Thérèse Commes

Transforming growth factor-β1 is an autocrine mediator of U937 cell growth arrest and differentiation induced by vitamin D3 and retinoids

Vitamin D and retinoids cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic U937 leukemia cells. In the present work, we investigated the role of TGF‐β as an endogenous mediator of this process. We found that the TGF‐β1 precursor began to accumulate in cell culture supernatants soon after the addition of 1α,25 dihydroxyvitamin D3 (VD) and retinoids. We used neutralizing antibodies (AbTGF‐β) and antisense oligonucleotide (AS Oligo) to inhibit its possible effects. Our data demonstrated that AbTGF‐β partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to the chemotactic peptide fMLP, and lysozyme secretion. AS Oligo was also inhibitory, and the effects of AS Oligo and AbTGF‐β were cumulative. Cell growth inhibition induced by VD and retinoids was completely reversed, and differentiation was reduced by about 75% when both inhibitors were associated. Time course experiments based on the delayed addition of AbTGF‐β and AS Oligo showed that TGF‐β1 was required for cell differentiation 24 h after the addition of inducers. Studies on TGF‐β receptors revealed that, while the expression of type II receptor was stable, the level of type I TGF‐β receptor mRNA and the expression of the protein began to decline early during the differentiation process. As a whole, these results support the notion that an autocrine TGF‐β pathway, activated by VD and retinoids in U937 cells, is involved in the early steps of the process leading to cell growth arrest and differentiation. J Cell Physiol 178:109–119, 1999.

Expression of P-glycoprotein and anionic glutathione S-transferase genes in non-hodgkin's lymphoma

Non-Hodgkins lymphomas (NHL) are usually sensitive to chemotherapy. A certain percentage of patients are primarily or subsequently resistant to chemotherapeutic agents. Several biological mechanisms are implicated in this phenomenon, including multidrug resistance (mdr1) and glutathione S-transferase (GST pi). We investigated these two systems, using dot blot analysis, in 41 patients who presented NHL with advanced disease. There were 15 patients with low grade, 22 with intermediate grade, and 4 patients with high grade using the Working Formulation for Clinical Usage. Twenty-five patients had not been previously treated and 16 had been treated, including 13 with refractory disease. Eleven out of 25 (44%) patients overexpressed mdr1 mRNA at diagnosis as compared to 6/16 (38%) in relapse, corresponding to 6/13 (46%) refractory patients. Nine out of 25 (36%) patients overexpressed GST pi mRNA at the time of diagnosis, and 6/16 (50%) in relapse. These data indicate that overexpression of these two messengers is not acquired after treatment in NHL. Furthermore, there is no relationship between the stage or histological grade and the overexpression of these two markers. This study shows that mdr1 and GST pi gene expressions are independent of one another. With regard to the clinical response, our results also demonstrated a higher level of treatment failure in the group co-expressing the two transcripts, 6/8 (75%) patients died in progressive disease as compared to 9/15 (60%) patients without overexpression, and 2/8 (25%) vs 6/15 (40%) responded to treatment. On the other hand, overexpression of only one of the two mRNAs did not allow us to observe a difference in the clinical response. Since it seems that coexpression of the mechanisms of resistance present a better clinical impact, it would be of interest to analyse simultaneously different mechanisms involved in the resistance phenomenon in NHL.

No detectable malignant B cells in the peripheral blood of patients with multiple myeloma.

Summary Previous studies have reported the presence of idiotypic B lymphocytes in the blood of patients with multiple myeloma (MM), suggesting that they may belong to the malignant clone. This led us to investigate by southern blot analyses the pesence of tumour‐specific immunoglobulin‐gene (Ig‐gene) rearrangements in the peripheral‐blood mononuclear cells of 21 MM patients. This method was shown to detect clonal cells when they represent as little as 2% of the cell population. B‐cell‐enriched fractions were also studied in nine cases. An occasional contamination by cruclatin malignant plasma cells was carefully evaluated using immunofluorescence. Clonal rearrangements were observed in only two cases, in which a contamination by myeloma cells was evident. In these cases the use of different endonucleases clearly demonstrated that these Igclonal rearrangements involved post‐switched cells. No clonal rearrangement was found when contamination by myeloma cells was absent. Our results demonstrate the absence of detectable B cells involved in the myeloma clone in the peripheral blood of patients with MM.

