A new agarose-based microsystem to investigate cell response to prolonged confinement.

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

The BMI1 polycomb protein represses cyclin G2-induced autophagy to support proliferation in chronic myeloid leukemia cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

Sex differences in the GSK3ß-mediated survival of adherent leukemic progenitors.

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3ß (GSK3ß) by serine 9-dephosphorylation. Strikingly, GSK3ß-mediated survival was restricted to leukemic progenitors from female patients. GSK3ß inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)ß(1)-integrin engagement, was specifically upregulated and controlled GSK3ß activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3ß-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3ß as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

TP63 P2 promoter functional analysis identifies ß-catenin as a key regulator of ΔNp63 expression.

The ΔNp63 protein, a product of the TP63 gene that lacks the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in several types of epithelial tissues. ΔNp63 is frequently overexpressed in squamous cell carcinoma (SCC) and in some other epithelial tumours. This overexpression may contribute to tumour progression through dominant-negative effects on the transcriptionally active (TA) isoforms of the p53 family (TAp63, TAp73 and p53), as well as through independent mechanisms. However, the molecular basis of ΔNp63 overexpression is not fully understood. Here, we show that the expression of ΔNp63 is regulated by the Wnt/ß-catenin pathway in human hepatocellular carcinoma (HCC) and SCC cell lines. This regulation operates in particular through TCF/LEF sites present in the P2 promoter of TP63. In addition, we show that ΔNp63 and ß-catenin are frequently coexpressed and accumulated in oesophageal SCC, but not in HCC. These results suggest that activation of the ß-catenin pathway may contribute to overexpression of ΔNp63 during tumour progression, in a cell type-specific manner.

Human placenta and fetal membranes express follistatin-related gene mRNA and protein.

Activin A is a placental glycoprotein and possible biological actions during pregnancy, suggested by experimental data, are the modulation of cytotrophoblast differentiation, placental hormonogenesis and uterotonins secretion. Follistatin-related gene (FLRG) is a 70 amino acids protein which binds activin A with high affinity, and which modulates its biological effects on target tissues by preventing the activin A interaction with its receptors. The present study investigated whether trophoblast, decidua and fetal membranes express FLRG mRNA (by RT-PCR) and peptide (by immunohistochemistry). Tissue specimens were collected at first and third trimester of pregnancy, from patients undergoing voluntary pregnancy interruption (no.=6; from 8 to 12 gestational weeks) and elective caesarean section at term (no.=6; 39-40 weeks of pregnancy). FLRG mRNA was expressed by the various gestational tissues both at early gestation and at term pregnancy. Immunoreactive protein was found in the trophoblast cells, epithelial amniotic and chorionic cells and maternal decidua; nevertheless, the most intense FLRG stain was detected in the walls of decidual and placental blood vessels. In conclusion, FLRG mRNA and peptide are expressed by placenta and fetal membranes. Its different immunolocalization with respect to follistatin and activin A supports a different role for FLRG in modulating activin A actions into gestational tissues.

FLRG, an activin-binding protein, is a new target of TGFbeta transcription activation through Smad proteins.

The FLRG gene encodes a secreted glycoprotein that binds to activin and is highly homologous to follistatin, an activin ligand. We cloned the promoter region of the human FLRG gene, and defined the minimal region necessary for transcription activation in a reporter-system assay. We showed that the fragment between positions -130 and +6, which consists of multiple consensus Sp1-binding sites, is required for the constitutive expression of the FLRG gene. We demonstrate here that FLRG mRNA expression is rapidly induced by TGFbeta or by transfection with Smad protein expression vectors in human HepG2 cells. We investigated the transcription-regulation mechanism of FLRG expression in HepG2 cells following treatment with TGFbeta. By deletion and point-mutation analysis of the FLRG promoter, we identified a Smad-binding element involved in the TGFbeta-inducible expression of the FLRG gene. Moreover, transactivation of the FLRG promoter by TGFbeta was compromised by dominant-negative mutants of Smad3 and Smad4 proteins. In addition, gel electrophoresis mobility-shift assays demonstrated the specific interaction of Smad3 and Smad4 proteins with the Smad-binding element consensus motif found in the FLRG promoter. Taken together, our data imply that Smad proteins participate in the regulation of expression of FLRG, a new target of TGFbeta transcription activation.

