Pascal Mossuz

Effects of two sex steroids (17β estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937

We investigated the effects of two sex steroids (17beta estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 microM for estrogen and 10 microM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (> 90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro.

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34+ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA‐2), fibronectin (VLA‐4 and VLA‐5) and laminin (VLA‐6) during the maturation of Mks. We have shown that a significant percentage of CFU‐MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM‐CD34+ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA‐2, VLA‐5 and VLA‐6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA‐4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA‐4 may be involved in the adhesion of CFU‐MK and immature Mks on fibronectin, then replaced by VLA‐5 in the final stages of maturation. The expression of VLA‐6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.

Circulating Cytokine Levels as Markers of Inflammation in Philadelphia Negative Myeloproliferative Neoplasms: Diagnostic and Prognostic Interest.

Cytokines are well known mediators of numerous physiological and pathological processes. They contribute to the regulation of normal hematopoiesis but increasing data suggest that they also have a clinical impact in some hematopoietic malignancies. In particular, there is evidence that cytokines are implicated in the functional symptoms of Philadelphia negative myeloproliferative neoplasms (Ph− MPNs), suggesting that evaluation of circulating levels of cytokines could be of clinical interest for the characterization of patients at the time of diagnosis and for disease prognosis. In this review, we present the current knowledge on alteration of circulating cytokine profiles in MPNs and their role in myelofibrosis pathogenesis. Phenotypic correlation, prognostic value of cytokines, and impact of JAK inhibitors are also discussed.

Apolipoprotein A1: A new serum marker correlated to JAK2 V617F proportion at diagnosis in patients with polycythemia vera

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) characterized by an acquired gain‐of‐function mutation of the JAK2 protein (JAK2 V617F). Allele‐specific quantitative PCR has showed a JAK2 V617F dosage effect on haematological and clinical parameters of PV at diagnosis, but it is unknown whether the level of certain serum proteins might correlate with the proportion of mutated JAK2. Taking into account that such proteins could represent useful prognostic marker, we investigated the serum protein profile of PV patients by SELDI‐TOF MS. We identified apolipoprotein A1 (Apo‐A1) as a serum marker correlated to the percentage of JAK2 V617F alleles; Apo‐A1 expression being the highest for PV patients with more than 75% of mutated alleles. Immuno‐assay on an automated random immuno‐analyser confirmed the correlation between Apo‐A1 concentrations and JAK2 V617F percentages, and showed that serum Apo‐A1 assay allowed the specific discrimination of PV patients with high levels of mutated alleles (≥75%). These data suggest that Apo‐A1 assay could be a useful assay for the stratification of PV patients at diagnosis.

Extracellular Matrix Receptors and the Differentiation of Human Megakaryocytes in vitro

We investigated the expression and functions of extracellular matrix receptors (or integrins) in the course of the differentiation of human megakaryocytes (Mks) leading to the formation of platelets. Integrins beta1 or Very Late Antigens (VLA) are specialized transmembrane receptors allowing the attachment of the cells to collagen (VLA-2), fibronectin (VLA-4 and -5) and laminin (VLA-6). A proportion of committed megakaryocytic progenitor cells (CFU-MK) adhere to fibronectin but not to collagen or laminin. The early immature Mks are retained on fibronectin (30%) and laminin (12%) but not on collagen whereas large mature Mks are still adherent to fibronectin and laminin and also acquired the capacity to adhere to collagen. The expression of the different VLA in the maturation of Mks correlates well with their adhesive properties. Hence, VLA-2 is not expressed on immature Mks but is present on the mature polyploid cells. VLA-4 is detected only on immature Mks which do not seem to bear VLA-5, while this last integrin appears on late Mks. VLA-6 showed a broad distribution from the early to late stages of Mks differentiation. Integrins beta3 of the cytoadhesin family are represented by alphaIIb beta3 that is the receptor for fibrinogen and alphaV beta3 which mediates adhesion to vitronectin. AlphaIIb beta3 is present on the CFU-MK and highly expressed throughout the Mks maturation stages while alphaV beta3 expression is much lower and seems to be detected only on the late Mks. The regulation of the expression of these receptors by cytokines and their respective roles in the maturation of Mks and the final production of platelets, are discussed. The development of efficient culture systems of human Mks in the presence of the recently cloned thrombopoietin will undoubtedly help to shed more light on the molecular mechanisms of their interactions via integrins with the BM microenvironment.

Reproducible Scoring of CFU-GM and BFU-E Grown in Collagen-Based Semisolid Medium After a Short (3 h) Training

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

Effects of retinoic acid on a new human erythro-megakaryocytic cell line AP-217

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis. The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line. The effects of inducers of differentiation were studied and focused on retinoic acid (RA). Uninduced AP-217 cells produced a low level of hemoglobin (Hb) that showed a moderate but significant dose-dependent increase after 13 cis-RA induction (four times above the control at 10(-5) M). To outline this effect, AP-217 cells were cloned at limiting dilution. A subclone (clone 2) was isolated which expressed glycophorin A on 12% of cells, and showed a marked sensitivity to RA. After a 4 day induction with increasing concentrations of RA (1-10 x 10(-6) M) Hb production by clone 2 cells was enhanced 12 times over the control at the highest concentration (10(-5) M). No effect of RA on the Hb production of K-562 and HEL was observed. This increased Hb production occurred simultaneously with a growth inhibition in clonogenic cultures (20% reduction) associated with a drastic reduction of the colony size. Moreover, we demonstrated the expression of mRNA for the beta globin gene in clone 2 and AP-217-cells. This is the first report of a positive effect of RA on the erythroid differentiation of a human leukemic cell line.

Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition.

Nanoparticle (NP)–protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP’s physicochemical features significantly impact on the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography–tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies.

Ph− myeloproliferative neoplasm red blood cells display deregulation of IQGAP1-Rho GTPase signaling depending on CALR/JAK2 status

Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph- MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patients genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph- MPN pathogenesis.

Biodiversity as a barrier to glioma cell invasion

Gliomas are extremely aggressive and lethal forms of brain cancer. Unlike many other cancer types, glioma cells rarely metastasize. They spread throughout the brain and invasiveness of glioma cells is a major cause of therapeutic failure. In plant ecosystem, biodiversity acts locally as a barrier to ecological invasion. By analogy, we hypothesize that the low cell diversity of differentiated tissues, a counterpart of their functional specificity, opens the way to local cancer cell invasion. Seeding the brain tumor microenvironment with heterogeneous cell populations could be a mean to limit cancer cell invasion by enhancing cell biodiversity.