Human natural killer cells suppress the proliferation of B cells

We have investigated the suppressive effect of human natural killer (NK) cells on autologous B-cell proliferation. Removal of NK cells by anti-NK-cell monoclonal antibodies (CD16, Leu 11b; Leu 7) increased by 2-3-fold the proliferative response of purified B cells activated by anti-mu and B-cell growth factor (BCGF). The inhibitory effect of NK cells was observed using recombinant IL-2 or semi-purified BCGF-I as sources of BCGF. Moreover NK cells, highly purified by centrifugation on a Percoll discontinuous density gradient, suppressed the proliferative response of purified autologous B cells activated by anti-mu and BCGF. These results show a suppressive effect of human NK cells on B-cell proliferation in vitro.

In Vitro Production of Beta2 Microglobulin by Human Myeloma Cells

AbstractSerum beta2 microglobulin (B2M) has recently been shown to be a powerful, although nonspecific, marker of multiple myeloma (MM) disease activity. Since the nature of cells producing high levels of B2M remains obscure in MM, we measured (in vitro) the spontaneous secretion of free B2M in 58 culture samples from 52 patients with MM. In comparison with samples from normal individuals and from individuals with monoclonal gammopathy of unknown significance, an abnormal secretion of B2M was found in 83% of samples containing myeloma cells. Levels of secretion significantly correlated with the percentage of tumor cells, tumor progression, and moreover, with the immunoglobulin type of the tumor. The highest levels of secretion were noted in IgG and IgA MM. Our present results would favor the hypothesis of a direct secretion of B2M by myeloma cells and emphasize the interest of B2M as a marker in a majority of (but not all) patients with MM.

The defect in peripheral blood B-cell activation in patients with multiple myeloma is not due to a deficiency in the production of B-cell growth and differentiation factors

The suppression of B lymphopoiesis is a major feature of multiple myeloma (MM). In this disease, there is a striking defect in the response of peripheral blood B cells to pokeweed mitogen (PWM). Normally, B-cell activation depends on B-cell growth factors (BCGFs) and B-cell differentiation factors (BCDFs), produced by peripheral blood mononuclear cells. We therefore evaluated whether the production of these cytokines was defective in patients with MM. We have studied the production of BCGFs (using the anti-μ assay) and, particularly, interleukin-2 and interferon-γ, two well-documented BCGFs. No defect in the production of BCGFs, interleukin-2, and interferon-γ was found in patients with active (N=14) or stable (N=10) MM, compared with healthy donors (N=13). The production of BCDFs (i.e., overall activity) was also evaluated and, more particularly, that of interleukin-6 (IL-6). This cytokine is a potent BCDF which is essential in the PWM-induced activation of B cells, acting at the terminal stages of B-cell differentiation. Again, no defect in the production of BCDFs and IL-6 was found in patients with MM. Therefore, the ability to secrete cytokines controlling the process of B-cell activation is not affected in such patients. This indicates that the profound failure of humoral immune response is not due to deficiency of peripheral blood mononuclear cells producing these factors.

