Myrtle Y. Gordon

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.

Factors influencing the false positive and negative rates of BCR-ABL fluorescence in situ hybridization.

BCR‐ABL fluorescence in situ hybridization has a useful role to play in experimental and clinical investigations of chronic myeloid leukaemia. However, the interpretation of results is complicated by variability in the false positive rate (FPR) and false negative rate (FNR). We therefore examined the effects on FNR and FPR of three factors, namely, the criteria used for defining a fusion signal, nucleus size, and the genomic position of the ABL breakpoint. We established two different criteria for BCR‐ABL positivity: by criterion A cells were scored as positive when BCR and ABL signals were overlapping or touching and by criterion B cells were positive if they satisfied criterion A or if the signals were separated by up to one signal diameter. We measured nucleus size and Philadelphia (Ph) positivity in 573 cells from normal persons and 787 cells from the Ph+ SD‐I cell line and related results to FNRs and FPRs. We also assessed the FNR in Ph+ CFU‐GM colonies from five patients with different ABL breakpoints. We showed that each of these factors influenced the FNR and FPR. The less strict criterion (B) for Ph positivity increased the FPR but reduced the FNR, the FPR increased as the nucleus size decreased, and the FNR was greatest in CML cells with a 5′ ABL breakpoint. We conclude that these factors should be considered when evaluating the results of FISH studies to detect the BCR‐ABL fusion gene and that analogous factors may influence results of FISH studies directed at other fusion genes. Genes Chromosom. Cancer 18:246–253, 1997.

Potential Mechanisms of Action of Interferon-α in CML

Treatment with interferon-alpha (IFN-alpha) adequately controls the leukemic cell mass in the majority of newly diagnosed patients with chronic myeloid leukemia (CML). However, the degree of response ranges from no hematologic response to complete suppression of the leukemic clone. The mechanism(s) by which IFN-alpha elicits these responses is unknown, but in vitro studies have indicated that IFN-alpha might function by (1) selective toxicity against the leukemic clone, (2) enhancement of immune regulation, and (3) modulation of bone marrow microenvironmental regulation of hematopoiesis. Using in vitro clonogenic assays we were unable to demonstrate that IFN-alpha selectively inhibited the proliferation of CML progenitor cells. We also found no difference in the expression of LFA-3 on normal or CML CD34+ cells. However, by panning and co-culturing hematopoietic cells on monolayers of bone marrow stromal cells, grown with and without IFN-alpha, we found that IFN-alpha enhanced the adhesion of CML progenitors to stromal cells, whereas adhesion by normal progenitor cells was essentially unaffected. This enhanced adhesion by CML progenitor cells was associated with a reduction in neuraminic acid levels in the extracellular matrix overlying stromal cells. Therefore, it is possible that one of the mechanisms by which IFN-alpha exerts its regulatory effect on the leukemic clone is through enhancement of hematopoietic cell-microenvironmental cell interactions.

Contact-mediated inhibition of human haematopoietic progenitor cell proliferation may be conferred by stem cell antigen, CD34

INTRODUCTIONnThe function of CD34, a transmembrane sialomucin expressed by human haematopoietic progenitor cells, is poorly understood. Its structure suggests it may act as a cell adhesion and signalling molecule.nnnMATERIALS AND METHODSnKGIa cells and primary CD34-positive marrow cells were tested for their ability to aggregate in the presence of the anti-CD34 antibody QBEND10; CFU-GM colonies were grown using standard methods and tested for their content of colony-forming cells by replating; haematons were isolated from marrow by filtration; the phosphorylation of CD34 was investigated by immunoprecipitation and Western blottingnnnDISCUSSIONnCD34-positive cells in human bone marrow, like KG1a cells, aggregate when incubated with QBEND10. Staining aggregates with anti-CD34-FITC revealed that aggregation involved co-localisation of CD34 at intercellular binding sites. We examined myeloid colonies (CFU-GM) grown from normal human bone marrow cells, and multicellular aggregates (haematons) separated from freshly aspirated marrow by filtration, and found CD34-positive cells bound together with co-localisation of the CD34 at the binding sites. This finding shows that CD34-positive cell-cell adhesion occurs physiologically in vitro and in vivo. QBEND10-induced aggregation of KG1a and CD34-positive cells was enhanced by staurosporine (a protein kinase C inhibitor) and inhibited by genistein (a protein tyrosine kinase inhibitor). Moreover, aggregated cells had increased phosphorylation of tyrosine on CD34 and translocation of protein kinase C (PKC) to the cytoplasm, compared with non-aggregated cells. We used the ability of primary colonies to produce secondary colonies on replating as a functional parameter and found that the replating ability of the colonies was increased by treatment with genistein (P=0.003). In addition, the ability of individual samples of primary CD34-positive cells to undergo QBEND10-induced aggregation and the ability of CD34-positive cell-derived colonies to produce secondary clones on replating were inversely related (r=0.86).nnnCONCLUSIONnOur results suggest that homotypic aggregation of haematopoietic progenitor cells may be an important mechanism for preventing inappropriate proliferation in vivo. Thus, regulation of expression of the CD34 molecule may play an important role in maintaining the normal level of haematopoietic activity by contact-mediated inhibition of progenitor cell proliferation.