Characterization of human hematopoietic cells with short-lived in vivo repopulating activity.

Recent studies with purified hematopoietic stem cells in vitro support a model of stem cell self-renewal control that involves distinct mechanisms regulating permissiveness to and execution of lineage restriction. Such a model predicts the existence of phenotypically separable populations of hematopoietic cells that are pluripotent and either capable or incapable of extensive self-renewal. Such populations have been previously described in the mouse. We describe here the first evidence that such cells can now be identified in humans using different types of immunodeficient mice as hosts.

Expression of FLRG, a novel activin A ligand, is regulated by TGF-beta and during hematopoiesis [corrected].

OBJECTIVE: The human gene FLRG, identified from a B-cell chronic lymphocytic leukemia bearing a t(11;19) translocation, encodes a secreted glycoprotein highly homologous with follistatin. Activin A is a TGF-beta family member involved in the regulation of growth and differentiation of various types of cells, such as those of the hematopoietic system. Its biological activity is antagonized by binding with follistatin. We investigated the binding of FLRG to activin A and the expression pattern of FLRG, follistatin, and activin A during hematopoiesis. MATERIALS AND METHODS: The binding of FLRG with activin A was investigated by immunoprecipitation and Far-Western blot analysis. Gene expression was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern Blot in purified hematopoietic populations. RESULTS: We demonstrate that FLRG, like follistatin, is able to bind to activin A. In bone marrow stromal cells, both mRNA and protein FLRG levels were found to be dramatically increased by TGF-beta. FLRG and activin A are expressed in the same cells, with a higher level of expression in the myeloid cells compared with the erythroid and megakaryocytic cells. FLRG and follistatin expression were different in the hematopoietic subpopulations tested. Moreover, we observed that FLRG and activin A expression was up-regulated during hematopoiesis. CONCLUSION: FLRG and activin A are expressed in the same hematopoietic cells and regulated by TGF-beta. Moreover, FLRG interacts with activin A, suggesting that FLRG, like follistatin, participates in the diverse regulatory functions of activin A, such as those in hematopoiesis.

Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates.

In this study, it is shown that short-term exposure of normal human marrow CD34(+)CD38(-) cells to low concentrations of tumor necrosis factor (TNF) in the presence of 100 ng/mL Flt3 ligand and Steel factor and 20 ng/mL interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor, in either bulk or single-cell serum-free cultures, markedly reduces their ability subsequently to generate colony-forming cells (CFCs) in 6-week stromal cell-containing long-term cultures without affecting their viability, mitogenic response, or short-term ability to produce CFCs. A similar differential effect on the functional attributes of CD34(+)CD38(-) cells was seen when C2- or C6-ceramide, but not dihydro-C2-ceramide (an inactive analog of ceramide), was substituted for TNF. The addition of D-erythro-MAPP (a specific inhibitor of intracellular ceramide degradation) enhanced the ability of TNF to selectively eliminate long-term culture-initiating cell (LTC-IC) activity. These findings indicate that TNF can directly modulate the ability of CD34(+)CD38(-) cells to maintain their LTC-IC function at doses below those required to initiate apoptosis, cell cycle arrest, or both, and they suggest that this may be mediated by the TNF-induced generation of intracellular ceramide. Identification of a signaling intermediate that can influence primitive hematopoietic cell fate decisions offers a new approach to the investigation of signaling mechanisms in normal stem cell populations and to how these may be altered in leukemic cells.

CML and apoptosis: the ceramide pathway.

