Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

Mitochondrial dynamics and metabolic regulation control T cell fate in the thymus.

Several studies demonstrated that mitochondrial dynamics and metabolic pathways control T cell fate in the periphery. However, little is known about their implication in thymocyte development. Our results showed that thymic progenitors (CD3-CD4-CD8- triple negative, TN), in active division, have essentially a fused mitochondrial morphology and rely on high glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). As TN cells differentiate to double positive (DP, CD4+CD8+) and single positive (SP, CD4+ and CD8+) stages, they became more quiescent, their mitochondria fragment and they downregulate glycolysis and OXPHOS. Accordingly, in vitro inhibition of the mitochondrial fission during progenitor differentiation on OP9-DL4 stroma, affected the TN to DP thymocyte transition by enhancing the percentage of TN and reducing that of DP, leading to a decrease in the total number of thymic cells including SP T cells. We demonstrated that the stage 3 triple negative pre-T (TN3) and the stage 4 triple negative pre-T (TN4) have different metabolic and functional behaviors. While their mitochondrial morphologies are both essentially fused, the LC-MS based analysis of their metabolome showed that they are distinct: TN3 rely more on OXPHOS whereas TN4 are more glycolytic. In line with this, TN4 display an increased Hexokinase II expression in comparison to TN3, associated with high proliferation and glycolysis. The in vivo inhibition of glycolysis using 2-deoxyglucose (2-DG) and the absence of IL-7 signaling, led to a decline in glucose metabolism and mitochondrial membrane potential. In addition, the glucose/IL-7R connection affects the TN3 to TN4 transition (also called ß-selection transition), by enhancing the percentage of TN3, leading to a decrease in the total number of thymocytes. Thus, we identified additional components, essential during ß-selection transition and playing a major role in thymic development.

Temporal Gene Expression Profiles Reflect the Dynamics of Lymphoid Differentiation.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

Intrathymic SIRPa cDC subsets organization in normal and stress conditions reveal another level of cDCs heterogeneity.

Three major subsets constitute the dendritic cells (DCs) pool in the thymus. They play key roles in self-antigen-specific thymocyte deletion and in the development of immunoregulatory T cells. Resident SIRPa- conventional DCs (cDCs, CD11c+ PDCA1lo ) are derived from intrathymic progenitors, whereas migratory SIRPa+ cDCs and plasmacytoid DCs (pDCs, CD11c+ PDCA1+ ) originate from extrathymic sites. Here, we describe the organization and the shaping of cDC populations at the steady state and under stress conditions in wild-type and mutant mice (CD3eKO, IL7RaKO, and Flt3LKO). In neonates, the thymus is mainly composed of SIRPa- -resident cDCs, whereas both cDC subsets are present in equal proportions in the adult. Upon thymus colonization, migratory SIRPa+ cDCs gain expression of phenotypic markers in a microenvironment dependent way. Here, we show that both processes are deeply impacted by mutations affecting T cell development. Under stress conditions such as sublethal irradiation, intrathymic resident SIRPa- cDCs are the first to regenerate the thymic cDC pool. Upon bone marrow transplantation, migratory SIRPa+ cDCs become the main source of thymic cDCs. These successive waves of regeneration eventually lead to a balance between resident and migratory DCs within the newly colonized thymus. These findings highlight an unrevealed division of labor between resident and migratory subsets for the organization/establishment of the thymic cDC compartment.

T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way.

Innate lymphoid cells (ILCs) have been shown to be significantly affected in the small intestine lamina propria and secondary lymphoid organs (SLOs) of conventional lymphopenic mice. How ILCs are regulated by adaptive immunity in SLOs remains unclear. In T cell-deficient mice, ILC2s are significantly increased in the mesenteric lymph nodes (MLNs) at the expense of CCR6+ ILC3s, which are nonetheless increased in the peripheral lymph nodes (PLNs). Here, we show that T cells regulate lymph node-resident ILCs in a tissue- and subset-specific way. First, reducing microbial colonization from birth restored CCR6+ ILC3s in the MLNs of T cell-deficient mice. In contrast, T cell reconstitution resulted in the contraction of both MLNs ILC2s and PLNs ILC3s, whereas antagonizing microbial colonization from birth had no impact on these populations. Finally, the accumulation of MLNs ILC2s was partly regulated by T cells through stroma-derived IL-33.

Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment.

Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.