Interleukin 2 production in bone marrow of normal individuals and patients associated with B-cell chronic lymphocytic leukaemia

T‐cells from patients with B‐cell chronic lymphocytic leukaemia (B‐CLL) have abnormal T4/T8 ratios and functions. Previously, we demonstrated that peripheral blood (PB) mononuclear cells from B‐CLL patients secrete significant amounts of interleukin 2 (IL2) with an apparent dysregulation of accessory cells controlling this production. In this study, IL2 production was investigated in PB and in bone marrow (BM) from patients with previously untreated B‐CLL, mostly in early stages of the disease, and compared to normal donors. A significant secretion was observed in both PB and BM from B‐CLL patients after stimulation by phyto‐haemagglutinin (PHA), with lower amounts in patients with nodular involvement of BM, as compared to interstitial or diffuse involvements. To explore the role of accessory cells in controlling IL2 production, we added phorbol ester or indomethacin to the culture system or irradiated the cells before culture. Phorbol ester significantly increased the IL2 secretion in both the PB and the BM of B‐CLL patients. The irradiation or the addition of indomethacin did not enhance the IL2 production in PB from B‐CLL patients. However, IL2 secretion increased in the BM cells from B‐CLL patients after addition of indomethacin or prior irradiation, in a similar way to that observed in PB and BM of normal controls, suggesting an apparent normal control of the IL2 production in BM from B‐CLL patients. In normal controls, we demonstrated that IL2 secretion per T‐cell from BM was 5·4‐fold greater than that from normal PB, suggesting a very efficient role of accessory cells controlling IL2 production in normal BM.

Differential long non-coding RNA expression profiles in human oocytes and cumulus cells

Progress in assisted reproductive technologies strongly relies on understanding the regulation of the dialogue between oocyte and cumulus cells (CCs). Little is known about the role of long non-coding RNAs (lncRNAs) in the human cumulus-oocyte complex (COC). To this aim, publicly available RNA-sequencing data were analyzed to identify lncRNAs that were abundant in metaphase II (MII) oocytes (BCAR4, C3orf56, TUNAR, OOEP-AS1, CASC18, and LINC01118) and CCs (NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, and VIM-AS1). These data were validated by RT-qPCR analysis using independent oocytes and CC samples. The functions of the identified lncRNAs were then predicted by constructing lncRNA-mRNA co-expression networks. This analysis suggested that MII oocyte lncRNAs could be involved in chromatin remodeling, cell pluripotency and in driving early embryonic development. CC lncRNAs were co-expressed with genes involved in apoptosis and extracellular matrix-related functions. A bioinformatic analysis of RNA-sequencing data to identify CC lncRNAs that are affected by maternal age showed that lncRNAs with age-related altered expression in CCs are essential for oocyte growth. This comprehensive analysis of lncRNAs expressed in human MII oocytes and CCs could provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

No preferential use of the VH(V) family in human multiple myeloma

A recently described immunoglobulin VH family (the VH(V) family) close to the DH and JH genes is preferentially rearranged in immature B‐cell tumours. The question of the emergence of multiple myeloma (MM) from a tumorous pre‐B cell is not yet resolved. To draw a comparison with chronic lymphocytic leukaemia (CLL), we studied the VH(V) rearrangements in 28 MM patients. A rearranged Hind III‐Bam HI fragment of 9.5 kb was detected in only one patient instead of the rearranged fragment of 8.5 kb described in CLL. Rearrangements of a member of the VH(V) family in a 9.5 kb fragment were also observed in two out of 20 lymphoblastoid cell lines obtained from peripheral blood of MM patients. We report here that the VH(V) family is not preferentially involved in this pathology and that the size of the only rearrangement obtained is larger than the 8.5 kb fragment observed in CLL. These results do not favour the hypothesis of a pre‐B cell involvement in MM.

New insights into diagnosis and therapeutic options for proliferative hepatoblastoma

Surgery and cisplatin‐based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%‐80% of patients. However, some important challenges remain in diagnosing high‐risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16‐gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four‐gene signature: hydroxysteroid 17‐beta dehydrogenase 6, integrin alpha 6, topoisomerase 2‐alpha, and vimentin, with topoisomerase 2‐alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT‐PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration–approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway–associated double‐strand DNA repair, and significantly impedes HB growth in vivo. Conclusion: The highly proliferating C2A subtype is characterized by topoisomerase 2‐alpha gene up‐regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89‐102).