Stephan Geley

CYP11B1 mutations causing congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

Accurate knowledge of the molecular basis of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency is a prerequisite for genetic counseling, prenatal diagnosis, and treatment. Analysis of nine patients suffering from severe manifestations of this disorder led to the identification of seven novel mutations in their CYP11B1 genes. A Caucasian patient was homozygous for the missense mutation R448H, previously found only in Jews of Moroccan origin. An Iranian patient was found to be homozygous for a different mutation in the same codon, R448C. Of four unrelated patients, two were homozygous for a nonsense mutation (W247X), whereas two others were compound heterozygotes for W247X in combination with either R448H or E371G. Two other patients were homozygous for either the missense mutation A331V or an in-frame CTG insertion adjacent to codon 464 (InsCTG464). One patient was a compound heterozygote for two mutations in exon 2, a 28-bp deletion (delta 28bpEx2) and the missense mutation V129M. All of the missense mutations and the CTG insertion caused a complete loss of steroid 11 beta-hydroxylating activity when expressed in cultured cells. These data support previous suggestions of mutational hot spots in CYP11B1 and confirm that severe clinical manifestations are associated with complete loss of enzymatic activity.

Ceramides induce a form of apoptosis in human acute lymphoblastic leukemia cells that is inhibited by Bcl-2, but not by CrmA

The generation of ceramides by the action of acidic and/or neutral sphingomyelinases has been implicated in many forms of apoptosis. We investigated whether exposure to ceramides is sufficient to induce apoptosis in human leukemia cells and, if so, what the characteristics of this form of apoptosis might be. Treatment of the acute lymphoblastic T‐cell line CEM‐C7H2 with short‐ and medium‐chain ceramide analogs (C2‐, C6‐, and C8‐ceramide) resulted in apoptosis, whereas the inactive C2‐dihydroceramide had no effect on cell survival. Induction of apoptosis was relatively slow (∼40% after 24 h) and required high concentrations of ceramide analogs (40–100 μM). To investigate a possible involvement of interleukin 1‐β‐converting enzyme (ICE) or ICE‐related proteases, we treated CEM‐C7H2 sublines constitutively expressing the vaccinia virus protease inhibitor crmA with ceramide analogs. Although such cells were completely resistant to apoptosis induced by antibodies to the Apo‐1/Fas surface receptor (a form of apoptosis known to be inhibitable by CrmA), they were not protected from ceramide‐induced cell death. In contrast, tetracycline‐regulated overexpression of Bcl‐2 protected CEM‐C7H2 sublines stably transfected with corresponding constructs from ceramide‐induced apoptosis. Thus, in these human leukemia cells, ceramides induce a relatively slow death response that can be prevented by Bcl‐2, but is independent of CrmA‐inhibitable proteases. These characteristics distinguish ceramide‐induced from other forms of apoptosis, such as Apo‐1/Fas‐induced cell death where ceramide production has been causally implicated.

High-throughput tissue microarray analysis of G1-cyclin alterations in classical Hodgkin's lymphoma indicates overexpression of cyclin E1

Deregulation of G1‐cyclins (CCN) plays a key role in the pathogenesis of many human malignancies, including non‐Hodgkins lymphomas (NHLs). In contrast to NHL, little is known about phenotypic and genotypic changes in the regulation of the cell cycle in classical Hodgkins lymphoma (cHL). To facilitate analysis of aberrant gene expression in cHL, a lymphoma tissue microarray (TMA) containing 752 cores of 330 different cHL samples was constructed. Direct comparison of Epstein–Barr virus (EBV) latent membrane protein 1 (LMP‐1) expression in Hodgkins and Reed–Sternberg (HRS) cells on conventional full sections with the corresponding duplicate/triplicate tumour cores on the TMA showed a concordance of 100%, indicating that cHL‐TMA is a reliable and representative method for evaluating gene expression profiles in situ. Using TMA technology, protein expression and gene amplification of different G1‐CCNs in cHL were analysed. Among the G1‐CCNs analysed, cyclin E (CCNE) was expressed in 212/253 cases (84%). In most of the individual tumours, over 75% of the HRS cells stained positive for CCNE, suggesting that CCNE is overexpressed in cHL. This overexpression was not due to CCNE gene amplification, as judged by fluorescence in situ hybridization, and did not correlate with EBV infection, as assessed by the expression of LMP‐1. Thus, the overexpression of CCNE could be caused by profound changes in HRS cell‐cycle regulation that could contribute to the malignant phenotype. Copyright

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1β-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells

AbstractGlucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27Kip1. Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27Kip1 accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.

Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells.

AbstractGlucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GRR477H ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GRR477H did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by ‘real-time’ RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction.

RNAi: ancient mechanism with a promising future

RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man. Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means. RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation. We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes. Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients.

Eutopic production of human chorionic gonadotropin β (hCGβ) and luteinizing hormone β (hLHβ) in the human testis

The classical pregnancy and tumor marker hCG has long been considered to be only accidentally expressed ectopically, e.g. by tumors. The biological functions of low levels of bCGβ, hCGα and holo‐hCG in the sera of nonpregnant healthy individuals remained unclear. Immunological analyses by our ultrasensitive time‐resolved fluoroimmunoassays revealed a concentration gradient from < 5 pg hCG in cubital vein serum versus up to 480 pg hCG in the corresponding benign testicular hydrocele fluids. Moreover, hCGβ and its cognate molecule luteinizing hormone β (LHβ) were present in cytosolic extracts of normal human testes. Both hCGβ and hLHβ are eutopically produced as proven by RT‐PCR and subsequent Southern and dot blot analyses. Thus, the view of a purely systemic hormonal function of hLH, and of hCG during pregnancy needs a reassessement as hCGβ and hLHβ are synthesized in the human testis and autocrine/paracrine actions seem to be likely.

Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl.

As a first step toward defining the molecular interactions between ligands and the IgE antigen-combining site, we report here the cDNA cloning and variable (V) region nucleic acid sequences of the heavy (H) and light (L) chains of 2 monoclonal mouse IgE antibodies to trinitrophenyl (ATCC-TIB142 = IGELa2 and ATCC-TIB141 = IGELb4). In all instances, full-length cDNA clones were obtained to facilitate future expression studies. The H chains were encoded by VH genes from the VH3660 and J558 gene families in context with DQ52 and DSP2.2 diversity (D) mini genes, and JH3 and JH4 joining (J) gene segments, respectively. Vk8/Jk2 and Vk1/Jk5 rearrangements encoded the respective L chain V-regions. Both antibodies exhibited considerable conservation of complementarity determining region (CDR) sequences, which will facilitate template-based computer modeling of the three-dimensional structures of complexes formed between various ligands and these antibodies. From sequence comparison between the dinitrophenyl (DNP)-binding myeloma protein MOPC-315 and these IgE antibodies likely candidates for hapten-contact residues within the binding sites of IGELa2 and IGELb4 have been suggested.

Detection and quantitation of genetically marked acute myeloid leukemia by competitive polymerase chain reaction after autologous bone marrow transplantation: a preclinical model for minimal residual disease.

Preclinical models and methods aimed at detecting and quantitating minimal residual disease (MRD) after autologous bone marrow transplantation (BMT) for acute myeloid leukemia (AML) could facilitate assessment of innovative therapeutic strategies for their antileukemic potential. Among the various techniques exploited to identify MRD, polymerase chain reaction (PCR) proved to be a valuable tool in instances in which clonogeneic markers are involved during the evolution of disease. In human AML, however, detection of MRD by PCR is limited to a minority of subgroups, as clonospecific markers are absent or presently unknown. Although gene labeling has proved to be efficient in detecting marker-devoid leukemia cells in preclinical models, detection and quantitation by PCR have not yet been considered. We therefore developed an experimental model in which detection and quantitation of genetically marked murine AML cells are based on a highly sensitive two-step nested PCR and competitive PCR protocol, respectively. We further demonstrated its applicability to a murine syngeneic BMT model that was designed to monitor minimal numbers of gene-tagged AML cells at various time intervals after transplantation. Our results showed that detection and quantitation could reproducibly be achieved at levels as low as one in 10(6) and 10(5) cells, respectively.