Single-cell analysis of thymocyte differentiation: identification of transcription factor interactions and a major stochastic component in αß-lineage commitment.

T cell commitment and αß/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αß/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αß/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αß-committed cells do not express the pre-TCR and revealed a major stochastic component in αß-lineage specification.

Cognate antigen stimulation generates potent CD8(+) inflammatory effector T cells.

Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88-deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8(+) inflammatory effector cells in a lymph node can mobilize 10(7) cells in 24 h, including lymphocytes, natural killer cells, and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

Chronic HIV infection affects the expression of the 2 transcription factors required for CD8 T-cell differentiation into cytolytic effectors.

CD8 T cells lose the capacity to control HIV infection, but the extent of the impairment of CD8 T-cell functions and the mechanisms that underlie it remain controversial. Here we report an extensive ex vivo analysis of HIV-specific CD8 T cells, covering the expression of 16 different molecules involved in CD8 function or differentiation. This approach gave remarkably homogeneous readouts in different donors and showed that CD8 dysfunction in chronic HIV infection was much more severe than described previously: some Ifng transcription was observed, but most cells lost the expression of all cytolytic molecules and Eomesodermin and T-bet by chronic infection. These results reveal a cellular mechanism explaining the dysfunction of CD8 T cells during chronic HIV infection, as CD8 T cells are known to maintain some functionality when either of these transcription factors is present, but to lose all cytotoxic activity when both are not expressed. Surprisingly, they also show that chronic HIV and lymphocytic choriomeningitis virus infections have a very different impact on fundamental T-cell functions, "exhausted" lymphocytic choriomeningitis virus-specific cells losing the capacity to secrete IFN-γ but maintaining some cytotoxic activity as granzyme B and FasL are overexpressed and, while down-regulating T-bet, up-regulating Eomesodermin expression.

Synergistic CD40 signaling on APCs and CD8 T cells drives efficient CD8 response and memory differentiation.

The role of CD4 help during CD8 response and memory differentiation has been clearly demonstrated in different experimental models. However, the exact mechanisms of CD4 help remain largely unknown and preclude replacement therapy to develop. Interestingly, studies have shown that administration of an agonist aCD40ab can substitute CD4 help in vitro and in vivo, whereas the targets of this antibody remain elusive. In this study, we address the exact role of CD40 expression on APCs and CD8 T cells using aCD40ab treatment in mice. We demonstrate that aCD40 antibodies have synergetic effects on APCs and CD8 T cells. Full efficiency of aCD40 treatment requires CD40 expression on both populations: if one of these cell populations is CD40-deficient, the CD8 T cell response is impaired. Most importantly, direct CD40 signaling on APCs and CD8 T cells affects CD8 T cell differentiation differently. In our model, CD40 expression on APCs plays an important but dispensable role on CD8 T cell expansion and effector functions during the early phase of the immune response. Conversely, CD40 on CD8 T cells is crucial and nonredundant for their progressive differentiation into memory cells. Altogether, these results highlight that CD40-CD40L-dependent and independent effects of CD4 help to drive a complete CD8 T cell differentiation.

c-Fos accelerates hepatocyte conversion to a fibroblastoid phenotype through ERK-mediated upregulation of paxillin-Serine178 phosphorylation.

Transforming growth factor beta (TGF-beta) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-beta, EGF or a combination of both. Cotreatment with EGF and TGF-beta, but neither cytokine alone, induced the conversion of hepatocytes to a fibroblastoid phenotype and increased their motility in Boyden chambers. EGF/TGF-beta cotreatment induced a higher effect on ERK phosphorylation compared to TGF-beta treatment alone. It also induced an increase in total and phosphorylated Ser(178) paxillin, a protein previously implicated in cell motility. This response was inhibited by two specific MEK inhibitors, indicating the involvement of the ERK pathway in paxillin activation. Overexpression of c-Fos correlated with increased cell scattering and motility, higher levels of ERK activation and phospho Ser(178) paxillin, increased levels of EGF receptor (EGF-R) mRNA and higher EGF-R phosphorylation levels following EGF/TGF-beta cotreatment. Conversely, siRNA-mediated invalidation of c-Fos delayed the appearance of fibroblastoid cells, decreased EGF-R mRNA and downregulated ERK and Ser(178) paxillin phosphorylations, indicating that c-Fos activates hepatocyte motility through an EGF-R/ERK/paxillin pathway. Since c-Fos is frequently overexpressed in hepatocarcinomas, this newly identified mechanism might be involved in the progression of hepatic tumors in vivo.

c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1.

