal blood is only 0.58/106. The absolute numbers of PΔ cells in CML patients are therefore greatly increased. The average numbers of CFU‐GM produced by individual PΔ cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU‐GM produced by individual CML PΔ cells was subnormal there was a relative increase in the number of day 7 CFU‐GM compared with the number of day 14 and 21 CFU‐GM, which agrees with the ‘discordant maturation hypothesis’. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU‐GM produced by PΔ cells or, alternatively, the production of CFU‐GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.

Restenosis after percutaneous transluminal angioplasty

Abstract Background: Determine the feasibility of studying the natural history of the atherosclerotic plaque following percutaneous transluminal angioplasty (PTA), using duplex scanning. Methods: Twenty-three patients with 40 stenoses (>70% and 2.0 was used to indicate >50% lumen diameter reduction. Results: Thirty stenoses were available for measurement and analysis. Mean reduction in plaque thickness after angioplasty was greater in echolucent plaques (2.33 ± 0.9 mm) than echogenic plaques (0.83 ± 0.6 mm; P 2.0) at 6 months was 12 of 30 (40%) remaining unchanged at 1 year; of the lesions that restenosed, 33% recurred before week 8 and the remainder between weeks 8 and 24, suggesting different mechanisms. During follow-up, all plaques showed “growth”; 2 mm in the remaining 13 (43%; group B). The incidence of restenosis (PSVR >2.0) was 4 of 17 (23%) in group A and 8 of 13 (61%) in group B ( P Conclusion: Duplex scanning provides valuable information on both luminal diameter and plaque thickness; it may be used to study the natural history of plaques following angioplasty and also the effects of therapeutic agents aimed at reducing restenosis.

Progenitor cells from patients with advanced phase chronic myeloid leukaemia respond to STI571 in vitro and in vivo

STI571 targets p210(BCR-ABL) in chronic myeloid leukaemia (CML). In vitro, STI571 reduces self-replication (replating ability) by chronic-phase CML CFU-GM. Here, we studied CFU-GM in advanced-phase (accelerated and blast crisis) CML. The numbers and self-replication of CFU-GM in advanced phase were greater than in the chronic phase. Self-replication by CFU-GM from advanced phase patients was reduced by STI571 or IFN alfa to the same extent as in the chronic phase. The reduced replating ability induced by STI571 correlated with that induced by IFN alpha (r=0.73). STI571 treatment in vivo also reduced replating ability and the numbers of CFU-GM/ml of blood.