In patients with Chronic Myeloid Leukemia (CML), the neoplastic (Bcr-Abl+) progenitors are characterised by an increased proliferative activity. These cells appear to become resistant to apoptosis following growth factor withdraw. We demonstrate that despite this property, Bcr-Abl transformed cells (primitive hematopoietic progenitors or cell lines) remains sensitive to apoptosis induced by Ceramides analogues. This effect is dose dependent and occurs faster in transformed cells as compared to their normal counterparts. In addition to the classical features of apoptosis, we observed that Ceramide-treated CML cells display a rapid and sequential activation of the Bcr-Abl and PI3 kinases. We then demonstrated the role of the Bcr-Abl kinase activity in the accelerated response observed in CML cells treated by Ceramide. The PI3 kinase seems to be partly involved in the accelerated Phosphatidyl-Serine exposure observed in Bcr-Abl transformed cells. Finally, we observed that Ceramide-induced apoptosis does not seem to implicate a Bcl2 protein modulation. Taken together these results support the hypothesis of at least two independent signaling pathways initiating programmed cell death: one will be involved in apoptosis mediated by signals such as cytokine-starving is blocked by the Bcr-Abl fusion protein while the other one initiated by Ceramide is accelerated by the Bcr-Abl protein.

Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein.

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

BCR-ABL accelerates C2-ceramide-induced apoptosis.

In patients with chronic myeloid leukemia (CML), the neoplastic (BCR-ABL+) progenitor cells are characterized by an increased proliferative activity. Whether these cells are also resistant to apoptosis and if so, under what conditions remains controversial. We now show that highly purified populations of very primitive neoplastic progenitor cells obtained directly from CML patients survive and proliferate in vitro for several weeks in the absence of any added growth factors (except insulin). In contrast, purified primary normal progenitors maintained under the same conditions die rapidly. Nevertheless, both primary CML cells and BCR-ABL+ BAF3 cells show the same dose-dependent sensitivity to TNF-alpha or ceramide-induced apoptosis as their respective normal counterparts. In fact, time course studies demonstrated an even faster onset of apoptosis in ceramide-treated BCR-ABL+ BAF3 cells as compared to normal controls. BCR-ABL+ cells treated with ceramide also showed a rapid and sequential increase in the tyrosine phosphorylation of p210(BCR-ABL), p46-56SHC and p120Cbl. These findings suggest growth factor deprivation and treatment with TNF-alpha or ceramide trigger different initial events both of which can lead to apoptosis in factor-dependent hematopoietic cells. However, in the first case, activation of apoptosis is blocked by the basal activity of p210(BCR-ABL), whereas in the second, the presence of p210(BCR-ABL) appears to accelerate the onset of apoptosis by a mechanism that may involve an activation of its kinase function.

Ex vivo cytokine expansion of peripheral blood Ph-negative cells in chronic myeloid leukaemia.

Hematopoietic progenitors can be expanded ex vivo in the presence of various cytokine combinations. Since normal early progenitor or stem cells persist in the blood and bone marrow of patients with Philadelphia chromosome [Ph]-positive chronic myeloid leukaemia (CML), the selection of normal (Ph-negative) progenitor cells from CML patients would be of considerable clinical value for ex vivo purging and autologous transplantation. To obtain these cells, CD34-positive (progenitor) cells from the peripheral blood (PB) of CML patients were either pretreated or not with 5-fluorouracil (5FU) and then grown in suspension culture for 7 days with a combination of cytokines. We compared different combinations of cytokines containing interleukin-1 alpha (IL1), interleukin-3 (IL3), stem cell factor (SCF), leukemia inhibitor factor (LIF), Flt3-ligand (FLT3L), and thrombopoietin (TPO). 5FU decreased cell proliferation in the liquid culture but concurrently increased the expansion of CFU-GM. While the addition of cytokines such as FLT3L and TPO improved CFU-GM expansion. FISH and RT-PCR analysis showed that this method significantly favored a higher frequency of Ph-negative cells after expansion in liquid culture. Therefore ex vivo expansion of putatively normal hematopoietic progenitor cells from cytapheresis is feasible in CML.

BCR-ABL expression in different subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic myeloid leukemia.