AIM: To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation. METHODS: IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3beta and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments. RESULTS: Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3beta, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3beta induced by serum stimulation and decreased Cyclin D1 stability in the nucleus. CONCLUSION: Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.

Jun D cooperates with p65 to activate the proximal kappaB site of the cyclin D1 promoter: role of PI3K/PDK-1.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the kappaB2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-kappaB proteins. Serum stimulation increased the expression of p65, p50, c-Fos, c-Jun and JunD and induced the recruitment of p65, p50 and JunD to the kappaB2 site of the promoter in DNA pull-down assays. Chromatin immunoprecipitation (ChIP) analysis confirmed the serum-induced recruitment of JunD to the promoter in vivo and showed that the presence of JunD was dependent on the presence of p65 and p50, indicating a protein-protein-dependent mechanism of JunD recruitment. Serum-induced activation of protein binding to kappaB2 correlated with high levels of phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylation. Both LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), and overexpression of a dominant-negative form of PDK-1 inhibited the JunD-stimulating effect in reporter assays. LY294002 also prevented the serum-induced recruitment of JunD, but not p65 or p50 to the promoter in ChIP assay. JunD-p65 complexes, identified in vivo by co-immunoprecipitation, were decreased by LY294002 and by small interfering RNA inhibition of PDK-1. Taken together, our data demonstrate a PI3K/PDK-1-dependent functional cooperation of NF-kappaB and JunD in the transcriptional regulation of CCND1 by serum.

In vivo altered unfolded protein response and apoptosis in livers from lipopolysaccharide-challenged cirrhotic rats.

BACKGROUND/AIMS: Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2alpha (eIF2alpha) to attenuate protein synthesis, including in NF-kappaB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFalpha-mediated apoptosis. Thus, we examined in vivo UPR and NF-kappaB activity in livers from cirrhotic and normal LPS-challenged rats. METHODS: Livers were harvested in rats that did or did not receive LPS. RESULTS: Under baseline conditions, no UPR was found in normal livers while PERK/eIF2alpha and ATF6 pathways were activated in cirrhotic livers. After LPS, in normal livers, the PERK/eIF2alpha pathway was transiently activated. ATF6 and IRE1 were activated. In cirrhotic livers, the PERK/eIF2alpha pathway remained elevated. ATF6 and IRE1 pathways were altered. LPS-induced, NF-kappaB-dependent antiapoptotic proteins increased in normal livers whereas their expression was blunted at the posttranscriptional level in cirrhotic livers. CONCLUSIONS: Cirrhotic livers exhibit partial UPR activation in the basal state and full UPR, although altered, after LPS challenge. Sustained eIF2alpha phosphorylation, a hallmark of cirrhotic liver UPR, is associated with a lack of LPS-induced accumulation of NF-kappaB-dependent antiapoptotic proteins which may sensitize cirrhotic livers to LPS/TNFalpha-mediated apoptosis.

Cartography of gene expression in CD8 single cells: novel CCR7- subsets suggest differentiation independent of CD45RA expression.

Understanding the distribution, function, and lineage relationship of CD8+ T-cell subpopulations is of fundamental value for the monitoring of the immune system in several experimental and clinical situations. However, the available data concerning the description of effector and memory CD8+ subsets in humans remain rather fragmentary because different studies favored the usage of distinct and restricted sets of cell surface markers and functional parameters. We associated multiple markers to subdivide CD8+ T cells into 14 different cell types, several of which were not described previously, and evaluated the coexpression of 18 genes simultaneously in individual cells from each subset. Our results show that each subset has a defined pattern of gene expression. Moreover, effector gene expression of CCR7- cells correlated only with CD27 expression levels and CD27/CD28 coexpression but not with CD45RA/R0 phenotypes. Our findings thus describe new CD8+ cell subsets, allow the identification of relatively homogeneous CD8+ subpopulations, provide a predictable and precise correlation between particular cell surface markers and CD8+ T-cell functional properties, and identify effector cells present in both CCR7-CD45RA+ and CCR7-CD45R0+ compartments. The results also indicate that activated cells might modulate the expression of CD45RA/R0 asynchronously rather than CCR7-CD45RA+ cells always issuing from CD45RA- precursors.

Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells.

AIM: To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment. METHODS: Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-X(L) and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots. RESULTS: Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-X(L) expression. CONCLUSION: Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.

