Dana Dvorakova

Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells.

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF‐2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF‐2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular‐mass isoforms of FGF‐2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF‐2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 → FGFR3 → FGFR4 → FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF‐2, extracellular signal‐regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF‐2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF‐2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF‐2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self‐renewal or differentiation of hESCs.

A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells.

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor‐2 (FGF‐2), which activates FGF receptors (FGFRs) and stimulates the mitogen‐activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin‐like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF‐2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF‐2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation‐related genes and downregulation of stem cell genes. Thus, exogenous FGF‐2 reinforced the pluripotency maintenance program of intracrine FGF‐2 signaling. Consistent with this hypothesis, expression of endogenous FGF‐2 decreased during hESC differentiation and FGF‐2 knockdown‐induced hESC differentiation. In addition, FGF‐2 signaling via FGFR2 activated MAPK kinase/extracellular signal‐regulated kinase and AKT kinases, protected hESC from stress‐induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self‐renewal, cell survival, and adhesion by exogenous and endogenous FGF‐2 may synergize to maintain the undifferentiated growth of hESCs. STEM CELLS 2009;27:1847–1857

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Fibroblast growth factor signaling in embryonic and cancer stem cells.

The fibroblast growth factor 2 (FGF‐2) pathway is one of the most significant regulators of human embryonic stem cell (hESC) self‐renewal and cancer cell tumorigenesis. Here we summarize recent data on the effects of FGF‐2 and its receptors on hESCs and leukemic stem/progenitor cells. Also, we discuss the similarities of these findings with stem cell renewal and differentiation phenotypes.

FGF-2 abnormalities in B cell chronic lymphocytic and chronic myeloid leukemias.

An elevated level of fibroblast growth factor-2 (FGF-2) in peripheral blood is considered to play a role in regulating the growth of leukemia cells. Here, we show that the level of plasma FGF-2 is increased in 54% of B cell chronic lymphocytic leukemias (B-CLL) and in 44% of chronic myeloid leukemias (CML). Notably, white blood cells (WBCs) from B-CLL patients contain 18, 22 and 24 kDa isoforms of FGF-2 whereas WBCs from CML patients contain only the 24 kDa isoform. Furthermore, as cultured B-CLL WBCs release 18 kDa FGF-2 into the medium, they constitute a potential source of FGF-2 in the blood. In a receptor binding assay, 125I-FGF-2 binds weakly to B-CLL WBCs, whereas the ligand binds more strongly to CML WBCs. Correspondingly, FGF-2 is unable to activate mitogen-activated protein kinase kinase (MEK) and its substrate, extracellular signal-regulated kinase (ERK), in B-CLL cells, whereas phosphorylation of both these cell growth-related kinases increases following treatment of CML WBCs. We conclude that B-CLL WBCs secrete FGF-2 with no apparent autocrine actions. In contrast, WBCs in CML bind FGF-2 provided by other FGF-2-hyperproducing cells and activate the MEK/ERK kinase cascade, possibly to modulate cell growth.

Inactivation of p53 and deletion of ATM in B-CLL patients in relation to IgVH mutation status and previous treatment.

Inactivation of p53 and deletion of ATM in B-CLL patients in relation to IgVH mutation status and previous treatment

Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient‐specific NPM1 mutations

Nucleophosmin (NPM1) mutations in exon 12 are the most common genetic alternation in cytogenetically normal AML (CN‐AML). Although mutation types A, B, and D represent the majority of cases, rare mutation variants of the NPM1 gene in individual patients do occur. In this study, we have evaluated a novel, DNA‐based real‐time quantitative polymerase chain reaction (RQ‐PCR) for the detection of three of the most commonly occurring mutations and for six rare patient‐specific mutation types, which represent 28% of all of the NPM1 mutations in our group of 25 CN‐AML patients. Furthermore, the prognostic relevance of NPM1‐based monitoring of minimal residual disease (MRD) in peripheral blood (PB), bone marrow (BM), and in specific cell subsets (CD34+, CD34−, CD34dim) of BM were evaluated. In 80% of the evaluable patients, a molecular relapse preceded a hematological relapse. Moreover, in this subset of patients, the molecular relapse occurred at a median of 97 days before the hematological relapse. Our compartment analysis showed a strong correlation between BM and PB (r = 0.907, P < 0.001) as well as a high copy number of mutated NPM1 in CD34+ BM cells. In conclusion, we have demonstrated applicability of our presented RQ‐PCR method for a large percentage of mutated NPM1 patients with CN‐AML as well as the usefulness for long‐term follow‐up monitoring of MRD and the prediction of hematological relapse. Am. J. Hematol., 2010.

