Myrtle Y. Gordon

During pregnancy, fetal cells enter the maternal bloodstream resulting in fetal cell microchimerism. The fetal cells persist in the mother for decades and colonize a variety of maternal organs. They are associated with maternal autoimmune diseases and may also participate in tissue repair. The identity of the microchimeric cells is not certain but they must be able to persist long-term and have potential for multitissue differentiation. Here we tested the hypothesis that the fetal microchimeric cells are primitive stem cells, represented by CD34+ adherent cells, which have a wide potential for differentiation. We isolated these stem cells from the blood of pregnant females (n = 25) and detected fetal cells of the correct gender, using fluorescence in situ hybridization, in a high proportion (71% male fetuses and 90% female fetuses; false positive rate 11%, false negative rate 29%) of cases. By RT–PCR, we demonstrated that the cells express Oct-4, Nanog and Rex-1. No fetal cells were detected in the mononuclear or total CD34+ cell populations but high frequencies (mean 11.8%) of fetal cells were detected in the adherent CD34+ cell population. These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

We analysed the subcellular distribution of p210^BCR-ABL protein using a junction-specific anti-BCR-ABL monoclonal antibody and confocal laser scanning microscopy (CLSM). Our studies have shown that p210^BCR-ABL is arranged in discrete foci in the cytoplasm of cell lines and primary CD34⁺ cells but not mononuclear cells suggesting the foci may be a feature of immature chronic myeloid leukaemia cells. We have devised a strategy to score the foci and found the mean number of foci varies between the cell types. The number of foci per cell is directly related to the level of p210^BCR-ABL expression. CLSM was also used to analyse the distribution and colocalization of CT10 regulator-like (CRKL) p210^BCR-ABL. CRKL-p210^BCR-ABL foci were completely or partially associated, touching or separate in different regions of the same cell. We also analysed the distribution of phosphorylated CRKL (pCRKL) with p210^BCR-ABL and unexpectedly found only a small proportion of pCRKL in complex with p210^BCR-ABL. The foci distribution and high levels of uncomplexed p210^BCR-ABL, pCRKL and CRKL protein suggested the possibility of a dynamic equilibrium. Imatinib promoted nuclear transport of p210^BCR-ABL-positive foci. It also disrupted complex formation between p210^BCR-ABL and casitas B-cell lymphoma and CRKL but not between p210^BCR-ABL and GRB2. Our observations of the CRKL and p210^BCR-ABL complex may be important for understanding the function of CRKL.

The control of symmetric and asymmetric division in the hematopoietic stem/progenitor cell population is critically important for the regulation of blood cell production. Asymmetric divisions depend on cell polarization, which may be conferred by location and/or interaction with neighboring cells. In this study, we sought evidence for polarization in CD34+ cells, which interact by binding to one another. In these cells, surface molecules became redistributed by mechanisms that included transport by lipid rafts, and the interacting cells were able to communicate via gap junctions. These changes were accompanied by modulation of cell cycle regulating proteins (p16Ink4a, p27kip1, cyclins D, and the retinoblastoma pathway proteins) and a reduction in progenitor cell proliferation in vitro. These results are consistent with an increase in asymmetric cell division kinetics. Accordingly, we found that interaction between CD34+ cells influenced the plane of cell division in a way that suggests unequal sharing of Notch-1 between daughter cell progeny. We conclude that interaction between CD34+ cells may coordinate cell function and participate in the control of hematopoietic stem/progenitor cell division kinetics. Disclosure of potential conflicts of interest is found at the end of this article.

Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR‐ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR‐ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR‐ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR‐ABL1, precluding accurate p210BCR‐ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid‐dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR‐ABL1‐degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR‐ABL1 and cytoplasmic effector proteins in primary CML material.

The biology of CML (chronic myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210BCR-ABL. Although CML manifests itself principally as unregulated expansion of the myeloid lineage, the lesion is present in the stem cell population and it has long been assumed that disregulated stem cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the myeloid progenitor cell population is increased. The ability to measure the PI as an endpoint of p210BCR-ABL expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.

Summary: We have investigated functional outcome of challenging primary chronic myeloid leukaemia (CML) cells with Bcr‐Abl fusion sequence‐directed RNA interference (RNAi). We targeted the Bcr‐Abl b3a2 variant, by RNAi, in primary chronic phase CML cells, and detected strikingly reduced proliferation of myeloid precursor cells expressing this variant. Lack of an effect in cells expressing a distinct Bcr‐Abl variant confirmed the specificity of the response. Through the functional targeting of an oncogene in primary human tumour cells, we have demonstrated that Bcr‐Abl enhances CML progenitor cell amplification, and that RNAi may be suitable for development as a specific anti‐leukaemia treatment.

Summary: 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 p210^Bcr‐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 p210^Bcr‐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 p210^Bcr‐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 patient's 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.