Inducible nitric oxide synthase (iNOS) activity could be responsible for resistance or sensitivity to IFN-gamma-induced apoptosis in several human hepatoma cell lines.

Response to interferon-gamma (IFN-gamma)-induced apoptosis of human hepatoma cell lines (HHCLs) is variable. We analyzed this different behavior in Hep3B, Chang-liver, HepG2, and HuH7 cells. We studied (1) IFN-gamma-induced apoptosis, (2) protein expression of Stat1, (3) binding of nuclear proteins to IFN-gamma activated sequence (GAS), (4) mRNA and expression of proteins acting in apoptosis, and (5) HuH7 sensitivity after inducible nitric oxide synthase (iNOS) siRNA transfection. IFN-gamma induced apoptosis in Hep3B and Chang-liver cells only. In all HHCLs, Stat1 protein increased. Binding of proteins and transactivation activity of GAS increased much more in HuH7. In all HHCLs, caspase activity and apoptotic proteins were not implicated in resistance or sensitivity. iNOS mRNA and protein expression increased in HuH7, disappeared in Hep3B, and remained unchanged in Chang-liver and HepG2. We compared the role of iNOS in Hep3B and HuH7. The iNOS inhibitor, L-NAME, sensitized HuH7 to IFN-gamma, Hep3B/HuH7 coculture partially inhibited Hep3B apoptosis, and HuH7 transfection with iNOS siRNA induced a 50% inhibition of iNOS protein and cell apoptosis. GAS activity and overexpression of iNOS in HuH7, but not in the other HHCLs, suggest that this enzyme could play an important role in the resistance of HuH7 to IFN-gamma-induced apoptosis, perhaps by the antiapoptotic action of NO.

Progesterone induces BRCA1 mRNA decrease, cell cycle alterations and apoptosis in the MCF7 breast cancer cell line.

BACKGROUND: Inherited mutations of the BRCA1 gene are responsible for hereditary breast and ovarian cancer syndrome. However, little is known of how disruption of BRCA1 functions preferentially increases cancer risk in hormone-dependent organs. We aimed to study whether BRCA1 was regulated by progesterone in the MCF7 breast cancer cell line. MATERIALS AND METHODS: MCF7 breast cancer cells were incubated with 10(-4) or 10(-10) M progesterone for 24 or 48 hours. BRCA1 expression, proliferation and apoptosis were analysed. RESULTS: 10(-4) M progesterone decreased cell proliferation, cell cycle progression and induced apoptosis. In addition, BRCA1 and cyclin A mRNA decreased. In contrast, none of these effects were observed in MCF7 cells incubated with 10(-10) M progesterone. CONCLUSION: The down-regulation of BRCA1 in MCF7 cells incubated with 10(-4) M progesterone seems to be a consequence of cell cycle alterations rather than a direct effect of the hormone on BRCA1.

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta.

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.

Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line.

p73, the first p53 gene homologue, encodes an array of p73 proteins including p73 alpha full-length (TAp73 alpha) and amino-truncated isoforms (Delta Np73 alpha), two proteins with opposite biological functions. TAp73 alpha can induce tumor suppressive properties, while Delta Np73 alpha antagonizes p53 as well as TAp73 in a dominant-negative manner. In human malignant neuroblasts, p53 protein is wild-type but known to be excluded from the nucleus, therefore disabling its function as a tumor suppressor. The present study investigates whether there is a functional link between p73 isoforms and p53 in neuroblastoma. Experiments were performed on two neuroblastoma cell lines differing in their p53 status, e.g. wild-type p53 SH-5Y5Y cells and mutated p53 IGR-N-91 cells. Data indicate that (i) both TA- and Delta N-p73 alpha enhance p53 protein level in SH-SY5Y cells, whereas level remains unchanged in IGR-N-91 cells; (ii) only in SH-SY5Y cells does forced TAp73 alpha overexpression markedly induce nuclear accumulation of p53 protein; (iii) p21 protein expression is increased in both cell lines infected with TAp73, suggesting that, in IGR-N-91 cells, p21 is induced by p73 through a p53-independent pathway; (iv) in the SHSY5Y cell line, Btg2 expression is strongly enhanced in cells overexpressing TA, and to a lesser extent in cells overexpressing Delta N. Taken together our results suggest that TAp73 may restore p53 function in NB with wild-type nonfunctional p53, but not in NB with mutated p53.