Changes in the expression of FGFR3 in patients with chronic myeloid leukaemia receiving transplants of allogeneic peripheral blood stem cells.

Fibroblast growth factor receptors (FGFR1–4) are implicated in various cellular events, including cell growth and transformation. Here, we showed that patients suffering from chronic myeloid leukaemia (CML) express high levels of FGFR3 mRNA in white blood cells (WBCs). After stem cell transplantation and reconstitution of haematopoiesis, the expression of FGFR3 decreased and was maintained at low levels that are typical of healthy individuals. However, FGFR3 expression became upregulated again in those patients that had accelerated BCR/ABL rearrangement and underwent relapse of leukaemia. Our findings suggest that, in CML, the changing levels of FGFR3 transcripts in WBCs may have prognostic significance.

Increased expression of fibroblast growth factor receptor 3 in CD34+ BCR-ABL+ cells from patients with chronic myeloid leukemia

Previously, we showed that expression of myeloma-associated (proto)oncogene fibroblast growth factor receptor 3 (FGFR-3) is increased in white blood cells from patients with chronic myeloid leukemia (CML). The abnormal expression was returned back to the normal levels as soon as these patients reconstituted their hematopoiesis following transplantation of allogeneic peripheral blood stem cells. The aims of this study were: (1) to define population(s) of cells overexpressing FGFR-3, and (2) to determine the expression of FGFR-3 during the clinical course of the disease. We show that the vast majority of FGFR-3 transcripts as well as FGFR-3 protein arise from CD34+ BCR-ABL+ cells. Although increased levels of FGFR-3 were found in majority of late chronic phase patients treated with interferon α or hydroxyurea, the expression of FGFR-3 was always lowered following treatment with BCR-ABL tyrosine kinase inhibitor STI571. Compared to unstimulated cells, high levels of FGFR-3 were also identified in CD34+ cells from granulocyte colony-stimulating factor-mobilized blood stem cell harvests from healthy donors, suggesting a potential growth factor-dependent basis for elevated expression of FGFR-3 in CML. These findings have implications for the involvement of FGFR-3 in malignant hematopoiesis and depict FGFR-3 tyrosine kinase in CD34+ leukemic cells as a possible target for tyrosine kinase inhibitors.

The assessment of human organic cation transporter 1 (hOCT1) mRNA expression in patients with chronic myelogenous leukemia is affected by the proportion of different cells types in the analyzed cell population

The monitoring of hOCT1 mRNA expression in patients with chronic myelogenous leukemia (CML) was used for predicting the response to imatinib treatment. However, different cell populations from patients who received various degrees of pretreatment were used for this analysis. Therefore, several biases in the results and their interpretation may arise. We investigated hOCT1 mRNA expression in different cell populations of peripheral blood (PB) from healthy volunteers and in imatinib nave de novo CML patients by analyzing changes in hOCT1 mRNA expression during the first 6 months of imatinib therapy. The hOCT1 mRNA expression was significantly higher in PB polymorphonuclears compared to mononuclears. The hOCT1 mRNA expression in total PB leukocytes is, therefore, preferentially determined by the percentage of polymorphonuclears. Expression in each analyzed group of cells was always significantly lower in imatinib nave de novo CML patients compared to healthy volunteers. This difference disappeared after the initiation of imatinib therapy, suggesting that CML tumor burden and the degree of pretreatment at the time of monitoring were both influencing factors.