Bernard Drenou

Transdifferentiation of hepatocyte‐like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte‐like and biliary‐like cells at confluence. If hepatocyte‐like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21CIP1 and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of β‐catenin localization and HNF3β expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. Conclusion: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors. (HEPATOLOGY 2007;45:957–967.)

The HLA-G gene is expressed at a low mRNA level in different human cells and tissues

Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.

HLA-G class I gene expression in normal and malignant hematopoietic cells

The class Ib HLA-G gene encodes for a molecule which is selectively expressed in fetal placental cells. Fetomaternal tolerance could be partially explained by the interactions between HLA-G molecules and KIR receptors of decidual NK cells. To determine whether the presence of HLA-G antigens might constitute a factor of immune tolerance during the tumoral process, we compared the expression of the HLA-G gene in normal and malignant hematopoietic cells. Despite a HLA-G transcriptional activity in several lymphocytes and monocytes, no antigens are found at the cell surface or in the cytosol using the specific HLA-G mAb, 87G. This lack of expression does not appear modified in malignant hematopoietic cells. However, treatment of the monohistiocytic cell line U937 with different cytokines enabled the expression of HLA-G antigens to be induced. We suggest that the potential induction of HLA-G molecules in monocytic malignant cells following secretion of cytokines may constitute a factor of immune tolerance in patients.

Inapparent Polycythemia Vera: An Unrecognized Diagnosis

PURPOSE The Polycythemia Vera Study Group (PVSG) has established useful criteria for the diagnosis of polycythemia vera. In some circumstances, an increase of plasma volume (PV) masks that of red cell mass (RCM), with hemoglobin (Hb) and hematocrit (Ht) remaining normal. This defines the concept of inapparent polycythemia. PATIENTS AND METHODS One hundred and three patients seen in the hematology unit with the diagnosis of polycythemia vera were studied. There were 55 males and 48 females with a median age of 59 years. Ninety-five patients fulfilled the PVSG criteria. Spontaneous erythroid colonies and low serum erythropoietin level confirmed the diagnosis in the 8 other cases. Patients were classified according to Hb and Ht level. RESULTS Group A consisted of 85 patients with increased Hb and Ht defined, respectively, by Hb > 18 g/dl, Ht > 0.52 in males and Hb > 16 g/dL, Ht>0.47 in females. Group B included 18 patients (17%) with inapparent polycythemia vera (IPV) defined by a normal Hb and Ht value at diagnosis. In this group, the reasons to perform RCM were as follows: splenomegaly associated with increased platelets and/or leucocytes counts (n = 8), portal vein thrombosis (n = 5), increased platelets or leucocytes counts without splenomegaly (n = 3), and isolated splenomegaly (n = 2). The two groups were balanced in terms of age, sex, leucocyte, serum iron, and platelet level. Hemoglobin and Ht levels were significantly different between the two groups. The difference between the PV was indeed highly significant. The mean PV increase was + 9.5% (nL < +20%) in group A versus + 36.3% in group B (P < 0.00005). Red cell mass was not different between the two groups. CONCLUSIONS Increased Hb or Ht should constitute the sole criteria for RCM determination. In the context of portal vein thrombosis, isolated hyperleucocytosis, thrombocytosis, or splenomegaly, a RCM should be performed. The frequency of IPV remains to be specified but the diagnosis of polycythemia vera is probably underestimated.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Summary.  Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy‐2′,7′‐dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0·002), CD34 positivity (P = 0·041) and P‐glycoprotein activity (P = 0·01). There was a lower rate of complete remission in MRP‐positive patients versus MRP‐negative patients (23% versus 81%; P = 0·001); overall survival was also better for MRP‐negative patients (P = 0·004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

Loss of HLA molecules in B lymphomas is associated with an aggressive clinical course

Major histocompatibility complex class I molecule expression is reduced in some malignant tumours permitting escape from immune surveillance and is therefore associated with a poor prognosis. Seven cases of non‐Hodgkin lymphomas out of 300 cases of malignant lymphoproliferative disorders totally lacked expression of class I molecules as determined by flow cytometry. Clinical data confirmed a particular aggressiveness of these cases with frequent extra‐nodal involvement, a poor international prognostic index, a histological high grade and a poor outcome leading to early death in five of the seven cases. A previous diagnosis of follicular lymphoma characterized by bcl‐2 rearrangements was made in four of these cases. HLA‐G (class Ib gene), which is reported to bind killer inhibitory receptors on NK cells, was absent from the cell surface. However, it was detected in three out of four cases at the mRNA level with transcripts encoding soluble forms. Additional analysis revealed other abnormalities: class II was negative in four out of the seven NHL cases and decreased expression of β2 microglobulin was observed in all cases. Peptide transporter proteins (TAP1) were detected in various degrees by immunocytochemistry. These observations showed that total lack of class I or class II molecules is a rare event in NHL and is associated with a poor prognosis. This could support a role for specific autologous T cells in immune surveillance.

HLA-G and lymphoproliferative disorders

The immunomodulatory properties of the HLA-G molecule explain its relevance in malignancies. Our investigations in lymphoproliferative disorders show (i) a frequent and variable distribution of alternatively spliced HLA-G mRNA isoforms, (ii) a rare cell surface expression in diffuse large cell lymphomas with HLA class I loss in half of cases, and (iii) an increased serum level of sHLA-G in half of cases. The potential role of the microenvironment and/or tumoral process in HLA-G expression is discussed in the light of these data. HLA-G rather through its soluble isoform might provide a new way of immune evasion for lymphoid proliferations.

Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

BACKGROUND/AIMS Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. METHODS The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. RESULTS In EGF/pyruvate-stimulated cultures, the level of [3H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [3H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However, the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [3H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. CONCLUSIONS Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma.

Differential expression of major histocompatibility complex class Ia, Ib and II molecules on monocytes and monocyte-derived dendritic and macrophagic cells

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.

Detection of P glycoprotein activity on normal and leukemic CD34+ cells

Rhodamine 123 is transported by the transmembrane efflux pump P glycoprotein (Pgp). We used this fluorescent dye to study multidrug resistance (MDR) activity in normal and leukemic CD34+ cells. These immature cells had a high degree of MDR activity. Among leukemic cells, CD34+ leukemias had significantly higher MDR activity as compared to CD34- leukemias. Heterogeneous results in cell subpopulations, however, indicate that prognosis should be interpreted in the light of MDR analysis.