In patients with chronic myeloid leukemia (CML), the leukemic (BCR-ABL+/Ph+) clone typically includes cells belonging to all of the myeloid lineages and frequently some B cells. From such observations it has been inferred that the initial BCR-ABL gene rearrangement event occurs in a pluripotent hematopoietic stem cell and that the clone subsequently generated is maintained by a subpopulation of neoplastic, BCR-ABL-expressing cells that retain at least some of the defining properties of normal hematopoietic stem cells. To test this hypothesis directly, we isolated various subpopulations of CD34+ cells from fresh or cryopreserved samples of peripheral blood from 5 CML patients with high white blood cell counts, 4 of which were selected because of their exclusive content of Ph+ progenitors (both colony-forming cells and long-term culture-initiating cells [LTC-IC]). Cells in each of the CD34+ subpopulations isolated were examined for the presence of BCR-ABL mRNA using a reverse transcriptase-polymerase chain reaction technique that reproducibly gave a positive signal from single K562 cells. BCR-ABL mRNA was detected in 117 of 147 samples (80%) in which actin mRNA was demonstrable. This included 60% to 90% of a large number of individually analyzed CD34+ cells including 46 single CD34+CD71-CD38- cells and 27 single CD34+CD71+CD38+ cells from 3 patients. In 2 of these cases, the same populations also contained a very high frequency of Ph+ LTC-IC. Our findings demonstrate BCR-ABL gene expression in neoplastic cells with functional as well as surface marker characteristics of very primitive normal hematopoietic cells. This implicates the BCR-ABL gene product directly in the acquisition by these cells of properties that alter their interactions with the microenvironment and deregulate their proliferation control.

Human immature thymocytes as target cells of the leukemogenic activity of human T-cell leukemia virus type I.

The risk of developing adult T-cell leukemia (ATL) associated with neonatal infection by human T-cell leukemia virus type I (HTLV-I) suggests that early events triggered by HTLV-I might be of crucial importance in initiating the multistep lymphoproliferative process leading several decades later to the development of leukemic disease. Thus, infection of thymocytes early in life might be directly correlated with the development of ATL. In the present study, we show that in vitro infection of mature (CD2+CD3+) or immature (CD2+CD3-) thymocytes resulted in the exogenous interleukin (IL)-2-dependent proliferation of HTLV-I-positive thymocytes, most of them displaying a CD2+CD3-CD4+ phenotype and expressing the CD25 molecule, the alpha chain of the IL-2 receptor. Furthermore, the CD80 and CD54 antigens, normally expressed by thymic stromal cells, were detected on these transformed thymocytes, indicating that HTLV-I infection may disturb the cooperation between thymocytes and their thymic environment. These HTLV-I-positive thymocytes were producing significant amounts of IL-6, which was found to be implicated in their proliferation and in the expression of CD25, as demonstrated by blocking experiments using a monoclonal antibody to IL-6. The present study suggests that immature thymocytes may provide an environment favorable to the unfolding of events leading to leukemia.

Pancreatic lymph nodes are early targets of T cells during adoptive transfer of diabetes in NOD mice.

The circulation pathway of diabetogenic T lymphocytes prior to insulitis was investigated using adoptive transfer of diabetes in the non-obese diabetic (NOD) mouse model. Transferred T cells were distinguished from recipient T cells using two strains of mice congenic at the Thy1 locus. They were monitored in the pancreas and in several lymphoid organs including thymus, spleen, and lymph nodes from pancreatic, mesenteric, axillary, inguinal and lomboaortic areas, from Day 0 to Day 15 after the adoptive lymphocytic transfer. Immunohistochemical studies showed that at Day 2 post-transfer the pancreatic lymph nodes (PLN) and to a lesser extent the spleen, are the first two organs to be infiltrated. The amount of T cells of donor origin using quantitative flow cytometric analysis was 4% and 2.6% respectively. This percentage increased to 19% in the PLN at Day 15 and did not exceed 7% in the spleen. Analysis of the expression of IL-2 receptor present at the surface of activated T lymphocytes showed that 73% of donor T cells were activated in the PLN within 3 days post-transfer in contrast to 0% in the spleen. The accumulation and activation of T cells in the PLN may imply a role of these lymphoid organs in harbouring the diabetogenic T cells during the early steps of the disease.