Stem cells as a treatment for chronic liver disease and diabetes

Advances in stem cell biology and the discovery of pluripotent stem cells have made the prospect of cell therapy and tissue regeneration a clinical reality. Cell therapies hold great promise to repair, restore, replace or regenerate affected organs and may perform better than any pharmacological or mechanical device. There is an accumulating body of evidence supporting the contribution of adult stem cells, in particular those of bone marrow origin, to liver and pancreatic islet cell regeneration. In this review, we will focus on the cell therapy for the diseased liver and pancreas by adult haematopoietic stem cells, as well as their possible contribution and application to tissue regeneration. Furthermore, recent progress in the generation, culture and targeted differentiation of human haematopoietic stem cells to hepatic and pancreatic lineages will be discussed. We will also explore the possibility that stem cell technology may lead to the development of clinical modalities for human liver disease and diabetes.

A Novel HSV-1 Virus, JS1/34.5−/47−, Purges Contaminating Breast Cancer Cells From Bone Marrow

Purpose: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5−/47−) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. Experimental Design: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5−/47− infection. Subsequently, the biological effects of JS1/34.5−/47− infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5−/47− purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. Results: We show effective killing of human breast cancer cell lines with the JS1/34.5−/47− virus. Furthermore, we show that treatment with JS1/34.5−/47− can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. Conclusion: Collectively, our data show that the JS1/34.5−/47− virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5−/47− can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.

CD34+ cell selection in chronic phase chronic myeloid leukaemia: a comparison of laboratory grade columns.

CD34 positive (CD34+) cell selection is increasingly used for a number of important applications including gene therapy studies, ex vivo expansion and purging. However there are no data regarding the use of different technologies for CD34+ cell selection in chronic myeloid leukaemia (CML). We therefore compared the performance of three laboratory grade CD34+ selection columns (MiniMACS, Cellpro Ceprate LC and Baxter Isolex 50), using CML chronic phase peripheral blood (PB) and bone marrow (BM). With different CML samples the CD34+ purity from the three columns was equivalent, but comparing five paired samples the Ceprate purity was greater than MiniMACS, at 92.5 and 80.9%, respectively, Pu2009=u20090.04. Combining results from paired and unpaired CML samples, MiniMACS (nu2009=u20097) gave a higher CD34+ yield than Ceprate LC (nu2009=u20098) or Isolex 50 (nu2009=u20094) with a mean of 51.1%, 24.3% and 13.2% respectively, (Pu2009=u20090.04 and 0.01). Cell losses with all columns were similar. Attempts to improve the yield from the Ceprate LC columns by modifying the method were unsuccessful. Following MiniMACS and Ceprate LC separation the clonogenic potentials of CD34+ cells in the pre- and positive cell fractions were the same. The proportion of CD34+38− or CD34+DR− cells was unchanged following column separation. These data suggest that the MiniMACS column may be the best column for CD34+ cell selection in CML but these results must be confirmed using large scale clinical columns once the MiniMACS column is licensed. It is possible that variations in CD34+ cell yields between the different columns reflect differences in antibody binding affinity to CML cells, or differences in column technologies.

Stem Cell Repair and Regeneration: Volume 2

Significant research activities in the tissue engineering or regenerative medicine (the term recently used) field started in the 1970s, and there is currently great excitement over the possibility of replacing damaged body parts through regenerative medicine. Potential strategies to replace, repair and restore the function of damaged tissues or organs include stem cell transplantation, transplantation of tissues engineered in the laboratory, and the induction of regeneration by the bodys own cells. It is believed that novel cellular therapeutics outperform any medical device, recombinant protein or chemical compound.

Stem Cells Handbook

Recent developments in stem cell research have crystallised the work of haematologists, embryologists, developmental biologists and cancer researchers, and have revealed the potential of adult stem cells from different sources for tissue regeneration and repair. The stated goal of The Stem Cells Handbook was to provide the background and current understanding of what stem cells are and what they can do. Accordingly, different chapters address stem cells from the liver, pancreas, nervous system, retina and lung as well as haemopoietic stem cells and bone marrow stromal or mesenchymal stem cells. The book is not divided into sections, but the arrangement of the chapters follows a logical progression. The first chapters cover different aspects of stem cell biology and, in particular, the chapter by Huang and Ingber introduces concepts about pattern formation, which will be crucial in the future in order to develop three-dimensional tissues from stem cells in vitro without a template. The next part of the book consists of the chapters concerning stem cells from different tissues, which also address the potential of each stem cell type for future therapies. Finally, Doyonnas and Blau provide a well-balanced summary of the current state-of-the-art and pose many questions that, as yet, have no answers. For such a wide-ranging volume, The Stem Cells Handbook is remarkably successful in meeting the editor’s goal of presenting a massive amount of information in a single resource.